NTP TECHNICAL REPORT
 

ON THE 

TOXICOLOGY AND CARCINOGENESIS
 

STUDIES OF PRIMIDONE
 

(CAS NO. 125-33-7)
 

IN F344/N RATS AND B6C3F1 MICE
 

(FEED STUDIES)
 

NATIONAL TOXICOLOGY PROGRAM
 

P.O. Box 12233
 

Research Triangle Park, NC 27709
 

September 2000 

NTP TR 476
 

NIH Publication No. 00-3966
 

National Toxicology Program
 

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 

Health and Human Services (DHHS): the National Cancer Institute (NCI), National Institutes of Health; the 

National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health; the National 

Center for Toxicological Research (NCTR), Food and Drug Administration; and the National Institute for 

Occupational Safety and Health (NIOSH), Centers for Disease Control and Prevention. In July 1981, the 

Carcinogenesis Bioassay Testing Program, NCI, was transferred to the NIEHS. The NTP coordinates the 

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 hazardous 

chemicals. This knowledge is used for protecting the health of the American people and for the 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 assurance audits 

before being presented for public review. 

These studies are designed and conducted to characterize and evaluate the toxicologic potential, including 

carcinogenic activity, of selected chemicals in laboratory animals (usually two species, rats and mice). 

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

human exposure, level of production, and chemical structure. The interpretive conclusions presented in this 

Technical Report are based only on the results of these NTP studies. Extrapolation of these results to other 

species and quantitative risk analyses for humans require wider analyses beyond the purview of these 

studies. Selection per se is not an indicator of a chemical’s carcinogenic potential. 

Listings of all published NTP reports and ongoing studies are available from NTP Central Data 

Management, NIEHS, P.O. Box 12233, MD E1-02, Research Triangle Park, NC 27709 (919-541-3419). 

The Abstracts and other study information for 2-year studies are also available at the NTP’s World Wide 

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

ON THE
 

TOXICOLOGY AND CARCINOGENESIS
 

STUDIES OF PRIMIDONE
 

(CAS NO. 125-33-7)
 

IN F344/N RATS AND B6C3F1 MICE
 

(FEED STUDIES)
 

NATIONAL TOXICOLOGY PROGRAM
 

P.O. Box 12233
 

Research Triangle Park, NC 27709
 

September 2000 

NTP TR 476
 

NIH Publication No. 00-3966
 

National Toxicology Program
 

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES
 

Public Health Service
 

National Institutes of Health
 
2 Primidone, NTP TR 476 

CONTRIBUTORS
 

National Toxicology Program 

Evaluated and interpreted results and reported findings 

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

D.A. Bridge, B.S. 

J.R. Bucher, Ph.D. 

J.K. Haseman, Ph.D. 

J. Heindel, Ph.D. 

R.A. Herbert, D.V.M., Ph.D. 

J.R. Leininger, D.V.M., Ph.D. 

R.R. Maronpot, D.V.M. 

D.P. Orzech, M.S. 

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

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

J.H. Roycroft, Ph.D. 

C.S. Smith, Ph.D. 

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

D.B. Walters, Ph.D. 

K.L. Witt, M.S., Integrated Laboratory Systems, Inc. 

Battelle Columbus Laboratories 

Conducted studies, evaluated pathology findings 

P.J. Kurtz, Ph.D., Principal Investigator 

M.R. Hejtmancik, Ph.D. 

J.D. Johnson, Ph.D. 

R.L. Persing, D.V.M. 

J.D. Toft, II, 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, M.S., D.V.M. 

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. 

NTP Pathology Working Group 

Evaluated slides, prepared pathology report on rats 

(18 July 1996) 

D.G. Goodman, V.M.D., Chairperson 

PATHCO, Inc. 

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

Experimental Pathology Laboratories, Inc. 

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

North Carolina State University 

R.A. Herbert, D.V.M., Ph.D. 

National Toxicology Program 

J.R. Leininger, D.V.M., Ph.D. 

National Toxicology Program 

C. Merrill, D.V.M., Observer 

North Carolina State University 

A. Nyska, D.V.M. 

National Toxicology Program 

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

National Toxicology Program 

Evaluated slides, prepared pathology report on mice 

(17 July 1996) 

P.K. Hildebrandt, D.V.M., Chairperson 

PATHCO, Inc. 

R. Cattley, V.M.D., Ph.D. 

Chemical Industry Institute of Toxicology 

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

Experimental Pathology Laboratories, Inc. 

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

National Toxicology Program 

R.A. Herbert, D.V.M., Ph.D. 

National Toxicology Program 

J.R. Leininger, D.V.M., Ph.D. 

National Toxicology Program 

A. Nyska, D.V.M. 

National Toxicology Program 

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

National Toxicology Program 

Biotechnical Services, Inc. 

Prepared Technical Report 

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

L.M. Harper, B.S. 

D.C. Serbus, Ph.D. 

J.E. Marshall, M.S. 

S.M. Swift, B.S. 
3 

CONTENTS
 

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

EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY ............ 11 
 

TECHNICAL REPORTS REVIEW SUBCOMMITTEE ............................... 12 
 

SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS ......... 13 
 

INTRODUCTION ........................................................ 15 
 

MATERIALS AND METHODS ............................................... 25 
 

RESULTS .............................................................. 35 
 

DISCUSSION AND CONCLUSIONS ........................................... 63 
 

REFERENCES .......................................................... 69 
 

APPENDIX A Summary of Lesions in Male Rats in the 2-Year Feed Study
 

of Primidone ................................................. 81 
 

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

of Primidone ................................................. 117 
 

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

of Primidone ................................................. 147 
 

APPENDIX D Summary of Lesions in Female Mice in the 2-Year Feed Study
 

of Primidone ................................................. 177 
 

APPENDIX E Genetic Toxicology ............................................. 209 
 

APPENDIX F Organ Weights and Organ-Weight-to-Body-Weight Ratios .................. 219 
 

APPENDIX G Hematology and Clinical Chemistry Results ............................ 223 
 

APPENDIX H Determinations of Primidone and Phenobarbital in Plasma ................. 229 
 

APPENDIX I Reproductive Tissue Evaluations and Estrous Cycle Characterization .......... 235 
 

APPENDIX J Chemical Characterization and Dose Formulation Studies .................. 239 
 

APPENDIX K Feed and Compound Consumption in the 2-Year Feed Studies of Primidone ...... 251 
 
Primidone, NTP TR 476 4 

APPENDIX L Ingredients, Nutrient Composition, and Contaminant Levels in NIH-07 Rat 

and Mouse Ration ............................................. 257 
 

APPENDIX M Sentinel Animal Program ........................................ 261 
 

APPENDIX N Single-Dose Toxicokinetic Studies
 

in F344/N Rats and B6C3F1 Mice ................................... 265 
 

APPENDIX O Continuous Breeding Study in Swiss (CD-1®) Mice ....................... 279 
 
H

O N

C2H5 NH

O

 

 

5 

ABSTRACT
 

PRIMIDONE 

CAS No. 125-33-7 

Chemical Formula: C12H14N2O2 Molecular Weight: 218.28 

Synonyms:	 5-Aethyl-5-phenyl-hexahydropyrimidin-4,6-dion; 2-deoxyphenobarbital; 2-desoxyphenobarbital; desoxyphenobarbitone; 

5-ethyldihydro-5-phenyl-4,6 (1H,5H)-pyrimidinedione; 5-ethylhexahydro-4,6-dioxo-5-phenylphrimidine; 5-ethylhexahydro5-phenylpyrimidine-4,6-dione; 5-ethyl-5-phenylhexahydropyrimidine-4,6-dione 

Trade names:	 Cyral; Hexadiona; Hexamidine; Lepimidin; Lepsiral; Majsolin; Midone; Milepsin; Misodine; Misolyne; Mizodin; Mizolin; 

Mylepsin; Mylepsinum; Mysedon; Mysoline; Prilepsin; Primacione; Primaclone; Primacone; Primakton; Primadon; 

Prysoline; Pyrimidone; ROE 101; Sertan 

Primidone is used alone or with other anticonvulsants 

in the control of grand mal, psychomotor, and focal 

epileptic seizures. It may control grand mal seizures 

refractory to other anticonvulsant therapy. Primidone 

was nominated by the National Cancer Institute for 

2-year toxicology and carcinogenicity studies due to 

its human use as an anticonvulsant. Male and female 

F344/N rats and B6C3F1 mice received primidone 

(greater than 99% pure) in feed for 14 days, 

14 weeks, or 2 years. Genetic toxicology studies 

were conducted in Salmonella typhimurium, cultured 

Chinese hamster ovary cells, and mouse bone marrow 

cells. 

14-DAY STUDY IN RATS 

Five male and five female rats were exposed to 0, 

1,250, 2,500, 5,000, 10,000 or 20,000 ppm 

primidone (equivalent to average daily doses of 

approximately 120, 240, 500, 970, or 1,100 mg 

primidone/kg body weight to males and 120, 240, 

500, or 900 mg/kg to females) in feed for 14 days. 

All 20,000 ppm females died before the end of the 

study as did one 10,000 ppm male and two 

20,000 ppm males. The mean body weights of 

10,000 ppm males and females and 20,000 ppm males 

were significantly less than those of the controls. 

Feed consumption by all exposed rats was generally 

similar to that by the controls. Males and females in 

the 10,000 and 20,000 ppm groups were observed to 

have eye discharge, ataxia, and abnormal posture and 

were thin and lethargic. 

14-DAY STUDY IN MICE 

Five male and five female mice were exposed to 0, 

625, 1,250, 2,500, 5,000 or 10,000 ppm primidone 

(equivalent to average daily doses of approximately 

100, 200, 400, or 800 mg/kg body weight to males 

and 100, 250, 500, or 900 mg/kg to females) in feed 

for 14 days. All mice in the 10,000 ppm groups and 

one male and one female mouse in the 5,000 ppm 

groups died on day 3 of the study. The mean body 

weights of mice in the 625, 1,250, 2,500, and 
 

 

 

 

 

6 Primidone, NTP TR 476 

5,000 ppm groups were similar to those of the 

controls. Feed consumption by all exposed mice was 

generally similar to that by the controls. Males and 

females in the 10,000 ppm groups were observed to 

have abnormal posture, ataxia, and lethargy. 

14-WEEK STUDY IN RATS 

Groups of 10 male and 10 female rats were exposed 

to 0, 300, 600, 1,300, 2,500, or 5,000 ppm 

primidone (equivalent to average daily doses of 

approximately 20, 40, 100, 200, or 400 mg/kg) in 

feed for 14 weeks. All rats survived to the end of the 

study. The mean body weights of male and female 

rats in the 2,500 and 5,000 ppm groups were 

significantly less than those of the controls. Feed consumption by all exposed rats was generally similar to 

that by the controls. 

A minimal to mild exposure-related thrombocytosis 

occurred on day 22 and at week 14 in all exposed 

groups of male rats and in females in the 1,300 ppm 

or greater groups. A minimal decrease in hemoglobin 

concentration occurred in 2,500 and 5,000 ppm male 

and female rats on day 22 and at week 14. 

The incidences of centrilobular hepatocyte hypertrophy in male rats exposed to 600 ppm or greater and 

in female rats exposed to 1,300 ppm or greater were 

significantly greater than those in the controls. The 

severity of chronic nephropathy in male rats exposed 

to 1,300 ppm or greater increased with increasing 

exposure concentration. 

14-WEEK STUDY IN MICE 

Groups of 10 male and 10 female mice were exposed 

to 0, 300, 600, 1,300, 2,500, or 5,000 ppm 

primidone (equivalent to average daily doses of 

approximately 50, 100, 200, 400, or 1,000 mg/kg to 

males and 60, 120, 220, 440, or 1,100 mg/kg to 

females) in feed for 14 weeks. Three male and two 

female mice in the 5,000 ppm group died during 

week 1 of the study. The final mean body weights of 

all exposed groups were similar to those of the 

controls. Feed consumption by male mice in the 

5,000 ppm group was slightly greater than that by the 

controls; this may have been due to feed spillage. 

Male and female mice in the 5,000 ppm groups were 

ataxic and lethargic. 

Compared to controls, the estrous cycle lengths of 

females exposed to 1,300, 2,500, or 5,000 ppm were 

significantly longer. The liver weights of male and 

female mice exposed to 600 ppm or greater were 

significantly greater than those of the controls. The 

incidences of centrilobular hepatocyte hypertrophy in 

all exposed males and in females exposed to 600 ppm 

or greater and the incidences of cytoplasmic alteration 

of the adrenal gland and hematopoietic cell proliferation of the spleen in 2,500 and 5,000 ppm males 

and in 5,000 ppm females were significantly greater 

than in the controls. 

2-YEAR STUDY IN RATS 

Groups of 50 male and 50 female F344/N rats were 

exposed to 0, 600, 1,300, or 2,500 ppm primidone 

(equivalent to average daily doses of approximately 

25, 50, or 100 mg/kg) in feed for 2 years. 

Survival, Body Weights, 

and Feed Consumption 

Survival of the 1,300 and 2,500 ppm males was significantly less than that of the controls. The mean 

body weights of males and females in the 2,500 ppm 

groups were less than those of the controls, beginning 

at week 29 for males and week 17 for females; the 

mean body weights of 1,300 ppm males and females 

were less than those of the controls during the second 

year of the study. Feed consumption by all exposed 

groups of rats was generally similar to that by the 

controls. 

Pathology Findings 

Male rats exposed to primidone had increased incidences of thyroid gland follicular cell neoplasms 

(adenoma and/or carcinoma). All exposed groups of 

male rats had follicular cell adenomas or carcinomas 

(combined) at incidences above the historical control 

range, with the highest incidence in the 1,300 ppm 

group. 

Hepatocyte cytoplasmic vacuolation and centrilobular 

hypertrophy were associated with primidone exposure 

in male and female rats. These changes were more 

severe in females than in males and the incidences in 

all exposed groups of females were significantly 

greater than those in the controls. Females in the 

2,500 ppm group had an increased incidence of hepatocellular eosinophilic foci. 
 

 

 

 

 

Primidone, NTP TR 476 7 

In 2,500 ppm males, the incidence of renal tubule 

hyperplasia was greater than that in the controls in the 

standard evaluation. Additional hyperplasias were 

found in the extended evaluation, and the incidences 

in exposed groups of males were significantly greater 

than that in the controls. In the extended evaluation, 

the incidence of renal tubule adenoma in 2,500 ppm 

males was significantly increased. The incidence of 

adenoma or carcinoma (combined) in 2,500 ppm 

males in the combined standard and extended evaluations were marginally increased over those in the 

controls. Male rats had an exposure-related increase 

in the severity of chronic nephropathy, which probably accounted for the reduced survival in the 1,300 

and 2,500 ppm groups. The incidences of kidney 

cysts were increased in 1,300 and 2,500 ppm males. 

Hyperparathyroidism, secondary to the loss of renal 

function, was present in many exposed male rats. 

The incidences of parathyroid gland hyperplasia in all 

groups of exposed males were significantly greater 

than that in the controls. 

2-YEAR STUDY IN MICE 

Groups of 50 male and 50 female mice were exposed 

to dietary levels of 0, 300, 600, or 1,300 ppm 

primidone (equivalent to average daily doses of 

approximately 30, 65, or 150 mg/kg to males and 25, 

50, or 100 mg/kg to females) in feed for 2 years. 

Survival, Body Weights, Feed Consumption, 

and Clinical Findings 

Survival of the 1,300 ppm males was significantly less 

than that of the controls. During the second year of 

the study, the mean body weights of 1,300 ppm male 

and female mice were less than those of the controls. 

The final mean body weights of 600 ppm males and 

females were less than those of the controls. Feed 

consumption by all exposed groups of mice was similar to that by the controls. During the latter part of 

the study, a treatment-related increase in the number 

of animals with swelling of the abdominal area was 

observed; necropsy revealed that the swelling was due 

to liver nodules/masses. 

Pathology Findings 

The liver was a target organ in both male and 

female mice. The incidences and multiplicities of 

hepatocellular neoplasms (hepatocellular adenoma, 

hepatocellular carcinoma, and hepatoblastoma) in all 

exposed groups of males and females (except hepatoblastoma in females) were significantly greater than 

those in the controls. The incidences of hepatocellular adenoma or carcinoma (combined) and hepatocellular adenoma, hepatocellular carcinoma, or 

hepatoblastoma (combined) in all exposed groups 

exceeded the historical control ranges in 2-year NTP 

studies. The incidences of centrilobular hepatocyte 

hypertrophy were increased in exposed groups of 

males and females, and the severities increased with 

increasing exposure concentration. The incidences of 

cytoplasmic vacuolization were increased in all 

exposed groups of females and in 300 ppm males. 

Incidences of eosinophilic focus in all exposed groups 

of females were significantly greater than those in the 

controls. 

Proliferative changes occurred in the thyroid gland 

in an exposure-related manner in male and female 

mice. Incidences of follicular cell hyperplasia were 

increased in all exposed groups of males and in 600 

and 1,300 ppm females, but incidences of follicular 

cell adenomas were increased only in male mice. 

GENETIC TOXICOLOGY 

Primidone was mutagenic in Salmonella typhimurium 

strain TA1535 in the absence of S9 activation only; no 

mutagenicity was detected in strain TA98, TA100, or 

TA1537, with or without S9. Primidone did not 

induce sister chromatid exchanges or chromosomal 

aberrations in cultured Chinese hamster ovary cells, 

with or without S9. The single in vivo study with 

primidone, a mouse bone marrow micronucleus test, 

also gave negative results. 
8 Primidone, NTP TR 476 

CONCLUSIONS 

Under the conditions of these 2-year feed studies, 

there was equivocal evidence of carcinogenic activity* 

of primidone in male F344/N rats based on a marginal 

increase in thyroid gland follicular cell neoplasms, 

primarily adenomas, and a marginal increase in renal 

tubule neoplasms. There was no evidence of carcinogenic activity of primidone in female F344/N rats 

exposed to 600, 1,300, or 2,500 ppm. There was 

clear evidence of carcinogenic activity of primidone in 

male B6C3F1 mice based on the increased incidences 

of hepatocellular neoplasms, and the increased incidence of thyroid gland follicular cell adenomas was 

also considered to be chemical related. There was 

clear evidence of carcinogenic activity of primidone 

in female B6C3F1 mice based on the increased incidences of hepatocellular neoplasms. 

Exposure of rats to primidone resulted in increased 

incidences of hepatocyte cytoplasmic vacuolization 

and centrilobular hypertrophy in males and females 

and eosinophilic foci in females. The increased severity of nephropathy and increased incidence of renal 

tubule hyperplasia in male rats were related to 

primidone exposure. Exposure of male mice to 

primidone resulted in hepatocyte centrilobular hypertrophy and thyroid gland follicular cell hyperplasia. 

Exposure of female mice to primidone resulted in 

hepatocyte centrilobular hypertrophy and cytoplasmic 

vacuolization, eosinophilic focus, and thyroid gland 

follicular cell hyperplasia. 

* Explanation of Levels of Evidence of Carcinogenic Activity is on page 11. A summary of the Technical Reports Review Subcommittee 

comments and the public discussion on this Technical Report appears on page 13. 
Primidone, NTP TR 476 9 

Summary of the 2-Year Carcinogenesis and Genetic Toxicology Studies of Primidone
 

Male Female Male Female
 

F344/N Rats F344/N Rats B6C3F1 Mice B6C3F1 Mice
 

Concentrations 

in feed 

Body weights 

2-Year survival 

rates 

Nonneoplastic 

effects 

Neoplastic 

effects 

Uncertain 

findings 

Level of evidence of 

carcinogenic activity 

0, 600, 1,300, or 

2,500 ppm 

1,300 and 2,500 ppm 

groups less than the 

control group 

13/50, 7/50, 4/50, 0/50 

Liver: hepatocyte 

cytoplasmic vacuolization 

(26/50, 28/50, 33/50, 

43/50); hepatocyte 

centrilobular hypertrophy 

(0/50, 14/50, 33/50, 

40/50) 

Kidney: severity of 

nephropathy (2.2, 2.9, 

3.4, 3.8); renal tubule 

hyperplasia (1/50, 2/50, 

4/50, 10/50) 

None 

Thyroid gland: follicular 

cell adenoma (1/50, 1/50, 

6/49, 3/49); follicular cell 

adenoma or carcinoma 

(2/50, 4/50, 7/49, 4/49) 

Kidney: renal tubule 

adenoma or carcinoma 

(standard and extended 

evaluations combined -

4/50, 2/50, 4/50, 7/50) 

Equivocal evidence 

0, 600, 1,300, or 

2,500 ppm 

1,300 and 2,500 ppm 

groups less than the 

control group 

24/50, 27/50, 31/50, 

28/50 

Liver: hepatocyte 

cytoplasmic vacuolization 

(25/50, 44/50, 46/50, 

44/50); hepatocyte 

centrilobular hypertrophy 

(1/50, 36/50, 38/50, 

35/50); eosinophilic focus 

(2/50, 0/50, 1/50, 18/50) 

None 

None 

No evidence 

0, 300, 600, or 1,300 ppm 

600 and 1,300 ppm groups 

less than the control group 

35/50, 34/50, 31/50, 19/50 

Liver: hepatocyte 

centrilobular hypertrophy 

(3/50, 30/50, 21/50, 18/50) 

Thyroid gland: follicular 

cell hyperplasia (8/49, 

20/48, 31/50, 42/50) 

Liver: hepatocellular 

adenoma (22/50, 41/50, 

39/50, 32/50); 

hepatocellular carcinoma 

(12/50, 31/50, 35/50, 

38/50); hepatocellular 

adenoma or carcinoma 

(31/50, 48/50, 47/50, 

46/50); hepatoblastoma 

(0/50, 17/50, 26/50, 7/50); 

hepatocellular carcinoma or 

hepatoblastoma (12/50, 

39/50, 40/50, 39/50); 

hepatocellular adenoma, 

hepatocellular carcinoma, or 

hepatoblastoma (31/50, 

49/50, 49/50, 46/50) 

Thyroid gland: follicular 

cell adenoma (0/49, 3/48, 

3/50, 6/50) 

None 

Clear evidence 

0, 300, 600, or 1,300 ppm 

600 and 1,300 ppm groups 

less than the control group 

41/50, 42/50, 44/49, 

39/50 

Liver: hepatocyte 

centrilobular hypertrophy 

(1/50, 11/50, 11/49, 

21/50); hepatocyte 

cytoplasmic vacuolization 

(3/50, 35/50, 39/49, 

28/50); eosinophilic focus 

(8/50, 23/50, 24/49, 

17/50) 

Thyroid gland: follicular 

cell hyperplasia (13/50, 

12/48, 28/48, 49/50) 

Liver: hepatocellular 

adenoma (15/50, 42/50, 

45/49, 47/50); 

hepatocellular carcinoma 

(3/50, 11/50, 19/49, 

38/50); hepatocellular 

adenoma or carcinoma 

(16/50, 42/50, 45/49, 

50/50); hepatoblastoma 

(1/50, 4/50, 4/49, 4/50); 

hepatocellular carcinoma 

or hepatoblastoma (4/50, 

12/50, 20/49, 39/50); 

hepatocellular adenoma, 

hepatocellular carcinoma, 

or hepatoblastoma (16/50, 

42/50, 46/49, 50/50) 

None 

Clear evidence 
10 Primidone, NTP TR 476 

Summary of the 2-Year Carcinogenesis and Genetic Toxicology Studies of Primidone
 

Genetic toxicology 

Salmonella typhimurium gene mutations: Positive in strain TA1535 without S9; negative in strains TA98, TA100, and 

TA1537 with and without S9 

Sister chromatid exchanges 

Cultured Chinese hamster ovary cells in vitro: Negative with and without S9 

Chromosomal aberrations 

Cultured Chinese hamster ovary cells in vitro: Negative with and without S9 

Micronucleated erythrocytes 

Mouse bone marrow in vivo: Negative 
Primidone, NTP TR 476 11 

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. 

•	 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. 

•	 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.
 

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

of neoplasms that may be chemical related. 

•	 No evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing no chemical-related
 

increases in malignant or benign neoplasms.
 

•	 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. These considerations should include: 

• adequacy of the experimental design and conduct;
 

• occurrence of common versus uncommon neoplasia;
 

• progression (or lack thereof) from benign to malignant neoplasia as well as from preneoplastic to neoplastic lesions; 

•	 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; 

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

or tissue;
 

• latency in tumor induction; 

•	 multiplicity in site-specific neoplasia; 

•	 metastases; 

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

lesion in another sex or species); 

• presence or absence of dose relationships; 

•	 statistical significance of the observed tumor increase; 

•	 concurrent control tumor incidence as well as the historical control rate and variability for a specific neoplasm; 

•	 survival-adjusted analyses and false positive or false negative concerns; 

•	 structure-activity correlations; and 

• in some cases, genetic toxicology. 
12 Primidone, NTP TR 476 

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 primidone on 

12 December 1996 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 the NTP studies: 

• to ascertain that all relevant literature data have been adequately cited and interpreted, 

• to determine if the design and conditions of the NTP studies were appropriate, 

• to ensure that the Technical Report presents the experimental results and conclusions fully and clearly, 

• to judge the significance of the experimental results by scientific criteria, and 

• to assess the evaluation of the evidence of carcinogenic activity and other observed toxic responses. 

Gary P. Carlson, Ph.D., Chairperson 

School of Health Sciences 

Purdue University 

West Lafayette, IN 

Arnold L. Brown, M.D. 

University of Wisconsin Medical School 

Madison, WI 

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., Principal Reviewer 

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., Principal Reviewer 

Division of Biostatistics 

Dana-Farber Cancer Institute 

Boston, MA 

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

Department of Pharmacology 

Howard University College of Medicine 

Washington, DC 

Frederick L. Tyson, Ph.D. 

St. Mary’s Hospital and Medical Center 

Cancer Research Institute 

Grand Junction, CO 

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

National Cancer Institute 

Frederick, MD 
 

 

 

Primidone, NTP TR 476 13 

SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS
 

On 12 December 1996, the draft Technical Report 

on the toxicology and carcinogenicity studies of 

primidone received public review by the National 

Toxicology Program’s Board of Scientific Counselors’ 

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 primidone by discussing 

the uses of the chemical and the rationale for study, 

describing the experimental design, reporting on 

survival and body weight effects, and commenting on 

compound-related neoplastic lesions in male and 

female mice and nonneoplastic lesions in male and 

female rats and mice. The proposed conclusions for 

the 2-year studies in mice and rats were equivocal 

evidence of carcinogenic activity in male rats, no 

evidence of carcinogenic activity in female rats, clear 

evidence of carcinogenic activity in male mice, and 

clear evidence of carcinogenic activity in female mice. 

Dr. Goldsworthy, a principal reviewer, agreed in 

principle with the proposed conclusions. He said the 

poor survival in 1,300 and 2,500 ppm male rats, as 

well as decreased weight gain, made the decision 

between equivocal evidence and some evidence 

unclear in male rats, even though the incidences of 

thyroid gland follicular cell and renal tubule 

neoplasms were above the historical control range. 

Dr. Goldsworthy asked whether it might have been 

appropriate with the male rat data to use the survivaladjusted “Poly-3” quantal response employed in the 

chloroprene study. Dr. J.K. Haseman, NIEHS, 

reported that the new “Poly-K” methods will be used 

routinely with the technical reports for the next 

review meeting. This and other newer methods have 

an advantage over current methods in that they do not 

require an assumption regarding whether a tumor is 

fatal or incidental. Dr. Goldsworthy thought that 

there was an overemphasis in the Introduction and 

Discussion sections on relating all of the neoplasm 

responses to a primary metabolite, phenobarbital, and 

that some discussion should be given to possible 

carcinogenic activity of primidone and the other primary metabolite, phenylethylmalonamide. 

Dr. Ryan, the second principal reviewer, agreed with 

the proposed conclusions. She liked the section 

dealing with plasma concentrations of primidone and 

phenobarbital and questioned whether markedly different plasma level patterns between rats and mice 

might explain differences in response between the 

species. Dr. Ryan noted the widespread human usage 

as an anticonvulsant and asked why some of these 

toxicology studies would not have been done as part 

of the FDA approval process. Dr. Dunnick said that 

primidone was developed in the 1950s and nominated 

because there were no long-term toxicology and carcinogenicity studies reported in the literature. 

Dr. LeBoeuf, the third principal reviewer, agreed 

with the proposed conclusions. He commented that 

the pharmacokinetics and toxicokinetics, although 

limited in scope, were extremely useful for cross 

comparisons to studies with phenobarbital and, 

further, that this type of data should be collected routinely to aid in interpretation of other bioassays. 

Dr. LeBoeuf said that the confirmation of an absence 

of Helicobacter in this study was comforting with 

regard to interpretation of the neoplasm results in 

mice. 

There was some discussion about the neoplasmpromoting activity of primidone/phenobarbital. 

Dr. J. Rice, International Agency for Research on 

Cancer, noted the markedly increased incidences of 

hepatoblastomas in exposed mice and said that agents 

capable of promoting hepatocarcinogenic effects in 

certain strains of mice, and especially in male mice, 

invariably generate a significant fraction of hepatoblastomas. This was consistently seen with phenobarbital. These neoplasms are highly malignant, 

metastasize readily, and are often lethal. 

Dr. Goldsworthy moved that the Technical Report on 

primidone be accepted with revisions discussed and 

the conclusions as written. Dr. LeBoeuf seconded the 

motion, which was accepted unanimously with eight 

votes. 
14 Primidone, NTP TR 476
 
H

O N

C2H5 NH

O

 

15 

INTRODUCTION
 

PRIMIDONE 

CAS No. 125-33-7 

Chemical Formula: C12H14N2O2 Molecular Weight: 218.28 

Synonyms:	 5-Aethyl-5-phenyl-hexahydropyrimidin-4,6-dion; 2-deoxyphenobarbital; 2-desoxyphenobarbital; desoxyphenobarbitone; 

5-ethyldihydro-5-phenyl-4,6 (1H,5H)-pyrimidinedione; 5-ethylhexahydro-4,6-dioxo-5-phenylphrimidine; 5-ethylhexahydro5-phenylpyrimidine-4,6-dione; 5-ethyl-5-phenylhexahydropyrimidine-4,6-dione 

Trade names:	 Cyral; Hexadiona; Hexamidine; Lepimidin; Lepsiral; Majsolin; Midone; Milepsin; Misodine; Misolyne; Mizodin; Mizolin; 

Mylepsin; Mylepsinum; Mysedon; Mysoline; Prilepsin; Primacione; Primaclone; Primacone; Primakton; Primadon; 

Prysoline; Pyrimidone; ROE 101; Sertan 

CHEMICAL AND 

PHYSICAL PROPERTIES 

Primidone, a desoxybarbiturate, was synthesized in 

1949 by Bogue and Carrington (1953). It was first 

reported to be clinically effective as an anticonvulsant by Handley and Stewart (1952). Primidone is a 

white crystalline substance with a molecular weight of 

218.28. It differs from phenobarbital by reduction of 

the carbonyl group at position 2 of the pyrimidine 

ring. The compound is tasteless and essentially 

neutral. It is sparingly soluble in water (0.6 g/L at 

37E C) and in most organic solvents with the exception of propylene glycol (20.0 g/L at 37E C) (Bogue 

and Carrington, 1953; Gallagher and Baumel, 1972). 

PRODUCTION, USE, 

AND HUMAN EXPOSURE 

Methods of synthesis include electrolytic reduction of 

phenobarbital, catalytic desulfuration of the corresponding 2-thiobarbituric acid, and ring closure of 

phenylethylmalonamide synthesized from benzyl chloride. The resulting mixture is then cooled and the 

precipitate product crystallized from ethanol:water 

(Sittig, 1979). Approximately 1,800,000 pounds 

(820,000 kg) of primidone were produced in the 

United States in 1982 (USITC, 1983). Approximately 

10,000 pounds (4,500 kg) of primidone were 

imported in 1981 and 90,000 pounds (41,000 kg) in 

1982 and 1983 (USITC, 1982, 1983, 1984). 
16 Primidone, NTP TR 476 

It is estimated that 50 million people worldwide have 

epilepsy, and the annual incidence ranges from 20 to 

70 cases per 100,000 (Shorvon, 1990; Brodie and 

Dichter, 1996). There are approximately 2 million 

cases of epilepsy in the United States (Hauser and 

Kurland, 1975; Mattson et al., 1985). Treatment 

with an antiepileptic drug usually begins when the 

patient has had more than one unprovoked seizure 

within a year (Brodie and Dichter, 1996). Various 

drugs used in reducing the frequency of partial seizures include carbamazepine, phenytoin, valproic 

acid, phenobarbital, and primidone (Brodie and 

Dichter, 1996). 

The starting oral dose of primidone for treatment 

of partial or generalized tonic-clonic seizures is 

10 mg/kg per day, with a maintenance oral dose of 2 

to 30 mg/kg per day. The target therapeutic plasma 

drug concentration is 5 to 12 μg/mL (PDR, 1996; 

Brodie and Dichter, 1996). 

Phenobarbital (a major metabolite of primidone) was 

introduced as an antiepileptic drug in 1912 (Watanabe 

et al., 1977). Target therapeutic plasma concentrations for phenobarbital are 10 to 40 μg/mL (Brodie 

and Dichter, 1996). 

ABSORPTION, DISTRIBUTION, 

METABOLISM, AND EXCRETION 

Experimental Animals 

Phenylethylmalonamide (PEMA) and phenobarbital 

have been identified as the major metabolites of 

primidone in most species of (Gallagher et al., 1972; 

Pisani et al., 1984; Martines et al., 1990; Sato et al., 

1992; Figure 1), mice (McElhatton et al., 1977), 

rabbits (Fujimoto et al., 1968; Hunt and Miller, 

1978), dogs (Frey et al., 1979; Yeary, 1980; Frey 

and Löscher, 1985), and rats (Baumel et al., 1973). 

Baumel et al. (1972, 1973) have shown that 

primidone possesses independent anticonvulsant 

activity, and that primidone and phenobarbital are 

more effective anticonvulsants than PEMA. 

Moriyama et al. (1994) have reported the pharmacokinetic parameters of primidone and its major 

metabolites in the rat (Table 1). The plasma half-life 

of primidone in the mouse has been described (Leal 

et al., 1979; Table 2). In all species, primidone has 

an elimination half-life (6 to 10 hours) that is 

considerably shorter than the elimination half-life for 

phenobarbital (3 to 4 days) (Eadie et al., 1981). 

The plasma levels of primidone and/or phenobarbital 

in the F344/N rat and B6C3F1 mouse in the current 

studies are described in Appendixes H and N. 
Phenylethylmalanomide

H H

O N O N O

C2H5 C2H5 NH NH

O O

Primidone Phenobarbital
 

O

C2H5 C NH2

C

C NH2
 

O
 

Phenylethylmalonamide 

Primidone, NTP TR 476 17 

FIGURE 1
 

Metabolites of Primidone (Baumel et al., 1972)
 
 

 

18 Primidone, NTP TR 476 

TABLE 1 

Pharmacokinetic Parameters of the Plasma Concentration of Primidone (PRM), Phenylethylmalonamide 

(PEMA), and Phenobarbital (PB) After Oral Administration of PRM in Ratsa 

Ka Tmax Cmax T1⁄2 AUCb 

(hour-1) (hour) (Fg/mL) (hour) (Fg@hour/mL) 

PRM 1.17 ± 0.26 1.36 ± 0.14 18.15 ± 1.62 1.64 ± 0.45 76.28 ± 1.35 

PEMA 5.70 ± 0.31 8.11 ± 0.45 4.29 ± 0.21 111.12 ± 4.70 

PB 6.55 ± 0.47 9.64 ± 0.42 4.96 ± 0.29 148.18 ± 8.01 

a Data from Moriyama et al. (1994). PRM at a dose of 50 mg/kg was administered orally. Each value indicates the mean ± standard 

error for six rats. 

b AUC values were calculated from 0 to 12 hours in PRM and 0 to 24 hours in PEMA and PRM after oral administration. 

TABLE 2 

Apparent Half-life in Mouse Plasma of Primidone and its Metabolitesa 

Drug Half-life 

(hour) 

Primidone 2.23 

Phenobarbital 4.26 

Phenylethylmalonamide 2.32 

a Data from Leal et al. (1979). Each compound was delivered transesophageally as a single 50 mg/kg dose. Each value represents the 

mean for five mice. 

Humans 

Primidone is metabolized primarily to PEMA by ring 

scission and by oxidation to phenobarbital by hepatic 

enzyme activity (Gallagher et al., 1972; Pisani et al., 

1984; Martines et al., 1990; Sato et al., 1992; 

Figure 1). Phenobarbital accumulates due to its prolonged serum half-life, and levels of phenobarbital 

equivalent to those obtained with treatment by phenobarbital alone are present in patients on a primidone 

dosage (Booker, 1972a). 

A summary of pharmacokinetic data of primidone in 

humans is presented in Table 3 (Martines et al., 

1990). The plasma half-life of primidone is estimated 

at 6.2 hours and that of phenobarbital at 84 hours. 

Analysis of the plasma concentration-time curve for 

primidone suggests the presence of two distinct 

absorption phases (Matzke et al., 1981). 
 

 

19 

c 

Primidone, NTP TR 476 

TABLE 3 

Parameters Describing the Disposition of Primidone and its Metabolites 

in Young and Elderly Study Groupsa 

Young (n=8) Elderly (n=10) P valueb 

Primidone terminal elimination rate constant (h-1) 0.0510 ± 0.0184 0.0655 ± 0.0244 NS 

Primidone half-life (h) 14.7 ± 3.5 12.1 ± 4.6 NS 

Primidone volume of distribution (1 kg-1)* 0.69 ± 0.18 0.56 ± 0.14 NS 

Primidone AUC (0, 12 h) (mg L-1 h)**c 112 ± 33 110 ± 28 NS 

Primidone total clearance (mL h-1 kg-1)* 33.2 ± 7.2 34.8 ± 9.0 NS 

Phenobarbitone AUC (0, 12 h) (mg L-1 h) 

PEMAd

 AUC (0, 12 h) (mg L-1 h)** 

78.7 ± 44.5 

33.7 ± 22.0 

111.6 ± 51.0 

57.1 ± 20.7 

NS 

<0.05 

Phenobarbitone AUC/Primidone AUC 0.71 ± 0.33 1.10 ± 0.63 NS 

PEMA AUC/Primidone AUC 0.29± 0.15 0.54 ± 0.21 <0.01 

Renal clearance of unchanged primidone 

 (mL h-1 kg-1) 15.3 ± 6.7 11.3 ± 4.8 NS 

Renal clearance of unchanged

 phenobarbitone (mL h-1 kg-1) 1.0 ± 0.2 0.8 ± 0.3 NS 

Renal clearance of PEMA (mL h-1 kg-1) 26.4 ± 8.0 18.3 ± 6.6 <0.05 

Proportion of dose recovered in urine as

 unchanged primidone (%)+ 45.9 ± 16.5 36.1 ± 22.7 NS 

Proportion of dose recovered in urine as

 phenobarbitone (%)+ 2.0 ± 0.8 2.1 ± 0.9 NS 

Proportion of dose recovered in urine as

 unconjugated p-OH-phenobarbitone (%) 2.3 ± 2.1 1.5 ± 0.6 NS 

Proportion of dose recovered in urine as total

 (unconjugated + conjugated) 

p-OH-phenobarbitone (%) 4.3 ± 4.1 3.5 ± 1.8 NS 

Proportion of dose recovered in urine as

 PEMA (%)+ 21.2 ± 8.6 27.1 ± 8.8 NS 

* Assuming complete oral availability 

** Normalized for a dose of 500 mg per day 

+ No conjugates of these compounds were detected. a Data from Martines et al. (1990). Elderly patients were 70 to 81 years and young patients were 18 to 26 years. b NS=Not significant 

AUC=Area under the curve 

d PEMA=Phenylethylmalonamide 

TOXICITY 

Experimental Animals 

In the rat, the oral LD50 is 1,500 mg/kg (Bogue and 

Carrinton, 1953) and the intraperitoneal LD50 is 

240 mg/kg (Chernobrovin et al., 1991). In the 

mouse, the oral LD50 is 280 mg/kg (Sullivan and 

McElhatton, 1975) and the intraperitoneal LD50 is 

332 mg/kg (Kozhevnikov et al., 1981). 

Carl et al. (1987a) hypothesized that chronic 

primidone treatment leads to folate depletion through 

interference with folate metabolism. When rats were 

treated chronically with primidone (100 mg/kg per 

12 hours, per os) for up to 8 weeks, there was an 

effect on folate-dependent metabolism in the liver as 

measured by an increase in the activity of the major 

one-carbon generating enzyme 5,10-hydroxymethyltransferase. The authors were not able to identify the 

primary site for interference with folate metabolism, 

but suggested that primidone interferes with the synthesis of folylpolyglutamates. 

Carl et al. (1987b) found that when primidone was 

given to rats (orally, 100 mg/kg, twice per day) for a 

week, pentaglutamate derivatives of folates (the major 

form in rat liver) were decreased in the liver, and 

total liver and plasma folate concentrations were 

decreased by 30%. Primidone had no effect on brain 

folate concentrations, at least within 8 weeks of 
 

 

 

 

 

 

20 Primidone, NTP TR 476 

treatment. Primidone has also been shown to cause 

folate depletion in clinical situations (Chanarin, 1979; 

Rosenberg et al., 1979; Reynolds, 1981). 

Primidone given to rats was protective against 

seizures induced by hexafluorodiethylether (Carl and 

Smith, 1988). Primidone has also been shown to 

cause changes in hepatic function and morphology in 

dogs receiving the drug for 6 months at 55 to 

165 mg/kg per day. Histologic findings included 

hepatocellular hypertrophy attributable to hyperplasia 

of the smooth endoplasmic reticulum. Single-cell 

necrosis and multifocal lipidosis were observed. 

Electron microscopy of the liver showed dilated bile 

canaliculi and damaged sinusoidal epithelium (Bunch 

et al., 1985). 

It has long been known that phenobarbital, a metabolite of primidone, induces the expression of drug- and 

steroid-metabolizing enzymes, particularly P450 2A, 

2B, and 2C, as well as aldehyde dehydrogenase, 

epoxide hydrolase, UDP-glucuronyl transferase, and 

several glutathione S-transferases (Imaoko et al., 

1989; Waxman and Azaroff, 1992; Honkakoski et al., 

1996). 

Humans 

Epileptic patients normally require long-term therapy 

with anticonvulsant drugs. With prolonged use, 

sporadic side effects reported include folic acid deficiency, induction of hepatic microsomal enzyme systems and hepatotoxic effects, rash, agranulocytosis, 

thrombocytopenia, lupus-like syndrome, and other 

nonspecific effects such as fatigue, listlessness, 

depression, psychosis, decreased libido, and impotence (Mattson et al., 1985; Braide and Davies, 1987; 

Brodie and Dichter, 1996). Primidone may have a 

sedative effect, but tolerance to the primidone sedative 

effect appears to develop even though serum levels of 

the drug/metabolites remain fairly constant. This 

tolerance is thought to represent a change in responsiveness rather than a change in metabolism or 

absorption of the drug (Booker, 1972b). 

Chronic anticonvulsant therapy has been associated 

with folate deficiency, and has been seen with 

primidone as well as some of the other antiepileptic 

drugs (e.g., phenytoin and phenobarbital). The mechanism of anticonvulsant-induced folate depletion is 

unknown. The involvement of these drugs in folate 

depletion has led to speculation that the folate depletion may be required for anticonvulsant action, but 

this concept has not been proven. Folate deficiency 

may cause megaloblastic anemia (Carl et al., 1987a). 

In the clinical situation, massive crystalluria has been 

reported, and the crystals identified as primidone. 

One case report describes a child that had been on a 

combination of primidone (250 mg, twice per day) 

and phenytoin (150 mg/day). One week prior to 

admission, a routine phenobarbital concentration was 

less than 4 μg/mL plasma (normal 15 to 40 μg/mL) 

and a routine phenytoin concentration was 2.5 μg/mL 

(normal 10 to 20 μg/mL). On admission, the 

primidone blood concentration was 150 μg/mL 

(normal 6 to 10 μg/mL), the phenobarbital level was 

8.5 μg/mL, and the phenytoin concentration was 

33 μg/mL. Kidney damage secondary to the crystalluria was not reported (Turner, 1980). 

REPRODUCTIVE TOXICITY 

AND TERATOGENICITY 

Experimental animals 

Because of the suggested association between teratogenic effects in epilepsy and drug treatment, a number 

of animal studies have been carried out. Sullivan and 

McElhatton (1975) evaluated the teratogenic potential 

of primidone in albino ICI mice. Primidone was 

administered either in feed at doses of 500, 1,250, 

2,000, or 2,500 mg primidone/kg feed on gestation 

days 6 through 16, or by gavage at 100, 150, 200, or 

250 mg/kg body weight on gestation days 12 through 

16. Maternal toxicity (ataxia and low pregnancy rate) 

was observed in mice fed 200 or 2,500 mg/kg. 

Dietary administration induced a dose-related increase 

in the incidence of cleft palates in fetuses. The percentages of fetuses with palatal defects in the feed 

controls were 0%; 500 mg/kg, 1.6%; 1,250 mg/kg, 

3.7%; 2,000 mg/kg, 5.3%; and 2,500 mg/kg, 15.1%. 

Oral administration induced submucosal cleft palates 

in 12 of 16 fetuses at 100 mg/kg and full-length cleft 

palates in 8 of 11 at 150 mg/kg and 6 of 15 at 

200 mg/kg. Except for an increase in cutaneous hemorrhages, no other drug-related malformations were 

observed. 

McElhatton et al. (1977) administered doses of 0, 25, 

50, 100, or 150 mg primidone/kg body weight by 
 

 

 

Primidone, NTP TR 476 21 

gavage to groups of 9 to 13 ICI albino mice on 

gestation days 6 through 16. Primidone induced fulllength clefts and submucosal and abnormally shaped 

palatal bones in the fetuses. The numbers of fetuses 

noted with palatal defects in the treated groups were 

16 of 84 (25 mg/kg), 18 of 117 (50 mg/kg), 19 of 102 

(100 mg/kg), and 17 of 92 (150 mg/kg), compared to 

0 of 85 in the controls. 

Oral administration of primidone at 30, 90, or 

180 mg/kg body weight to groups of 19 CD-1 mice 

on gestation days 6 through 16 induced a variety of 

abnormalities in the fetuses. These included cleft 

palates (90 and 170 mg/kg), enlarged cerebral ventricles (all doses), club foot (30 and 180 mg/kg), open 

eyes (180 mg/kg), and hemorrhage in the subarachnoid space (90 mg/kg). In addition, there was an 

increase in advance fetal resorptions (19.4%) at the 

90 mg/kg level when compared to either the vehicle 

(8.1%) or untreated controls (11.8%). The overall 

incidence of fetuses with major defects in all treated 

groups regardless of dose was 4.8% compared to 

1.3% in the untreated controls (Sullivan and 

McElhatton, 1975). 

Rao et al. (1986) reported that daily dosing of 

male Swiss mice with primidone at 4.4, 8.8, or 

13.1 mg/mouse for 5 consecutive days caused a significant dose-dependent increase in the incidence of 

sperm-head abnormalities. 

The teratogenic effects of primidone may be due to 

alterations in folic acid metabolism (Carl et al., 

1987b). 

Folate-deficient epithelial tissues are at increased risk 

for DNA damage, probably due to the role that folate 

plays in DNA synthesis, repair and methylation 

(Blount and Ames, 1995). Recent studies in plants 

have shown that methylation of DNA is one way of 

controlling gene activity, and turns off genes during 

normal development (Ronemus et al., 1996). 

Humans 

There have been a number of epidemiological surveys 

in various parts of the world reporting increases in the 

incidence of congenital malformation in the children 

of women with epilepsy (Elshove and van Eck, 1971; 

Spiedel and Meadow, 1972; Fedrick, 1973; Monson 

et al., 1973; Annegers et al., 1974; Rating et al., 

1982), but a cause and effect relationship between 

exposure to the drug and an adverse effect has not 

been established. It has not been possible to determine whether the increased malformation rate is due 

to the disease or to the drugs used in its treatment. 

Some studies have suggested that the incidence of 

malformations is higher in epileptics receiving drug 

treatment during pregnancy (Janz and Fuchs, 1964; 

Spiedel and Meadow, 1972; Monson et al., 1973; 

Lindhout et al., 1992). 

Primidone has been found in cord blood and in breast 

milk (Martinez and Snyder, 1973; Nau et al., 1980). 

CARCINOGENICITY 

Experimental Animals 

There have been no previous carcinogenicity studies 

of primidone in rodents. However, carcinogenicity 

studies of phenobarbital, a metabolite of primidone, 

have been reported and are summarized below. 

When phenobarbital is given alone to certain strains of 

rodents without prior exposure to an initiator, it eventually elicits a low to moderate yield of hepatocellular 

neoplasms, presumably by promotion of naturally 

(“spontaneously”) initiated cells (IARC, 1976, 1987; 

Feldman et al., 1981; Ward, 1983). Many of these 

early studies of phenobarbital alone did not differentiate between liver tumor types. Other studies have 

shown that phenobarbital (500 ppm in drinking water) 

given to C3H/HeNCr mice at 12 months of age significantly increased the number of gross tumors or 

microscopic foci and adenomas or carcinomas in the 

liver (Ward et al., 1988a). 

Phenobarbital sodium, administered to CFA mice for 

109 weeks at a concentration of 500 mg/kg in feed, 

increased the incidence of liver tumors: liver tumors 

occurred in 24/30 exposed males and 21/28 exposed 

females, compared with 11/45 control males and 

10/44 control females (Thorpe and Walker, 1973). In 

C3H mice exposed to 500 mg/kg phenobarbital in 

feed for 12 months, the incidence of liver tumors was 

16/17 in exposed males and 10/16 exposed females, 

compared with 7/17 in control males and 1/16 in control females (Peraino et al., 1973a,b). In CF1 mice 

receiving 0.05% phenobarbital in drinking water for 

life (maximum duration, 120 weeks), the incidence of 

liver tumors was 77/98 in treated males and 45/73 
 

 

 

 

 

 

22 Primidone, NTP TR 476 

in treated females compared with 12/44 untreated 

males and 0/47 untreated females (Ponomarkov et al., 

1976). When phenobarbital was administered to 

Wistar rats for their lifespan at 500 mg/L in drinking 

water, 13/36 treated males and 9/34 treated females 

had liver tumors, compared to none in 36 male and 

35 female control rats (Rossi et al., 1977). (These 

studies did not classify the liver tumors according to 

the classifications used today.) In contrast, phenobarbital fed to male Fischer rats at 500 ppm for 

1 week followed by 1,000 ppm for 103 weeks 

resulted in foci of nodular hyperplasia in 11/33 rats 

and parenchymal cell damage in all treated rats, but 

no evidence for phenobarbital-induced neoplasms 

(Butler, 1978). Additional studies in CD-1 and 

B6C3F1 mice administered 10, 75, or 150 mg phenobarbital per kg body weight daily resulted in doserelated increases in the incidences of hepatocellular 

adenomas and carcinomas, with a predominant 

eosinophilic phenotypic appearance (McClain, 1993). 

Hepatoblastomas were also observed in treated 

B6C3F1 males, and a few hepatocellular carcinomas 

and hepatoblastomas metastasized to the lung. A 

clear stepwise progression of lesions was observed in 

phenobarbital-treated mice from focal areas of altered 

hepatocytes to focal hyperplasia, adenomas, carcinomas, and metastases. With few exceptions, the 

carcinomas were found within adenomas, strongly 

indicating that these arise from adenomas. 

Phenobarbital is thought to exert its carcinogenic 

activity (or “tumor promoting activity”) in part by the 

induction of DNA synthesis in rodents initiated 

with a genotoxic carcinogen (Klaunig, 1993). Phenobarbital “promotes” liver tumors in mice after 

initiation by diethylnitrosamine (Klaunig, 1993) or 

N-nitrosodiethylamine (Rice et al., 1992, 1994; 

Weghorst et al., 1993/1994) as well as N-nitrosodimethylamine and N-ethoyl-N-nitrosourea (IARC, 

1987). Phenobarbital “promotes” liver tumors in rats 

after initiation by tamoxifen (Carthew et al., 1995) or 

2-acetylaminofluorene (Peraino et al., 1971, 1973b, 

1980) as well as N-nitrosodiethylamine, 2-methylN,N-dimethyl-4-aminoazobenzene, benzo["]pyrene, 

cycasin, N-hydroxy-N-formyl- or -acetylaminobiphenyl, N-nitroso-N-(4-hydroxybutyl)butylamine, or 

N-nitrosomorpholine (IARC, 1987). Phenobarbital 

has also been shown to “promote” liver tumors in a 

nonhuman primate (Patas monkey) after initiation with 

diethylnitrosamine (Palmer et al., 1984). The liver 

tumor promoting effects of phenobarbital on hepatocellular carcinogenesis in the rodent are markedly 

dependent on the underlying genotype of the strain; 

DBA/2 and C3H mice are sensitive to phenobarbitalinduced hepatocarcinogenesis, C57BL/6 are resistant 

and Syrian golden hamsters are generally resistant 

(Rice et al., 1992). 

In a study comparing the responsiveness of three 

strains of rats to phenobarbital induction of liver 

enzymes, it was found that the DA and F344/NCr rats 

showed markedly increased induction of enzymes in 

comparison to the Zucker rat (Lubet et al., 1992). 

Phenobarbital promotes hepatocarcinogenesis in more 

than a single rodent species and in nonhuman 

primates. 

In a study comparing the roles of Aroclor-1254, 

dichlorodiphenyltrichloroethane, and phenobarbital 

on the promotion of liver tumors in D2B6F1 mice 

receiving N-nitrosodiethylamine as the inducing 

agent, phenobarbital was the most effective agent in 

stimulating the evolution of hepatocellular neoplasms 

to hepatoblastomas (Diwan et al., 1994). 

Phenobarbital fed to rats at 500 ppm has been shown 

to induce hepatic P4502B1 by 40 fold. Phenobarbital 

induces this enzyme in the mouse and Patas monkey 

but not in the Syrian hamster (Rice et al., 1992), suggesting that induction of this enzyme may be predictive of susceptibility to liver tumor promotion among 

species. Other studies have suggested that the 

phenobarbital-induced liver monoxygenase system 

may potentiate the formation of reactive oxygen in 

neoplastic liver nodules (Scholz et al., 1990). 

It has been hypothesized that exposure to phenobarbital causes chronic cytotoxicity that may result in 

increased cell turnover, which leads to increased susceptibility to cancer. However, several chronic 

hepatotoxins (e.g., butylated hydroxytoluene, diallylphthalate, acetaminophen) are not carcinogenic in 

mice or rats. In order to further understand the 

mechanisms in phenobarbital liver carcinogenicity in 

the mouse, phenobarbital was given to male B6C3F1 

mice in drinking water at 500 ppm for 40 weeks. 

This treatment regimen was evaluated for its effects 

on histopathological lesions and cell turnover. It was 

found that phenobarbital caused increases in liver/ 

body weight ratios and centrilobular cytomegaly, but 
 

 

 

 

 

 

 

Primidone, NTP TR 476 23 

not thymidine kinase activity or DNA synthesis as 

measured by tritiated thymidine autoradiography or 

bromodeoxyuridine immunohistochemistry. Thus, in 

these studies there were no consistent chronic effects 

on cell turnover (as measured by label index and 

thymidine kinase activity) that correlated with the 

carcinogenic or tumor-promoting capability of phenobarbital (Ward et al., 1988b). 

Humans 

There have been a number of studies to investigate the 

possible influence of anticonvulsant treatment on 

cancer risk. Olsen et al. (1993) reported that, in a 

nested case-control study of 104 lung cancers and 

18 bladder cancers in patients taking a variety of anticonvulsant drugs (including phenobarbital, phenytoin, 

primidone, ospolate, carbamazepine, oxazolidine, 

etosuximide and other drugs) and 322 cancer-free 

controls, there was no indication that anticonvulsant 

treatment was associated with these cancers. There 

was some suggestion that phenobarbital treatment 

reduced the incidence of bladder cancer. [In a previous study done by the same group in Denmark, an 

association was reported between anticonvulsant drug 

therapy and an increase in lung cancer and a decrease 

in bladder cancer (Olsen et al., 1989), but this was 

not confirmed in the 1991 study.] Olsen et al. (1995) 

also found that antiepileptic treatment was not associated with an increased risk for hepatobiliary cancer. 

GENETIC TOXICITY 

Primidone was mutagenic in Salmonella typhimurium 

strain TA1535 when tested in the absence of S9 exogenous metabolic activation; no mutagenic activity 

was detected in the presence of S9, or in any of 

the other tester strains employed, with or without S9 

(Mortelmans et al., 1986). No increase in the 

frequency of sex-linked recessive lethal mutations was 

noted in male Drosophila melanogaster treated as 

larvae by feeding on solutions of 6 to 12 mM 

primidone (Zolotareva et al., 1979). No increases in 

sister chromatid exchanges or chromosomal aberrations were induced in cultured Chinese hamster ovary 

cells after primidone exposure with and without S9 

activation (Riedel and Obe, 1984), and chromosomal 

aberrations were not induced in human peripheral 

lymphocytes treated in vitro in the absence of S9 

(Stenchever and Allen, 1973; Bishun et al., 1975). In 

two independent tests, primidone did not induce dominant lethal mutations in germ cells of male mice 

treated by single intraperitoneal injection (maximum 

doses of 90 or 400 mg/kg, respectively) (Epstein 

et al., 1972; Zolotareva et al., 1979). No induction of chromosomal aberrations was reported in 

bone marrow cells of male mice treated with up to 

400 mg/kg primidone by intraperitoneal injection 

(Zolotareva et al., 1979). There is one report of 

increased frequencies of micronucleated polychromatic erythrocytes in bone marrow of mice 

administered 13.11 mg primidone (approximately 

500 mg/kg) twice at an interval of 24 hours (Rao 

et al., 1986); however, this study had protocol 

deficiencies that make the interpretation of the data 

questionable. Chromosomal effects from primidone 

(somatic or germinal) in vivo have been suggested by 

Buckel (1975, 1976); these reports are abstracts that 

contain no data, statistics, or methods. The current 

NTP studies and other published reports do not support these claims of in vivo mammalian chromosomal 

effects. 

In summary, the genetic toxicity data for primidone 

are limited in scope and amount, but suggest that the 

mutagenic action of the chemical is highly specific: 

clear demonstration of the mutagenic activity of 

primidone was limited to a single report of mutation 

induction in S. typhimurium strain TA1535 in the 

absence of S9 metabolic activation only (Mortelmans 

et al., 1986). 

A greater amount of mutagenicity test data exists for 

phenobarbital, a major metabolite of primidone. The 

activity profile of this drug is similar to that of the 

parent compound. In vitro studies gave isolated positive results with weak mutagenic activity demonstrated in S. typhimurium strain TA1535 in 

the presence of S9 (Zeiger and Haworth, 1985), 

induction of aneuploidy observed in Saccharomyces 

cerevisiae without S9 activation (Albertini et al., 

1985), and weak activity demonstrated in the cultured 

Chinese hamster ovary cell chromosomal aberrations 

assay (Gulati et al., 1985). Negative results were 

reported in tests for induction of DNA single strand 

breaks in hamster V79 cells (Swenberg et al., 1976), 

sister chromatid exchanges and chromosomal aberrations in Chinese hamster ovary cells (Riedel and Obe, 

1984) and human fetal fibroblasts (Stenchever and 
 

24 Primidone, NTP TR 476 

Jarvis, 1971), with and without S9. No initiation of 

unscheduled DNA synthesis, indicative of DNA 

damage, was detected in rat cell cultures treated with 

phenobarbital (Ide et al., 1981; Althaus et al., 1982). 

In vivo, no induction of DNA single strand breaks 

was noted in NMRI mice administered 0.1% phenobarbital in drinking water for 5 days (Schwarz et al., 

1979) or Sprague-Dawley rats given 0.05% phenobarbital in feed for 5 weeks (Brambilla et al., 1986). 

The frequency of micronucleated erythrocytes in 

mouse bone marrow was not increased after two intraperitoneal injections of 116 mg/kg phenobarbital 

(King et al., 1979). Two laboratories reported positive results with phenobarbital in cytogenetic tests 

conducted in vivo with mice (Nandan and Rao, 1981, 

1982, 1983) and human epilepsy patients (Kulkarni 

et al., 1984); however, both investigations contained 

unusual protocols and the results require independent 

confirmation. 

STUDY RATIONALE 

Primidone was nominated by the National Cancer 

Institute for study because there were no previous 

toxicity or carcinogenicity studies reported in the literature. The chemical was administered in the feed 

because the drug is given orally in the clinical 

situation. 
 

 

 

25 

MATERIALS AND METHODS
 

PROCUREMENT AND 

CHARACTERIZATION OF PRIMIDONE 

Primidone was obtained from Siegfried, LTD 

(Zofingen, Switzerland) in one lot (G041889). 

Identity, purity, and stability analyses were conducted 

by the analytical chemistry laboratory, Research 

Triangle Institute (Research Triangle Park, NC) 

(Appendix J). Reports on analyses performed in 

support of the primidone studies are on file at the 

NIEHS. 

The chemical, a white crystalline powder, was identified as primidone by melting point, infrared, 

ultraviolet/visible, nuclear magnetic resonance spectroscopy, and low- and high-resolution mass spectrometry. The purity of lot G041889 was determined 

by Karl Fischer water analysis, thin-layer chromatography, and high-performance liquid chromatography. Karl Fischer water analysis indicated 

0.27% ± 0.01% water. Thin-layer chromatography 

by two systems indicated a major product spot and no 

impurities. High-performance liquid chromatography 

revealed a major peak and one impurity with an area 

of 0.06% by one system or 0.32% by a second system 

relative to the major peak area. The overall purity 

was determined to be greater than 99%. 

Accelerated stability studies of the bulk chemical were 

performed by the analytical chemistry laboratory 

using high-performance liquid chromatography. 

These studies indicated that primidone was stable as 

a bulk chemical for 2 weeks when stored protected 

from light at 22E to 27E C. To ensure stability, the 

bulk chemical was stored at room temperature protected from light in plastic bags in metal containers. 

Stability was monitored over the course of the 14-day, 

14-week, and 2-year studies using high-performance 

liquid chromatography. No degradation of the bulk 

chemical was detected. 

PREPARATION AND ANALYSIS 

OF DOSE FORMULATIONS 

The dose formulations were prepared once during the 

14-day studies, five times during the 14-week studies, 

and approximately every 6 weeks during the 2-year 

studies by mixing primidone with feed (Table J1). 

Homogeneity and stability studies of the 300 and 

5,000 ppm dose formulations for the 14-week studies 

and of 300 and 2,500 ppm dose formulations for 

the 2-year studies were performed by the study laboratory using high-performance liquid chromatography. 

Homogeneity was confirmed, and the stability of the 

dose formulations was confirmed for at least 28 days 

at –14E to –19E C when stored in sealed glass bottles. 

The study laboratory confirmed stability for dose formulations stored in plastic containers at 5E C for at 

least 35 days. 

Periodic analyses of the dose formulations of 

primidone were conducted at the study laboratory 

using high-performance liquid chromatography. For 

the 14-week studies, dose formulations were analyzed 

three times during the studies (Table J3). During the 

2-year studies, dose formulations were analyzed 

approximately every 8 weeks (Table J4). Of the dose 

formulations analyzed and used during the 14-day 

studies, all formulations were within 10% of the 

target. Of the dose formulations analyzed and used 

during the 14-week studies, all formulations were 

within 10% of the target concentration; all animal 

room samples for rats and 14 of 15 animal room 

samples for mice were within 10% of the target concentration. Of the dose formulations analyzed and 

used during the 2-year studies, 57 of 58 dose formulations for rats and 37 of 39 dose formulations for 

mice were within 10% of the target concentration; all 

animal room samples for rats and 9 of 12 animal 

room samples for mice were also within 10% of the 

target concentration. One dose formulation that 

was 111% of the target concentration was fed to 

mice; all dose formulations given to rats were within 

10% of the target concentration. 
 

 

26 Primidone, NTP TR 476 

14-DAY STUDIES 

Male and female F344/N rats and B6C3F1 mice were 

obtained from Taconic Farms (Germantown, NY). 

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

Animals were quarantined for 11 or 12 days and were 

42 or 43 days old on the first day of the studies. 

Groups of five male and five female rats were fed 

diets containing 0, 1,250, 2,500, 5,000, 10,000, or 

20,000 ppm primidone. Groups of five male and 

five female mice were fed diets containing 0, 625, 

1,250, 2,500, 5,000, or 10,000 ppm primidone. Feed 

and water were available ad libitum. Rats were 

housed five per cage and mice were housed individually. Clinical findings were recorded twice daily for 

rats and mice. Feed consumption was recorded twice 

weekly for rats and weekly for mice by cage. The 

animals were weighed initially, weekly, and at the end 

of the studies. Details of the study design and animal 

maintenance are summarized in Table 4. 

14-WEEK STUDIES 

The 14-week studies were conducted to evaluate the 

cumulative toxic effects of repeated exposure to 

primidone and to determine the appropriate exposure 

concentrations to be used in the 2-year studies. 

Male and female F344/N rats and B6C3F1 mice were 

obtained from Simonsen Laboratories (Gilroy, CA). 

On receipt, the rats and mice were 4 weeks old. Animals were quarantined for 11 or 12 days and were 40 

to 42 days old on the first day of the studies. Before 

the studies began, five male and five female rats and 

mice were randomly selected 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 control rats and mice 

using the protocols of the NTP Sentinel Animal 

Program (Appendix M). 

Groups of 20 male and 20 female rats and 10 male 

and 10 female mice were fed diets containing 0, 300, 

600, 1,300, 2,500, or 5,000 ppm primidone. Ten 

male and ten female rats were designated as special 

study animals for hematology and clinical chemistry 

evaluations. Feed and water were available 

ad libitum. Rats were housed five per cage and mice 

were housed individually. Clinical findings were 

recorded weekly for rats and mice. Feed consumption was recorded twice weekly for rats and weekly 

for mice by cage. The animals were weighed initially, weekly, and at the end of the studies. Details 

of the study design and animal maintenance are 

summarized in Table 4. 

At all time points, rats were anesthetized with CO2 

or a CO2/O2 mixture for blood collection. Blood 

for hematology was collected in tubes containing 

potassium EDTA as the anticoagulant. Blood for 

serum analyses was collected in containers without 

anticoagulant, allowed to clot at room temperature, 

and centrifuged to separate serum. Hematology 

determinations, including erythrocyte and leukocyte 

counts, hemoglobin concentration, hematocrit, mean 

cell volume, mean cell hemoglobin, and mean cell 

hemoglobin concentration, were performed on a 

Serono-Baker System 9000 hematology analyzer 

(Baker Instruments, Allentown, PA). Differential 

leukocyte counts and morphologic evaluation of blood 

cells were performed by light microscopic examination of blood films stained with Wright-Giemsa. 

Reticulocyte counts were performed by light microscopy, using smears prepared from blood stained by 

incubating equal volumes of whole blood and new 

methylene blue for at least 20 minutes. A Miller disc 

was used for reticulocyte quantitation. All clinical 

chemistry evaluations were performed on a Hitachi 

704® chemistry analyzer (Boehringer Mannheim, 

Indianapolis, IN) using reagents obtained from the 

manufacturer. Reagents for sorbitol dehydrogenase 

and total bile salts analyses were obtained from Sigma 

Diagnostics (St. Louis, MO). The parameters 

measured are listed in Table 4. 

At the end of the 14-week studies, samples were 

collected for sperm motility and vaginal cytology 

evaluations on rats and mice exposed to 0, 1,300, 

2,500, or 5,000 ppm. The parameters evaluated are 

listed in Table 4. Methods used were those described 

in the NTP’s sperm morphology and vaginal cytology 

evaluations protocol (NTP, 1987). For 12 consecutive days prior to scheduled terminal sacrifice, the 

vaginal vaults of the females were moistened with 

saline, if necessary, and samples of vaginal fluid and 

cells were stained. Relative numbers of leukocytes, 

nucleated epithelial cells, and large squamous epithelial cells were determined and used to ascertain 

estrous cycle stage (i.e., diestrus, proestrus, estrus, 

and metestrus). Male animals were evaluated for 

sperm count and motility. The left testis and left epididymis were isolated and weighed. The tail of the 
 

Primidone, NTP TR 476 27 

epididymis (cauda epididymis) was then removed 

from the epididymal body (corpus epididymis) and 

weighed. Test yolk (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 the numbers 

of motile and nonmotile spermatozoa were counted 

for five fields per slide by two observers. Following 

completion of sperm motility estimates, each left 

cauda epididymis was placed in buffered saline solution. Caudae were finely minced, and the tissue was 

incubated in the saline solution and then heat fixed at 

65E C. Sperm density was then determined microscopically with the aid of a hemacytometer. To quantify spermatogenesis, the testicular spermatid head 

count was determined by removing the tunica albuginea and homogenizing the left testis in phosphatebuffered saline containing 10% dimethyl sulfoxide. 

Homogenization-resistant spermatid nuclei were 

counted with a hemacytometer. 

A necropsy was performed on all core study animals. 

The heart, right kidney, liver, lung, right testis, 

thymus, and thyroid gland were weighed. Tissues for 

microscopic examination were fixed and preserved 

in 10% neutral buffered formalin, processed and 

trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 μm, and stained with hematoxylin and 

eosin. A complete histopathologic examination was 

performed on 0 and 5,000 ppm rats and mice. 

Table 4 lists the tissues and organs routinely 

examined. 

2-YEAR STUDIES 

Study Design 

Groups of 50 male and 50 female rats were fed diets 

containing 0, 600, 1,300, or 2,500 ppm primidone for 

104 weeks. Groups of 50 male and 50 female mice 

were fed diets containing 0, 300, 600, or 1,300 ppm 

primidone for 104 to 105 weeks. 

Source and Specification of Animals 

Male and female F344/N rats and B6C3F1 mice were 

obtained from Taconic Farms (Germantown, NY) for 

use in the 2-year studies. Rats and mice were quarantined for 9 or 13 days before the beginning of the 

studies. Five male and five female rats and mice 

were randomly selected for parasite evaluation and 

gross observation of disease. Rats were 41 or 42 days 

old and mice 38 or 39 days old at the beginning of the 

studies. 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 and female mice were housed five per cage and 

male mice were housed individually. Feed and water 

were available ad libitum. Feed consumption was 

measured over a 7-day period during study weeks 1 

and 4, then once every 4 weeks thereafter by cage. 

Cages for rats and female mice were changed twice 

weekly; cages for male mice were changed weekly. 

Further details of animal maintenance are given in 

Table 4. Information on feed composition and contaminants is provided in Appendix L. 

Clinical Examinations and Pathology 

All animals were observed twice daily. Clinical 

findings were recorded initially and then monthly 

thereafter; body weights were recorded initially, then 

monthly, and at the end of the studies. 

On the last 2 days of the 2-year studies, blood was 

collected from the retroorbital sinus of three anesthetized female rats in each group or via cardiac puncture of three male and female mice per group. There 

were not sufficient numbers of male rats alive at the 

end of the study for determination of plasma concentrations of primidone and phenobarbital. Blood 

samples were collected at five time points (8:30 a.m., 

2:30 p.m., 8:00 p.m., 10:00 p.m., and 8:30 a.m.) for 

the determination of plasma concentrations of 

primidone and/or phenobarbital. Feed was available 

ad libitum during the collection period. The plasma 

was stored at )20E C or lower until analysis. Plasma 

samples were analyzed using a procedure for the 

analysis of concentrations of primidone and phenobarbital developed during the single-dose toxicokinetic 

studies (Appendix N). Plasma was separated via centrifugation and was analyzed using high-performance 

liquid chromatography. The average plasma concentrations of primidone and phenobarbital and standard 

deviations were calculated. The logarithms of these 

values were plotted as a function of time. Results of 

analyses of plasma concentrations of primidone and/or 

phenobarbital at the end of the 2-year studies are 

given in Appendix H. 
 

 

 

 

 

28 Primidone, NTP TR 476 

Complete necropsies and microscopic examinations 

were performed on all rats and mice. At necropsy, 

all organs and tissues were examined for grossly 

visible lesions, and all major tissues were fixed and 

preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned 

to a thickness of 5 to 6 μm, and stained with 

hematoxylin and eosin for microscopic examination. 

For extended evaluation of renal tubule proliferative 

lesions in male rats, kidneys were step-sectioned at 

1 mm intervals to obtain a maximum of four additional sections per kidney. For all paired organs 

(e.g., adrenal gland, kidney, ovary), samples from 

each organ were examined. Tissues examined microscopically are listed in Table 4. 

Microscopic evaluations were completed by the 

study laboratory pathologist, and the pathology data 

were entered into the Toxicology Data Management 

System. The microscopic slides, paraffin blocks, and 

residual wet tissues were sent to the NTP Archives 

for inventory, slide/block match, and wet tissue audit. 

The slides, individual animal data records, and pathology tables were evaluated by an independent quality 

assessment laboratory. The individual 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 

cortex and medulla of male and female rats, bone 

marrow of female rats, heart of male rats, kidney of 

male rats, liver, ovary of female mice, pancreatic 

islets of male and female mice, pituitary gland (pars 

distalis) of female mice, spleen of male rats and male 

and female mice, thyroid gland of male rats and male 

and female mice, and uterus of female mice. 

The quality assessment report and the reviewed slides 

were submitted to the NTP Pathology Working Group 

(PWG) chairperson, who reviewed the selected tissues 

and addressed any inconsistencies in the diagnosis 

made by the laboratory and quality assessment pathologists. Representative histopathology slides containing examples of lesions related to chemical 

administration, examples of disagreements in diagnoses between the laboratory and quality assessment 

pathologists, or lesions of general interest were 

presented by the chairperson to the PWG for review. 

The PWG consisted of the quality assessment pathologist and other pathologists experienced in rodent 

toxicologic pathology. This group examined the 

tissues without any knowledge of dose groups or 

previously rendered diagnoses. When the PWG 

consensus differed from the opinion of the laboratory 

pathologist, the diagnosis was changed. Final diagnoses for reviewed lesions represent a consensus 

between the laboratory pathologist, reviewing 

pathologist(s), and the PWG. Details of these review 

procedures have been described, in part, by Maronpot 

and Boorman (1982) and Boorman et al. (1985). For 

subsequent analyses of the pathology data, the 

decision of whether to evaluate the diagnosed lesions 

for each tissue type separately or combined was 

generally based on the guidelines of McConnell et al. 

(1986). 
Primidone, NTP TR 476 29 

TABLE 4 

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

14-Day Studies 14-Week Studies 2-Year Studies 

Study Laboratory 

Battelle Columbus Laboratories 

(Columbus, OH) 

Battelle Columbus Laboratories 

(Columbus, OH) 

Battelle Columbus Laboratories 

(Columbus, OH) 

Strain and Species 

Rats: F344/N 

Mice: B6C3F1 

Rats: 

Mice: 

F344/N 

B6C3F1 

Rats: 

Mice: 

F344/N 

B6C3F1 

Animal Source 

Taconic Farms 

(Germantown, NY) 

Simonsen Laboratories, Inc. 

(Gilroy, CA) 

Taconic Farms 

(Germantown, NY) 

Time Held Before Studies 

Rats: 11 days 

Mice: 12 days 

Rats: 

Mice: 

11 days 

12 days 

Rats: 

Mice: 

13 days 

9 days 

Average Age When Studies Began 

Rats: 42 days 

Mice: 43 days 

Rats: 

Mice: 

40 days (males) or 41 days (females) 

41 days (males) or 42 days (females) 

Rats: 

Mice: 

41 days (males) or 42 days (females) 

38 days (males) or 39 days (females) 

Date of First Dose 

Rats: 17 September 1990 

Mice: 18 September 1990 

Rats: 

Mice: 

17 December 1990 (males) 

18 December 1990 (females) 

11 December 1990 (males) 

12 December 1990 (females) 

Rats: 

Mice: 

6 April 1992 (males) 

7 April 1992 (females) 

16 April 1992 (males) 

17 April 1992 (females) 

Duration of Dosing 

14 days (7 days/week) 92 days (core study animals) 

22 days (special study rats) (7 days/week) 

Rats: 

Mice: 

104 weeks (7 days/week) 

104-105 weeks (7 days/week) 

Date of Last Dose 

Rats: 1 October 1990 

Mice: 2 October 1990 

Rats: 

Mice: 

18 March 1991 (males) 

19 March 1991 (females) 

12 March 1991 (males) 

13 March 1991 (females) 

Rats: 

Mice: 

29 March 1994 (males 

29-31 March 1994 (females) 

11–12 April 1994 (males) 

13–15 April 1994 (females) 

Necropsy Dates 

Rats: 1 October 1990 

Mice: 2 October 1990 

Rats: 

Mice: 

18 March 1991 (males) 

19 March 1991 (females) 

12 March 1991 (males) 

13 March 1991 (females) 

Rats: 

Mice: 

29 March 1994 (males) 

29-31 March 1994 (females) 

11–12 April 1994 (males) 

13–15 April 1994 (females) 

Average Age at Necropsy 

8 weeks 19 weeks 110 weeks 

Size of Study Groups 

5 males and 5 females Rats: 

Mice: 

20 males and 20 females 

10 males and 10 females 

50 males and 50 females 

Method of Distribution 

Animals were distributed randomly into 

groups of approximately equal initial mean 

body weights. 

Same as 14-day studies Same as 14-day studies 

Animals per Cage 

Rats: 5 

Mice: 1 

Rats: 

Mice: 

5 

1 

Rats: 

Mice: 

5 

1 (males) or 5 (females) 
30 Primidone, NTP TR 476 

TABLE 4 

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

14-Day Studies	 14-Week Studies 2-Year Studies 

Method of Animal Identification 

Tail tattoo 

Diet 

NIH-07 open formula meal diet (Zeigler 

Brothers, Inc., Gardners, PA), available 

ad libitum 

Water 

Tap water (City of Columbus municipal 

supply) via automatic watering system 

(Edstrom Industries, Inc., Waterford, WI), 

available ad libitum 

Cages 

Polycarbonate (Lab Products, Inc., 

Maywood, NJ), changed twice per week in 

rat studies and changed weekly in mice 

studies 

Bedding 

Sani-Chips (P.J. Murphy Forest Products 

Corp., Montville, NJ), changed twice per 

week in rat studies and weekly in mice 

studies 

Cage Filters 

DuPont 2024 spun-bonded polyester (Snow 

Filtration Co., Cincinnati, OH), changed 

every other week 

Animal Room Environment 

Temperature: 21.1E–23.3E C 

Relative humidity: 52%–65% 

Room fluorescent light: 12 hours/day 

Room air changes: 10/hour 

Doses 

Rats:	 0, 1,250, 2,500, 5,000, 10,000, or 

20,000 ppm in feed, available 

ad libitum. 

Mice:	 0, 625, 1,250, 2,500, 5,000, or 

10,000 ppm in feed, available 

ad libitum 

Type and Frequency of Observation 

Observed twice daily; animals were 

weighed initially, weekly, and at the end of 

the studies; clinical findings were recorded 

twice daily. Feed consumption was 

recorded twice weekly for rats and weekly 

for mice by cage. 

Tail tattoo 

Same as 14-day studies 

Same as 14-day studies 

Same as 14-day studies 

Same as 14-day studies 

Same as 14-day studies 

Temperature: 21.1E–23.9E C (rats) or

 21.1E–25.0E C (mice) 

Relative humidity: 40%–62% (rats) or

 20%–97% (mice) 

Room fluorescent light: 12 hours/day 

Room air changes: 10/hour 

0, 300, 600, 1,300, 2,500, or 5,000 ppm in 

feed, available ad libitum 

Observed twice daily; animals were weighed 

initially, weekly, and at the end of the 

studies; clinical findings were recorded 

weekly. Feed consumption was recorded 

twice weekly for rats and weekly for mice by 

cage. 

Tail tattoo 

Same as 14-day studies 

Same as 14-day studies 

Same as 14-day studies, except female mouse 

cages changed twice per week 

Same as 14-day studies, except female mouse 

bedding changed twice per week 

Same as 14-day studies 

Temperature: 20.6E–25.6E C (rats) or

 19.4E–25.0E C (mice) 

Relative humidity: 31%–62% (rats) or

 30%–63% (mice) 

Room fluorescent light: 12 hours/day 

Room air changes: 10/hour 

Rats: 0, 600, 1,300, or 2,500 ppm in feed, 

available ad libitum 

Mice: 0, 300, 600, or 1,300 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 initially and 

then monthly thereafter. Feed consumption 

was recorded over a 7-day period during study 

weeks 1 and 4, then once every 4 weeks 

thereafter. 
Primidone, NTP TR 476 31 

TABLE 4 

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

14-Day Studies 14-Week Studies 2-Year Studies 

Method of Sacrifice 

Anesthetization with carbon dioxide 

followed by exsanguination by cardiac 

puncture 

Necropsy 

None 

Clinical Pathology 

None 

Histopathology 

None 

Same as 14-day studies 

Necropsy performed on all core study 

animals. Organs weighed were heart, right 

kidney, liver, lung, right testis, thymus, and 

thyroid gland. 

Blood was collected from the retroorbital 

sinus of special study rats on study days 4 

and 22 and of core study rats surviving to the 

end of the study for hematology and clinical 

chemistry. 

Hematology: hematocrit, hemoglobin 

concentration, erythrocyte, reticulocyte, and 

nucleated erythrocyte counts, mean cell 

volume, mean cell hemoglobin, mean cell 

hemoglobin concentration, platelet count, 

and total leukocyte count and differentials. 

Clinical chemistry: urea nitrogen, 

creatinine, total protein, albumin, alanine 

aminotransferase, alkaline phosphatase, 

creatine kinase, sorbitol dehydrogenase, and 

bile salts. 

Complete histopathology was performed on 

0, and 5,000 ppm rats and mice. In addition 

to gross lesions and tissue masses, the 

following tissues were examined: adrenal 

gland, brain, clitoral gland, esophagus, 

femur, gallbladder (mice), harderian gland 

(mice), heart, large intestine (cecum, colon, 

rectum), small intestine (duodenum, jejunum, 

ileum), kidney, liver, lung, lymph nodes 

(mandibular and mesenteric), mammary 

gland with adjacent skin, nasal cavity and 

turbinates, ovary, pancreas, parathyroid 

gland, pituitary gland, preputial gland, 

prostate gland, salivary gland, spleen, 

stomach (forestomach and glandular), testes 

with epididymis and seminal vesicle, thymus, 

thyroid gland, trachea, urinary bladder, and 

uterus. In rats, the liver and kidney (males) 

were examined to a no-effect-level. In mice, 

the adrenal gland, liver, and spleen were 

examined to a no-effect-level. 

Same as 14-day studies 

Necropsy performed on all animals. 

None 

Complete histopathology was performed on all 

rats and mice. In addition to gross lesions and 

tissue masses, the following tissues were 

examined: adrenal gland, brain, clitoral 

gland, esophagus, femur, gallbladder (mice), 

heart, large intestine (cecum, colon, rectum), 

small intestine (duodenum, jejunum, ileum), 

kidney, liver, lungs, lymph nodes (mandibular 

and mesenteric), mammary gland with 

adjacent skin, nasal cavity and turbinates, 

ovary, pancreas, parathyroid gland, pituitary 

gland, preputial gland, prostate gland, salivary 

gland, spleen, stomach (forestomach and 

glandular), testes with epididymis and 

seminal vesicle, thymus, thyroid gland, 

trachea, urinary bladder, and uterus. 
32 Primidone, NTP TR 476 

TABLE 4 

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

14-Day Studies 14-Week Studies 2-Year Studies 

Sperm Motility and Vaginal Cytology 

None At the end of the studies, sperm samples None 

were collected from all male animals in the 

0, 1,300, 2,500, and 5,000 ppm groups for 

sperm motility evaluations. The parameters 

evaluated included spermatid heads, 

spermatid count, motility, and concentration. 

The left cauda, left epididymis, and left testis 

were weighed. Vaginal samples were 

collected for up to 12 consecutive days prior 

to the end of the studies from all females 

exposed to 0, 1,300, 2,500, or 5,000 ppm 

for vaginal cytology evaluations. The 

parameters evaluated included relative 

frequency of estrous stages and estrous cycle 

length. 

Determinations of Primidone 

and Phenobarbital in Plasma 

None None On the last 2 days of the studies, blood was 

collected from the retroorbital sinus of three 

female rats or by cardiac puncture from three 

male and three female mice from each group 

at five time points (8:30 a.m., 2:30 p.m., 

8:00 p.m., 10:00 p.m., and 8:30 a.m.) for the 

determination of plasma concentrations of 

primidone and/or phenobarbital. 

STATISTICAL METHODS 

Survival Analyses 

The probability of survival was estimated by the 

product-limit procedure of Kaplan and Meier (1958) 

and is presented in the form of graphs. Animals 

found dead of other than natural causes or missing 

were censored from the survival analyses; animals 

dying from natural causes were not censored. Statistical analyses 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 

are presented in Tables A1, A5, B1, B5, C1, C5, D1, 

and D5 as the numbers of animals bearing such 

lesions at a specific anatomic site and the numbers of 

animals with that site examined microscopically. For 

calculation of statistical significance, the incidences of 

most neoplasms (Tables A3, B3, C3, and D3) and all 

nonneoplastic lesions are given as the numbers of 

animals affected at each site examined microscopically. However, when macroscopic examination 

was required to detect neoplasms in certain tissues 

(e.g., harderian gland, intestine, mammary gland, and 

skin) before microscopic evaluation, or when neoplasms had multiple potential sites of occurrence 

(e.g., leukemia or lymphoma), the denominators 

consist of the number of animals 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., the KaplanMeier estimate of 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). 
 

 

 

 

Primidone, NTP TR 476 33 

Analysis of Neoplasm Incidences 

The majority of neoplasms in these studies were considered to be incidental to the cause of death or not 

rapidly lethal. Thus, the primary statistical method 

used was logistic regression analysis, which assumed 

that the diagnosed neoplasms were discovered as the 

result of death from an unrelated cause and thus did 

not affect the risk of death. In this approach, neoplasm prevalence was modeled as a logistic function 

of chemical exposure and time. Both linear and quadratic terms in time were incorporated initially, and 

the quadratic term was eliminated if the fit of the 

model was not significantly 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 of adjusting for intercurrent mortality is the prevalence analysis of Dinse and Lagakos (1983), further described 

and illustrated by Dinse and Haseman (1986). When 

neoplasms are incidental, this comparison of the 

time-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 summarized in the appendixes. These methods include the life table test (Cox, 1972; Tarone, 

1975), appropriate for rapidly lethal neoplasms, and 

the Fisher exact test and the Cochran-Armitage trend 

test (Armitage, 1971; 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 selected nonneoplastic lesions. For further 

discussion of these statistical methods, refer to 

Haseman (1984). 

Analysis of Nonneoplastic Lesion Incidences 

Because all nonneoplastic lesions in this study were 

considered to be incidental to the cause of death or not 

rapidly lethal, the primary statistical analysis used was 

a logistic regression analysis in which non-neoplastic 

lesion prevalence was modeled as 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. 

Organ and body weight data, which historically have 

approximately normal distributions, were analyzed 

with the parametric multiple comparison procedures 

of Dunnett (1955) and Williams (1971, 1972). Hematology, clinical chemistry, spermatid, and epididymal 

spermatozoal data, which have typically skewed 

distributions, were analyzed using the nonparametric 

multiple comparison methods of Shirley (1977) and 

Dunn (1964). Jonckheere’s test (Jonckheere, 1954) 

was used to assess the significance of the dose-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 dose-related trend (Dunnett’s or Dunn’s 

test). Prior to statistical analysis, extreme values 

identified by the outlier test of Dixon and Massey 

(1951) were examined by NTP personnel, and 

implausible values were eliminated from the analysis. 

Average severity values were analyzed for significance with the Mann-Whitney U test (Hollander and 

Wolfe, 1973). Because vaginal cytology data are proportions (the proportion of the observation period that 

an animal was in 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 of neoplasm incidence in certain 

instances. Consequently, neoplasm incidences from 

the NTP historical control database, which is updated 

yearly, are included in the NTP reports for neoplasms 

appearing to show compound-related effects. 
 

 

 

 

 

 

34 Primidone, NTP TR 476 

QUALITY ASSURANCE METHODS 

The 14-week and 2-year studies were conducted in 

compliance with Food and Drug Administration Good 

Laboratory Practice Regulations (21 CFR, Part 58). 

In addition, as records from the 2-year studies were 

submitted to the NTP Archives, these studies were 

audited retrospectively by an independent quality 

assurance contractor. Separate audits covering 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 in the 

reports and are on file at NIEHS. The audit findings 

were reviewed and assessed by NTP staff, so all 

comments had been resolved or were otherwise 

addressed during the preparation of this Technical 

Report. 

GENETIC TOXICOLOGY 

The genetic toxicity of primidone was assessed by 

testing the ability of the chemical to induce mutations 

in various strains of Salmonella typhimurium, sister 

chromatid exchanges and chromosomal aberrations in 

cultured Chinese hamster ovary cells, and increases in 

the frequency of micronucleated polychromatic bone 

marrow erythrocytes in mice. The protocols for these 

studies and the results are given in Appendix E. 

The genetic toxicity studies of primidone are part of 

a larger effort by the NTP to develop a database that 

would permit the evaluation of carcinogenicity in 

experimental animals from the molecular structure 

and the effect of the chemical in short-term in vitro 

and in vivo genetic toxicity tests. These genetic toxicity tests were originally developed to clarify mechanisms of chemical-induced DNA damage growing out 

of the earlier electrophilicity/mutagenicity relationship 

proposed by Miller and Miller (1977) and the somatic 

mutation theory of cancer (Straus, 1981; Crawford, 

1985). Therefore, the information obtained from 

these tests applies only to mutagenic carcinogens. 

There is a strong correlation between a chemical’s 

potential electrophilicity (structural alert to DNA reactivity), mutagenicity in Salmonella, and carcinogenicity in rodents. The combination of electrophilicity 

and Salmonella mutagenicity is highly correlated 

with the induction of carcinogenicity in rats and mice 

and/or at multiple tissue sites (Ashby and Tennant, 

1991). Other in vitro genetic toxicity tests correlate 

less 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 the most 

predictive in vitro test for rodent carcinogenicity (89% 

of the Salmonella mutagens are rodent carcinogens), 

and that there is no complementarity among the 

in vitro genetic toxicity tests. That is, no battery of 

tests that included the Salmonella test improved the 

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

bone marrow chromosome aberration or micronucleus 

tests appears to be less than the Salmonella test 

(Shelby et al., 1993; Shelby and Witt, 1995). Positive responses in long-term peripheral blood micronucleus tests have not been formally evaluated for 

their predictivity for rodent carcinogenicity. But, 

because of the theoretical and observed associations 

between induced genetic damage and adverse effects 

in somatic and germ cells, the determination of in vivo 

genetic effects is important to the overall understanding of the risks associated with exposure to a 

particular chemical. 
 

 

 

35 

RESULTS
 

RATS 

14-DAY STUDY 

One 10,000 ppm male, two 20,000 ppm males, and 

all 20,000 ppm females died before the end of 

the study (Table 5). The final mean body weights 

and mean body weight gains of 10,000 ppm males 

and females and of 20,000 ppm males were significantly lower than those of the controls; the final mean 

body weight of 20,000 ppm males was less than the 

initial mean body weight. Feed consumption by the 

20,000 ppm males was less than that by the controls. 

Feed consumption by all other exposed groups was 

generally similar to that by the controls. Dietary 

levels of 1,250, 2,500, 5,000, 10,000, or 20,000 ppm 

primidone resulted in average daily doses of approximately 120, 240, 500, 970, or 1,100 mg primidone/ 

kg body weight to males and 120, 240, 500, or 

900 mg/kg to females. The average daily dose for 

20,000 ppm females was not calculated due to high 

mortality. Males and females in the 10,000 and 

20,000 ppm groups were observed to have eye discharge, ataxia, and abnormal posture and were thin 

and lethargic. 

TABLE 5 

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

Dose 

(ppm) 

Survivala Initial 

Mean Body Weightb

 (g) 

Final Change 

Final Weight 

Relative 

to Controls

(%) 

Feed 

Consumptionc 

 Day 7 Day 14 

Male 

0 

1,250 

2,500 

5,000 

10,000 

20,000 

5/5 

5/5 

5/5 

5/5 

4/5d 

3/5e 

136 ± 7 

133 ± 7 

138 ± 7 

137 ± 8 

133 ± 9 

139 ± 7 

211 ± 5 

208 ± 8 

212 ± 9 

207 ± 9 

175 ± 7** 

123 ± 9** 

75 ± 4 

75 ± 1 

74 ± 3 

70 ± 3 

46 ± 4** 

!25 ± 4** 

99 

100 

98 

83 

58 

16.3 

15.4 

15.9 

15.9 

15.1 

6.8 

16.8 

17.4 

17.1 

17.9 

14.9 

7.6 

Female 

0 

1,250 

2,500 

5,000 

10,000 

20,000 

5/5 

5/5 

5/5 

5/5 

5/5 

0/5f 

103 ± 3 

101 ± 2 

104 ± 2 

105 ± 2 

104 ± 2 

103 ± 3 

140 ± 4 

135 ± 2 

137 ± 2 

138 ± 3 

125 ± 2** 

— 

36 ± 2 

34 ± 2 

32 ± 2 

34 ± 1 

20 ± 2** 

— 

97 

98 

99 

89 

— 

11.3 

10.4 

11.2 

12.2 

9.4 

4.4 

12.1 

11.4 

11.5 

12.0 

11.3 

— 

** Significantly different (P#0.01) from the control group by Williams’ or Dunnett’s test a Number of animals surviving at 14 days/number initially in group. Subsequent calculations are based on animals surviving to the end of 

the study. b Weights and weight changes are given as mean ± standard error. No data were calculated for groups with 100% mortality. 

Feed consumption is expressed as grams of feed consumed per animal per day. d Day of death: 9 e Day of death: 9, 12 f Day of death: 5, 6, 9 

c 
 

 

 

36 

c 

Primidone, NTP TR 476 

14-WEEK STUDY
 

All rats survived to the end of the study (Table 6). concentrations of 300, 600, 1,300, 2,500, or 

The final mean body weights and mean body weight 5,000 ppm primidone resulted in average daily doses 

gains of male and female rats in the 2,500 and of approximately 20, 40, 100, 200, or 400 mg/kg to 

5,000 ppm groups were significantly lower than those males and females. Clinical findings that may have 

of the controls. Feed consumption by all exposed rats been related to primidone exposure included nasal 

was generally similar to that by the controls. Dietary and/or eye discharge. 

TABLE 6 

Survival, Body Weights, and Feed Consumption of Rats in the 14-Week Feed Study of Primidone 

Dose 

(ppm) 

Survivala Initial 

Mean Body Weightb

 (g) 

Final Change 

Final Weight 

Relative 

to Controls

(%) 

Feed 

Consumptionc 

 Week 1 Week 14 

Male 

0 

300 

600 

1,300 

2,500 

5,000 

10/10 

10/10 

10/10 

10/10 

10/10 

10/10 

110 ± 3 

113 ± 3 

112 ± 3 

112 ± 3 

112 ± 3 

112 ± 3 

348 ± 6 

363 ± 8 

358 ± 2 

356 ± 4 

325 ± 8* 

321 ± 2** 

238 ± 4 

251 ± 7 

246 ± 3 

244 ± 3 

213 ± 7** 

204 ± 5** 

104 

103 

103 

93 

92 

14.4 

15.2 

14.9 

15.0 

14.4 

15.4 

17.4 

17.5 

17.6 

15.6 

20.2 

18.5 

Female 

0 

300 

600 

1,300 

2,500 

5,000 

10/10 

10/10 

10/10 

10/10 

10/10 

10/10 

101 ± 2 

100 ± 2 

102 ± 2 

101 ± 2 

101 ± 2 

102 ± 2 

201 ± 3 

198 ± 3 

197 ± 4 

194 ± 3 

187 ± 3** 

188 ± 2** 

99 ± 4 

97 ± 2 

95 ± 3 

93 ± 2 

86 ± 3** 

86 ± 3** 

99 

98 

97 

93 

94 

11.4 

11.6 

11.5 

12.0 

12.4 

12.4 

10.4 

10.7 

10.7 

9.8 

10.1 

9.4 

* Significantly different (P#0.05) from the control group by Williams’ or Dunnett’s test 

** P#0.01 a Number of animals surviving at 14 weeks/number initially in group. b	 Weights and weight changes are given as mean ± standard error. Final mean body weights and mean body weight changes of 2,500 and 

5,000 ppm males are based on five animals per group due to dehydration in some animals. 

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

The hematology and clinical chemistry data for rats in 

the 14-week study are listed in Table G1. A minimal 

to mild exposure-related thrombocytosis, evidenced 

by increased platelet counts, occurred on day 22 and 

at week 14 in all exposed groups of male rats and in 

females exposed to 1,300 ppm or greater. A minimal 

decrease in hemoglobin concentration, suggesting an 

anemic tendency, occurred in 2,500 and 5,000 ppm 

male and female rats on day 22 and at week 14. 

There were, however, no alterations of other markers 

of anemia (hematocrit values or erythrocyte counts) to 

support the decreases in hemoglobin. A minimal to 

mild hyperproteinemia (increased total protein concentration) occurred on day 22 and at week 14 in male 

rats exposed to 600 ppm or greater and 2,500 and 

5,000 ppm female rats. In the male rats, albumin 

concentrations also were increased at week 14 in the 

600 ppm or greater groups. There were minimal to 

mild decreases of alkaline phosphatase activity in the 

1,300 ppm females and 2,500 ppm or greater male 

and female rats on day 22 and in the 1,300 ppm or 

greater males and all exposed groups of females at 
 

 

 

Primidone, NTP TR 476 37 

week 14. Changes in other hematological and clinical 

chemistry variables did not appear to be exposure 

related and were not considered toxicologically 

relevant. 

No significant differences in sperm motility or vaginal 

cytology parameters between exposed groups and 

control groups were observed (Table I1). 

The liver weights of males exposed to 600 ppm or 

greater and females exposed to 300 ppm or greater 

were significantly greater than those of the controls 

(Table F1). 

The incidences of centrilobular hepatocyte hypertrophy in male rats exposed to 600 ppm or greater and in 

female rats exposed to 1,300 ppm or greater were 

significantly greater than those in the controls 

(Table 7). Hepatocytes most closely associated with 

central veins were enlarged, with a gradual decrease 

in size closer to the portal triad. This enlargement 

was due exclusively to an increase in cell cytoplasm 

which tended to stain intensely eosinophilic, suggesting an increased size or number of organelles. There 

was chemical-related hepatocellular hypertrophy at 

exposure concentrations of 600 ppm or greater, but 

the morphology at the 5,000 ppm concentration 

included vacuolization. The severity of chronic 

nephropathy of the kidney in male rats exposed to 

1,300 ppm or greater increased with increasing exposure concentration (Table 7). Exposed males also 

exhibited dilation of one or more of the renal cortical 

tubules (principally in the distal nephron), which were 

filled with an eosinophilic proteinaceous fluid (hyaline 

casts), which is an indication of increased severity 

of nephropathy. The severity grading scheme for 

nephropathy in this 14-week study was somewhat 

unusual in that the background incidence of chronic 

nephropathy in control males was graded as minimal, 

rather than not graded. This made all severities for 

this lesion higher than usually observed for 14-week 

studies. A post-PWG review of the kidney step 

sections showed little difference in the severity of 

nephropathy between the 600, 1,300, and 2,500 ppm 

groups. 

Exposure Concentration Selection Rationale: Based 

on lower mean body weights, increased liver weights, 

and severity of nephropathy at 5,000 ppm, the highest 

primidone exposure concentration for the 2-year feed 

study in rats was set at 2,500 ppm. 

TABLE 7 

Incidences of Selected Nonneoplastic Lesions in Rats in the 14-Week Feed Study of Primidone 

0 ppm 300 ppm 600 ppm 1,300 ppm 2,500 ppm 5,000 ppm 

Male 

Livera 

Hepatocyte Centrilobular,

 Hypertrophyb 

10 

0 

10 

0 

10 

10** (1.0)c 

10 

10** (2.1) 

10 

10** (3.0) 

10 

10** (3.0) 

Kidney 

Nephropathy

10 

9 (1.0) 

10 

10 (1.0) 

10 

10 (1.2) 

10 

10 (1.3) 

10 

10 (1.9)** 

10 

10 (3.0)** 

Female 

Liver 

Hepatocyte Centrilobular,

 Hypertrophy

10 

0 

10 

0 

10 

1 (1.0) 

10 

10** (2.0) 

10 

10** (3.0) 

10 

10** (3.0) 

** Significantly different (P#0.01) from the control group by the Fisher exact test (incidences) or the Mann-Whitney U test (severity of 

nephropathy) a Number of animals with organ/tissue examined microscopically b Number of animals with lesion 

Average severity grade of lesions in affected animals: 1=minimal; 2=mild; 3=moderate; 4=marked c 
 

 

38 Primidone, NTP TR 476 

2-YEAR STUDY 

Survival 

Estimates of 2-year survival probabilities for male and 

female rats are shown in Table 8 and in the KaplanMeier survival curves (Figure 2). Survival of the 

1,300 and 2,500 ppm males was significantly lower 

than that of the controls; survival of all other exposed 

groups was similar to that of the controls. 

Body Weights, Feed and Compound 

Consumption, and Clinical Findings 

The mean body weights of males and females in the 

2,500 ppm groups were lower than those of the 

controls beginning at week 29 for males and week 17 

TABLE 8 

for females; the mean body weights of 1,300 ppm 

males and females were lower than those of the 

controls during the second year of the study (Tables 9 

and 10 and Figure 3). Feed consumption by all 

exposed groups of rats was generally similar to that 

by the controls (Tables K1 and K2). Dietary levels of 

600, 1,300, or 2,500 ppm primidone resulted in 

average daily doses of approximately 25, 50, or 

100 mg/kg to males and females. Male rats in the 

1,300 and 2,500 ppm groups were observed to be 

lethargic during the second year of the study; no other 

clinical findings related to primidone exposure were 

observed. 

Survival of Rats in the 2-Year Feed Study of Primidone
 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

Male 

Animals initially in study	 50 50 50 50 

Moribund 33 33 39 36 

Natural deaths 4 10 7 14 

Animals surviving to study termination 13 7 4 0 

Percent probability of survival at end of studya 26 14 8 0 

Mean survival (days)b 639 645 631 558 

Survival analysisc	 P<0.001 P=0.324 P=0.016 P<0.001 

Female 

Animals initially in study	 50 50 50 50 

Moribund 21 18 14 19 

Natural deaths 5 5 5 3 

Animals surviving to study termination 24 27 31 28d 

Percent probability of survival at end of study 48 54 62 56 

Mean survival (days) 673 650 684 672 

Survival analysis	 P=0.450N P=0.968N P=0.219N P=0.597N 

a Kaplan-Meier determinations b	 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 group columns. A negative trend or lower mortality in an exposure group is indicated by N. d	 Includes one animal that died during the last week of the study 

c 
Primidone, NTP TR 476 39 

Figure 2
 

Kaplan-Meier Survival Curves for Male and Female Rats
 

Exposed to Primidone in Feed for 2 Years
 
40 Primidone, NTP TR 476 

TABLE 9 

Mean Body Weights and Survival of Male Rats in the 2-Year Feed Study of Primidone 

Weeks 0 ppm 600 ppm 1,300 ppm 2,500 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 (g) Survivors (g) controls) Survivors (g) controls) Survivors (g) controls) Survivors 

1 151 50 151 100 50 150 100 50 150 99 50 

2 191 50 192 100 50 191 100 50 190 99 50 

3 222 50 227 102 50 223 100 50 227 102 50 

4 256 50 258 101 50 256 100 50 258 101 50 

5 271 50 276 102 50 276 102 50 278 103 50 

6 294 50 299 102 50 299 102 50 296 101 50 

7 308 50 309 100 50 306 99 50 305 99 50 

8 327 50 330 101 50 329 101 50 324 99 50 

9 336 50 342 102 50 337 100 50 329 98 50 

10 344 50 351 102 50 349 101 50 339 99 50 

11 350 50 359 103 50 355 101 50 348 99 50 

12 363 50 369 102 50 364 100 50 358 99 50 

13 372 50 378 102 50 376 101 50 369 99 50 

17 395 50 401 101 50 398 101 50 386 98 50 

21 421 50 424 101 50 423 100 50 405 96 50 

25 440 50 442 100 50 438 100 50 418 95 50 

29 459 50 456 99 50 450 98 50 432 94 50 

33 471 50 467 99 50 458 97 50 440 94 50 

37 480 50 474 99 50 466 97 50 441 92 50 

41 490 50 485 99 50 476 97 50 454 93 49 

45 493 48 489 99 50 479 97 50 458 93 49 

49 506 48 498 98 49 488 96 50 455 90 49 

53 514 48 508 99 49 488 95 49 464 90 49 

57 517 48 510 99 49 496 96 49 471 91 48 

60 518 48 509 98 49 494 95 49 468 90 48 

65 521 48 515 99 49 500 96 48 468 90 45 

69 521 46 505 97 49 493 95 48 460 88 45 

73 522 45 510 98 46 492 94 48 459 88 44 

77 521 43 505 97 45 485 93 47 444 85 40 

81 513 42 503 98 43 481 94 44 443 86 29 

85 510 37 500 98 40 471 93 39 390 77 18 

89 498 36 479 96 38 469 94 33 

93 485 33 477 98 29 445 92 23 

97 483 24 462 96 22 432 89 14 

101 468 18 444 95 14 

Mean for weeks 

1-13 291 295 101 293 101 290 100 

14-52 462 460 100 453 98 432 94 

53-101 507 494 97 479 94 452 89 
Primidone, NTP TR 476 41 

TABLE 10 

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

Weeks 0 ppm 600 ppm 1,300 ppm 2,500 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 (g) Survivors (g) controls) Survivors (g) controls) Survivors (g) controls) Survivors 

1 118 50 118 99 50 117 99 50 118 99 50 

2 133 50 133 100 50 135 102 50 135 101 50 

3 143 50 143 100 50 146 102 50 145 101 50 

4 160 50 158 99 50 159 99 50 157 98 50 

5 167 50 166 99 50 166 99 50 165 99 50 

6 176 50 173 98 50 175 99 50 172 97 50 

7 185 50 183 99 50 182 98 50 178 96 50 

8 186 50 184 99 50 182 98 50 182 98 50 

9 190 50 186 98 50 187 99 50 184 97 50 

10 194 50 192 99 50 191 98 50 189 97 50 

11 200 50 197 98 50 193 96 50 192 96 50 

12 204 50 199 98 50 197 96 50 195 95 50 

13 207 50 201 97 50 199 96 50 198 96 50 

17 219 50 212 97 50 210 96 50 206 94 50 

21 229 50 223 97 50 219 96 50 212 93 50 

25 236 50 230 98 50 225 95 50 218 92 50 

29 247 50 242 98 50 237 96 50 225 91 50 

33 255 50 252 99 50 243 95 50 233 91 50 

37 265 50 259 98 50 252 95 50 238 90 50 

41 273 50 268 98 50 258 95 50 244 89 50 

45 280 50 274 98 50 268 96 50 251 90 50 

49 294 50 284 97 50 278 94 50 260 88 50 

53 301 50 288 96 50 284 94 50 266 89 50 

57 308 49 296 96 49 291 94 49 276 90 50 

60 317 48 304 96 47 300 95 49 281 89 50 

65 322 48 309 96 45 304 94 49 291 90 49 

69 328 48 317 97 43 310 95 48 295 90 48 

73 339 48 326 96 43 319 94 47 302 89 46 

77 341 48 328 96 42 322 95 47 304 89 46 

81 342 45 328 96 42 325 95 45 310 91 42 

85 350 44 330 94 38 327 93 45 308 88 42 

89 347 44 329 95 36 330 95 43 314 90 38 

93 358 37 340 95 34 338 94 40 320 89 37 

97 363 32 347 96 33 338 93 39 315 87 37 

101 361 29 348 96 29 337 93 34 317 88 33 

Mean for weeks 

1-13 174 172 99 171 98 170 98 

14-52 255 249 98 243 95 232 91 

53-101 337 322 96 317 94 300 89 
 

 

 

42 Primidone, NTP TR 476 

Figure 3
 

Growth Curves for Male and Female Rats
 

Exposed to Primidone in Feed for 2 Years
 
 

 

Primidone, NTP TR 476 43 

Determinations of Primidone 

and Phenobarbital in Plasma 

On the last 2 days of the study, blood was collected 

from the retroorbital sinus of three female rats at five 

time points for the determination of plasma concentrations of primidone and phenobarbital. The plasma 

concentrations of primidone and phenobarbital and 

their standard deviations are given in Tables H1 and 

H2. For the female rats, peak primidone and phenobarbital plasma concentrations increased with increasing exposure concentration. The peak concentrations 

were observed from 8:00 p.m. until 10:00 p.m. 

Pathology and Statistical Analyses 

This section describes the statistically significant or 

biologically noteworthy changes in the incidences of 

neoplasms and/or nonneoplastic lesions of the thyroid 

gland, liver, kidney (with parathyroid gland, bone, 

forestomach, glandular stomach, and lung), and testis 

and the incidences of mononuclear cell leukemia. 

Summaries of the incidences of neoplasms and nonneoplastic lesions, individual animal tumor diagnoses, 

statistical analyses of primary neoplasms that occurred 

with an incidence of at least 5% in at least one animal 

group, and historical incidences for the neoplasms 

mentioned in this section are presented in Appendix A 

for male rats and Appendix B for female rats. 

Thyroid Gland: Male rats exposed to primidone 

had increased incidences of follicular cell neoplasms 

(adenoma and/or carcinoma) (Tables 11 and A3). All 

exposed groups of male rats had follicular cell adenomas or carcinomas (combined) at incidences above the 

historical control range (Table A4), with the highest 

incidence in the 1,300 ppm group; however, there 

was no significant trend or pairwise effect. One follicular cell carcinoma was observed in a control 

female. In some cases, these neoplasms have been 

attributed to increased liver metabolism of thyroxin, 

secondary to a chemical-induced increase in microsomal glucuronidation activity. Usually, however, 

increased metabolism of thyroxin leads to diffuse 

thyroid follicular cell hyperplasia as well as neoplasia 

(Capen, 1996). In this study, only the neoplasms 

were present; diffuse follicular cell hyperplasia 

occurred only rarely. The reason for the lack of a 

significant increase in the incidence of hyperplasia 

was not apparent. Follicular cell adenomas were 

well-differentiated and well-circumscribed masses that 

compressed the adjacent parenchyma. They were 

composed of variably sized follicles and/or cystic 

spaces with papillary fronds projecting into the lumen. 

The cells were more basophilic than normal, but 

otherwise resembled normal follicular epithelial cells. 

Follicular cell carcinomas were more varied in 

appearance than adenomas and showed some invasion 

into surrounding parenchyma or into the fibrous 

capsule. In some areas the cells were arranged in 

solid sheets, as well as in follicles and cysts. 
 

 

44 Primidone, NTP TR 476 

TABLE 11 

Incidences of Neoplasms and Nonneoplastic Lesions of the Thyroid Gland in Rats 

in the 2-Year Feed Study of Primidone 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

Male 

Number Examined Microscopically 

Follicle, Cysta 50 1 (2.0)b 

50 

3 (2.3) 

49 

1 (3.0) 

49 

6 (2.8) 

Follicular Cell, Hyperplasia 2 (1.5) 1 (2.0) 2 (2.0) 1 (1.0) 

Follicular Cell Adenomac 

Overall rated 1/50 (2%) 1/50 (2%) 6/49 (12%) 3/49 (6%) 

Adjusted ratee 

Terminal ratef 

5.0% 

0/13 (0%) 

4.5% 

0/7 (0%) 

21.7% 

0/4 (0%) 

10.7% 

0/0 (0%) 

First incidence (days) 697 673 354 529 

Logistic regression testg P=0.227 P=0.754 P=0.047 P=0.280 

Follicular Cell Carcinomah 

Overall rate 1/50 (2%) 3/50 (6%) 1/49 (2%) 1/49 (2%) 

Adjusted rate 7.7% 20.6% 5.6% 5.6% 

Terminal rate 1/13 (8%) 1/7 (14%) 0/4 (0%) 0/0 (0%) 

First incidence (days) 723 (T) 641 667 589 

Logistic regression test P=0.461 P=0.281 P=0.662 P=0.583 

Follicular Cell Adenoma or Carcinomai 

Overall rate 2/50 (4%) 4/50 (8%) 7/49 (14%) 4/49 (8%) 

Adjusted rate 12.3% 24.2% 26.1% 15.7% 

Terminal rate 1/13 (8%) 1/7 (14%) 0/4 (0%) 0/0 (0%) 

First incidence (days) 697 641 354 529 

Logistic regression test P=0.266 P=0.309 P=0.076 P=0.207 

Female 

Number Examined Microscopically 49 50 50 50 

Follicular Cell, Hyperplasia 0 0 1 (2.0) 0 

Follicular Cell Carcinoma 1 0 0 0 

(T)Terminal sacrifice 

a Number of animals with lesion 

b Average severity of lesions in affected animals: 1=minimal; 2=mild; 3=moderate; 4=marked c Historical incidence for 2-year NTP feed studies with untreated control groups (mean ± standard deviation): 11/1,295 (0.9% ± 1.2%); 

range, 0%-4% d	 Number of animals with neoplasm per number of animals with thyroid gland examined microscopically e Kaplan-Meier estimated neoplasm incidence after adjustment for intercurrent mortality f	 Observed incidence in animals surviving until the end of the study 

g	 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 neoplasms in animals dying prior 

to terminal kill as nonfatal. 

h	 Historical incidence: 11/1,295 (0.9% ± 1.2%); range, 0%-4% i	 Historical incidence: 22/1,295 (1.7% ± 1.6%); range, 0%-6% 
 

 

 

Primidone, NTP TR 476 45 

Liver: Hepatocyte cytoplasmic vacuolation and centrilobular hypertrophy were associated with primidone 

exposure in male and female rats (Tables 12, A5, and 

B5). These changes were more severe in females than 

males and the incidences in all exposed groups of 

females were significantly greater than those in the 

controls. Vacuolation was often observed within the 

enlarged cells. Females in the 2,500 ppm group had 

TABLE 12 

an increased incidence of hepatocellular eosinophilic 

foci. Male rats in the 600 and 1,300 ppm groups had 

marginal increases in the incidences of eosinophilic 

foci. The lesions consisted of areas of hepatocytes 

with distinct cytoplasmic eosinophilia, although some 

of the cells had clear cytoplasm or pale granular cytoplasm. The foci retained features of the normal liver 

lobule. 

Incidences of Selected Nonneoplastic Lesions of the Liver of Rats in the 2-Year Feed Study of Primidone 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

Male 

Number Examined Microscopically 50 50 50 50 

Eosinophilic Focusa 1 6 5 2 

Hepatocyte, Centrilobular Hypertrophy 0 14** (1.4)b 33** (2.0) 40** (2.4) 

Hepatocyte, Vacuolization Cytoplasmic 26 (1.6) 28 (1.7) 33 (2.0) 43** (2.3) 

Female 

Number Examined Microscopically 50 50 50 50 

Eosinophilic Focus 2 0 1 18** 

Hepatocyte, Centrilobular Hypertrophy 1 (2.0) 36** (1.0) 38** (2.1) 35** (3.0) 

Hepatocyte, Vacuolization Cytoplasmic 25 (1.8) 44** (2.0) 46** (1.8) 44** (2.1) 

** Significantly different (P#0.01) from the control group by the logistic regression test a Number of animals with lesion 

b Average severity of lesions in affected animals: 1=minimal; 2=mild; 3=moderate; 4=marked 

Kidney (with parathyroid gland, bone, forestomach, 

glandular stomach, and lung): The incidence of renal 

tubule hyperplasia in 2,500 ppm males was greater 

than that in the controls in the standard evaluation 

(Table 13). Additional hyperplasias were found in the 

extended evaluation, and the incidences in all groups 

of exposed males were significantly greater than that 

in the controls. The incidences of renal tubule 

adenoma or carcinoma (combined) in exposed groups 

of male rats were similar to those in the controls at 

the standard evaluation. In the extended evaluation, 

the incidence of renal tubule adenoma in 2,500 ppm 

males was significantly greater than in the controls. 

The incidence of adenoma or carcinoma (combined) 

in 2,500 ppm males in the combined standard and 

extended evaluations was marginally increased over 

that in the controls. Male rats had an exposurerelated increase in the severity of chronic nephropathy. In 2,500 ppm male rats, nephropathy was 

considered severe in most animals, with marked fibrosis, loss of nephrons, and mineralization. Chronic 

nephropathy was responsible for the decreased survival of 1,300 and 2,500 ppm male rats. The 

incidences of cysts were increased in 1,300 and 

2,500 ppm males. 

Hyperparathyroidism, secondary to the loss of renal 

function, was present in many exposed male rats. 

The incidences of parathyroid gland hyperplasia in all 

groups of exposed males were significantly greater 

than that in the controls (2/45, 16/41, 26/46, 40/49). 

The incidences of bone fibrous osteodystrophy (0/50, 
46 

c 

Primidone, NTP TR 476 

TABLE 13 

Incidences of Neoplasms and Nonneoplastic Lesions of the Kidney of Male Rats 

in the 2-Year Feed Study of Primidone 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

Single Section (Standard Evaluation) 

Number Examined Microscopically 

Cysta 50 

2 (3.0)b 

50 

2 (3.0) 

50 

12** (2.3) 

50 

9* (2.8) 

Nephropathy, Chronic 49 (2.2) 48 (2.9) 50 (3.4) 50 (3.8) 

Renal Tubule, Hyperplasia 1 (1.0) 2 (3.0) 4 (2.3) 10* (2.5) 

Renal Tubule, Adenoma or Carcinomac 2 1 1 2 

Step Sections (Extended Evaluation) 

Renal Tubule, Hyperplasia 4 11* 22** 27** 

Renal Tubule, Adenoma 

Overall rated 2/50 (4%) 1/50 (2%) 4/50 (8%) 5/50 (10%) 

Adjusted ratee 

Terminal ratef 

13.8% 

1/13 (8%) 

4.5% 

0/7 (0%) 

20.9% 

0/4 (0%) 

54.9% 

0/0 (0%) 

First incidence (days) 715 673 613 585 

Logistic regression testg P=0.012 P=0.557N P=0.264 P=0.030 

Single Sections and Step Sections (Combined) 

Renal Tubule, Hyperplasia 5 13* 26** 37** 

Renal Tubule, Adenoma 

Overall rate 4/50 (8%) 2/50 (4%) 4/50 (8%) 6/50 (12%) 

Adjusted rate 23.2% 8.0% 20.9% 56.0% 

Terminal rate 2/13 (15%) 0/7 (0%) 0/4 (0%) 0/0 (0%) 

First incidence (days) 619 654 613 529 

Logistic regression test P=0.056 P=0.355N P=0.563 P=0.098 

Renal Tubule, Carcinoma 0 0 0 1 

Renal Tubule, Adenoma or Carcinoma 

Overall rate 4/50 (8%) 2/50 (4%) 4/50 (8%) 7/50 (14%) 

Adjusted rate 23.2% 8.0% 20.9% 62.3% 

Terminal rate 2/13 (15%) 0/7 (0%) 0/4 (0%) 0/0 (0%) 

First incidence (days) 619 654 613 529 

Logistic regression test P=0.025 P=0.355N P=0.563 P=0.050 

* Significantly different (P#0.05) from the control group by the logistic regression test 

** P#0.01 a Number of animals with lesion 

b Average severity of lesions in affected animals: 1=minimal; 2=mild; 3=moderate; 4=marked 

Historical incidence in 2-year NTP feed studies with untreated controls (mean ± standard deviation): 12/1,301 (0.9% ± 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 after adjustment for intercurrent mortality f	 Observed incidence in animals surviving until the end of the study 

g	 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 neoplasms in animals dying 

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

 

 

Primidone, NTP TR 476 47 

10/50, 26/50, 37/50) and incidences of mineralization 

of the forestomach (0 ppm, 0/50; 600 ppm, 3/50; 

1,300 ppm, 3/50; 2,500 ppm, 12/49), glandular 

stomach (0/50, 6/50, 15/50, 36/49), and lung 

interstitium (0/50, 3/50, 7/50, 25/50) in 2,500 ppm 

males were significantly greater than those in the 

controls (Table A5). These lesions were considered 

to be related to chronic nephropathy. 

Mononuclear Cell Leukemia: The incidence of 

mononuclear cell leukemia in 2,500 ppm females was 

marginally increased relative to the controls (13/50, 

13/50, 13/50, 22/50; Table B3). The incidence in the 

2,500 ppm group was well within the historical control range (14% to 52%; Table B4), and this slight 

increase was not considered to be chemical related. 

Testis: There was a significant increase in the 

incidences of testicular interstitial cell adenoma in the 

2,500 ppm males due primarily to an earlier onset 

of this neoplasm (41/50, 44/50, 42/50, 43/48; 

Table A3). Testicular adenoma is a common neoplasm that generally occurs in nearly 100% of animals 

surviving to at least 18 months. In this study, 98% 

(42/43) of 2,500 ppm males surviving beyond day 440 

had testicular interstitial cell adenoma compared with 

85% (41/48) of the controls. This slight increase was 

not considered chemical related. 
 

 

48 Primidone, NTP TR 476 

MICE 

14-DAY STUDY 

All male and female mice in the 10,000 ppm groups 5,000 ppm primidone resulted in average daily doses 

and one male and one female mouse in the 5,000 ppm of approximately 100, 200, 400, or 800 mg/kg to 

groups died on day 3 of the study (Table 14). The males and 100, 250, 500, or 900 mg/kg to females. 

final mean body weights and mean body weight gains The average daily dose for 10,000 ppm male and 

of mice in the 625, 1,250, 2,500, and 5,000 ppm female groups was not calculated due to high 

groups were similar to those of the controls. Feed mortality. Male and female mice in the 10,000 ppm 

consumption by exposed and control mice was gener- groups were observed to have abnormal posture, 

ally similar. Dietary levels of 625, 1,250, 2,500, or ataxia, and lethargy. 

TABLE 14 

Survival, Body Weights, and Feed Consumption of Mice in the 14-Day Feed Study of Primidone 

Final Weight Feed 

Mean Body Weightb (g) Relative Consumptionc 

Dose Survivala Initial Final Change to Controls Day 7 Day 14 

(ppm) (%) 

Male 

0 5/5 22.5 ± 0.5 23.6 ± 0.4 1.1 ± 0.4	 3.8 4.3 

625 5/5 22.5 ± 0.6 24.0 ± 0.5 1.5 ± 0.2 102 4.0 3.7 

1,250 5/5 23.0 ± 0.4 23.9 ± 0.4 0.9 ± 0.2 102 3.7 4.1 

2,500 5/5 23.0 ± 0.5 24.2 ± 0.4 1.2 ± 0.3 103 4.8 4.4 

5,000 4/5d 22.9 ± 0.5 24.7 ± 0.5 1.4 ± 0.2 105 3.8 3.7 

10,000 0/5d 23.3 ± 0.4 — — — — — 

Female 

0 5/5 18.1 ± 0.4 18.8 ± 0.4 0.8 ± 0.3	 3.8 4.7 

625 5/5 17.9 ± 0.2 18.3 ± 0.3 0.4 ± 0.3 97 2.8 3.4 

1,250 5/5 17.9 ± 0.4 18.8 ± 0.4 0.9 ± 0.4 100 3.4 3.9 

2,500 5/5 18.0 ± 0.4 19.1 ± 0.4 1.1 ± 0.2 102 2.7 5.2 

5,000 4/5d 18.6 ± 0.3 19.6 ± 0.4 0.8 ± 0.3 104 3.3 3.6 

10,000 0/5d 18.4 ± 0.3 — — — — — 

a Number of animals surviving at 14 days/number initially in group. Subsequent calculations are based on animals surviving to the end of 

the study. b	 Weights and weight changes are given as mean ± standard error. No final mean body weights were calculated for groups with 100% 

mortality. Differences from the control group are not significant by Williams’ or Dunnett’s test. 

Feed consumption is expressed as grams of feed consumed per animal per day. d	 Day of death: 3 

c 
 

Primidone, NTP TR 476 49 

14-WEEK STUDY 

Three male and two female mice in the 5,000 ppm 

groups died during week 1 of the study (Table 15). 

The final mean body weights of all exposed groups 

were similar to those of the controls. Feed consumption by male mice in the 5,000 ppm group was 

slightly greater than that by the controls; this may 

have been due to feed spillage. Dietary levels of 300, 

600, 1,300, 2,500, or 5,000 ppm primidone resulted 

in average daily doses of approximately 50, 100, 200, 

400, or 1,000 mg primidone/kg body weight to males 

and 60, 120, 220, 440, or 1,100 mg/kg to females. 

Male and female mice in the 5,000 ppm groups displayed signs of ataxia and lethargy. 

The estrous cycle lengths of females exposed to 

1,300, 2,500, or 5,000 ppm were significantly longer 

than that of the controls (Table H2). No differences 

in sperm motility were observed. 

The liver weights of male and female mice exposed to 

600 ppm or greater were significantly greater than 

those of the controls (Table F2). 

TABLE 15 

Survival, Body Weights, and Feed Consumption of Mice in the 14-Week Feed Study of Primidone 

Final Weight 

Mean Body Weightb (g) Relative Feed 

Dose Survivala Initial Final Change to Controls Consumptionc 

(ppm) (%) Week 2 Week 14 

Male 

0 10/10 19.8 ± 0.3 31.3 ± 0.8 11.5 ± 0.6 3.6 4.8 

300 10/10 20.0 ± 0.4 31.2 ± 0.8 11.2 ± 0.9 100 3.5 4.5 

600 10/10 20.2 ± 0.3 31.6 ± 1.3 11.5 ± 1.1 101 3.2 5.0 

1,300 10/10 20.3 ± 0.4 31.7 ± 0.6 11.4 ± 0.4 101 3.0 4.8 

2,500 10/10 20.1 ± 0.3 31.7 ± 0.8 11.6 ± 0.7 101 3.5 4.9 

5,000 7/10d 20.4 ± 0.3 29.1 ± 0.7 8.8 ± 0.8 93 3.5 6.8 

Female 

0 10/10 17.6 ± 0.3 27.4 ± 0.5 9.8 ± 0.4 4.1 6.9 

300 10/10 17.7 ± 0.2 28.5 ± 0.5 10.8 ± 0.4 104 3.3 5.5 

600 10/10 17.4 ± 0.4 29.7 ± 0.7 12.3 ± 0.6* 108 3.4 5.7 

1,300 10/10 17.4 ± 0.2 28.6 ± 1.1 11.1 ± 1.0 104 2.7 5.0 

2,500 10/10 17.7 ± 0.3 28.7 ± 0.5 10.9 ± 0.4 105 3.1 5.0 

5,000 8/10d 17.9 ± 0.2 26.7 ± 0.7 8.7 ± 0.5 98 3.3 6.7 

* Significantly different (P#0.05) from the control group by Williams’ or Dunnett’s test a Number of animals surviving at 14 weeks/number initially in group. Subsequent calculations are based on animals surviving to the end 

of the study. b Weights and weight changes are given as mean ± standard error. 

Feed consumption is expressed as grams of feed consumed per animal per day. d Week of death: 1 

c 
 

50 Primidone, NTP TR 476 

The incidences of hepatocyte centrilobular hypertrophy of the liver in all exposed males and in females 

exposed to 600 ppm or greater were significantly 

greater than in the controls (Table 16). Hepatocytes 

most closely associated with central veins were 

enlarged, with a gradual decrease in size the closer to 

the portal triad. The enlargement was due almost 

exclusively to an increase in cell cytoplasm. The 

cytoplasm of enlarged hepatocytes tended to stain 

more intensely eosinophilic, suggesting an increased 

size and/or number of organelles. The incidences of 

cytoplasmic alteration of the adrenal cortex in males 

exposed to 2,500 or 5,000 ppm and in 5,000 ppm 

females were significantly greater than in the controls 

(Table 16). Enlargement of adrenal cortical cells was 

due to an increase in cell cytoplasm, with the zona 

fasciculata cells being principally affected. The cause 

and significance of the adrenal gland change was not 

determined. The incidences of hematopoietic cell 

proliferation of the spleen in 2,500 and 5,000 ppm 

males and in 5,000 ppm females were significantly 

greater than in the controls (Table 16). 

Exposure Concentration Selection Rationale: Based 

on reduced survival, adrenal cortical cytoplasmic 

alteration, and increased severity of hepatocellular 

centrilobular hypertrophy at higher feed concentrations in the 14-week studies, primidone exposure concentrations selected for the 2-year feed study in mice 

were 300, 600, and 1,300 ppm. 

TABLE 16 

Incidences of Selected Nonneoplastic Lesions in Mice in the 14-Week Feed Study of Primidone 

0 ppm 300 ppm 600 ppm 1,300 ppm 2,500 ppm 5,000 ppm 

Male 

Livera 

Hepatocyte Centrilobular,

 Hypertrophyb 

10 

0 

10 

10** (1.0)d 

10 

10** (1.0) 

10 

10** (2.0) 

10 

10** (3.0) 

7c 

7** (3.0) 

Adrenal Gland 

Cytoplasmic Alteration

10 

0 

—e — 10 

0 

10 

9** (1.0) 

7c 

7** (1.9) 

Spleen 

Hematopoietic Cell Proliferation 

10 

0 

— — 10 

0 

10 

5* (1.0) 

7c 

7** (1.1) 

Female 

Liver 

Hepatocyte Centrilobular,

 Hypertrophy

10 

0 

10 

0 

10 

10** (1.0) 

10 

10** (2.0) 

10 

10** (3.0) 

8c 

8** (3.0) 

Adrenal Gland 

Cytoplasmic Alteration

10 

0 

— — — 10 

0 

8c 

8** (1.0) 

Spleen 

Hematopoietic Cell Proliferation 

10 

1 (1.0) 

— 1 

1 (1.0) 

10 

2 (1.0) 

10 

4 (1.5) 

8c 

8** (1.1) 

* Significantly different (P#0.05) from the control group by the Fisher exact test 

** P#0.01 a Number of animals with organ/tissue examined microscopically b Number of animals with lesion 

Does not include animals dying during week 1 d Average severity grade of lesions in affected animals: 1=minimal; 2=mild; 3=moderate; 4=marked e Tissue not examined at this exposure concentration 

c 
 

Primidone, NTP TR 476 51 

2-YEAR STUDY 

Survival 

Estimates of 2-year survival probabilities for male and 

female mice are shown in Table 17 and in the 

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

the 1,300 ppm males was significantly lower than that 

of the controls; survival of all other groups of mice 

was similar to that of the controls. 

Body Weights, Feed and Compound 

Consumption, and Clinical Findings 

During the second year of the study, the mean body 

weights of 1,300 ppm male and female mice were less 

than those of the controls (Tables 18 and 19, 

Figure 5). The final mean body weights of 600 ppm 

males and females were less than those of the 

controls. Feed consumption by all exposed groups of 

mice was similar to that by the controls (Tables K3 

and K4). Dietary concentrations of 300, 600, or 

1,300 ppm primidone resulted in average daily doses 

of approximately 30, 65, or 150 mg/kg to males and 

25, 50, or 100 mg/kg to females. During the latter 

part of the study, a treatment-related increase in the 

number of animals with swelling of the abdominal 

area was observed; necropsy revealed that the 

swelling was due to liver nodules/masses. No other 

clinical findings related to primidone exposure were 

observed. 

Determinations of Primidone 

and Phenobarbital in Plasma 

During the last 2 days of the study, blood was 

collected by cardiac puncture from three male and 

three female mice from each group at five time points 

for the determination of plasma concentrations of 

phenobarbital. The plasma concentrations of phenobarbital and their standard deviations are given in 

Tables H3 and H4. For exposed females, the only 

primidone plasma concentration value not below the 

limit of detection (0.5 μg/mL) was 1.72 μg/mL in the 

1,300 ppm group. Phenobarbital plasma concentrations increased with increasing exposure concentration 

for male and female mice; peak plasma concentrations 

were observed from 10:00 p.m. until 8:30 a.m. 
52 

c 

Primidone, NTP TR 476 

TABLE 17 

Survival of Mice in the 2-Year Feed Study of Primidone 

0 ppm 300 ppm 600 ppm 1,300 ppm 

Male 

Animals initially in study	 50 50 50 50 

Moribund 6 7 6 17 

Natural deaths 9 9 13 14 

Animals surviving to study termination 35 34d 31 19 

Percent probability of survival at end of studya 70 68 62 38 

Mean survival (days)b 686 701 688 651 

Survival analysisc	 P<0.001 P=1.000N P=0.611 P=0.005 

Female 

Animals initially in study	 50 50 50 50 

Missinge 0 0 1 0 

Moribund 7 1 3 8 

Natural deaths 2 7 2 3 

Animals surviving to study termination 41 42 44 39 

Percent probability of survival at end of study 82 84 90 78 

Mean survival (days) 704 722 716 712 

Survival analysis	 P=0.642 P=0.864N P=0.359N P=0.884 

a Kaplan-Meier determinations b	 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 group columns. A lower mortality in an exposure group is indicated by N. d	 Includes one animal that died during the last week of the study e Censored from the survival analyses 
Primidone, NTP TR 476 53 

Figure 4
 

Kaplan-Meier Survival Curves for Male and Female Mice
 

Exposed to Primidone in Feed for 2 Years
 
54 Primidone, NTP TR 476 

TABLE 18 

Mean Body Weights and Survival of Male Mice in the 2-Year Feed Study of Primidone 

Weeks 0 ppm 300 ppm 600 ppm 1,300 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 (g) Survivors (g) controls) Survivors (g) controls) Survivors (g) controls) Survivors 

1 22.4 50 22.0 98 50 22.0 98 50 21.9 98 50 

2 23.8 50 23.8 100 50 23.7 100 50 23.6 99 50 

3 25.4 50 25.7 101 50 25.6 101 50 25.7 101 50 

4 27.3 50 27.3 100 50 27.0 99 50 27.6 101 49 

5 28.0 50 28.7 103 50 28.6 102 50 28.6 102 49 

6 29.8 50 30.3 102 50 30.1 101 50 29.8 100 49 

7 30.9 50 31.9 103 50 31.4 102 50 30.7 99 49 

8 31.9 50 33.0 103 50 32.1 101 50 31.4 98 49 

9 32.6 50 33.8 104 50 32.9 101 50 32.5 100 49 

10 33.9 50 35.2 104 50 34.5 102 50 33.8 100 49 

11 35.2 50 35.9 102 50 35.2 100 50 34.3 97 49 

12 36.3 50 36.5 101 50 35.9 99 50 34.8 96 49 

13 37.8 50 38.0 101 50 36.9 98 50 36.3 96 49 

17 42.3 50 42.2 100 50 41.2 97 50 39.1 92 49 

21 44.5 50 45.3 102 50 43.7 98 50 41.8 94 49 

25 46.0 50 46.6 101 50 45.4 99 50 43.7 95 49 

29 47.2 50 47.2 100 50 46.1 98 50 44.3 94 49 

33 47.5 50 48.3 102 50 47.3 100 50 45.2 95 49 

36 47.8 50 48.4 101 50 47.6 100 50 45.4 95 49 

41 49.9 50 49.6 99 50 48.5 97 50 46.8 94 49 

45 49.2 50 49.1 100 50 48.2 98 50 47.5 97 49 

49 50.4 50 51.3 102 50 50.8 101 50 49.5 98 49 

53 50.3 50 51.4 102 50 51.0 101 50 48.8 97 49 

57 50.5 50 52.3 104 50 51.8 103 50 49.3 98 48 

61 49.7 50 51.3 103 50 50.7 102 50 48.3 97 48 

65 50.7 49 52.0 103 50 52.2 103 49 48.4 96 48 

69 49.9 49 51.7 104 50 51.5 103 49 46.4 93 47 

73 50.3 49 52.1 104 50 52.4 104 49 45.3 90 46 

77 49.7 47 51.5 104 50 50.7 102 49 43.4 87 45 

81 50.2 45 51.8 103 50 49.5 99 49 41.8 83 43 

84 50.8 41 52.2 103 50 48.9 96 46 41.9 83 41 

88 51.2 41 52.0 102 47 46.9 92 44 40.7 80 40 

93 49.6 39 50.7 102 40 44.1 89 39 39.5 80 33 

97 49.1 38 51.1 104 37 43.9 89 35 38.3 78 30 

101 48.9 37 48.3 99 36 43.0 88 32 37.9 78 24 

Mean for weeks 

1-13 30.4 30.9 102 30.5 100 30.1 99 

14-52 47.2 47.6 101 46.5 99 44.8 95 

53-101 50.1 51.4 103 49.0 98 43.8 87 
Primidone, NTP TR 476 55 

TABLE 19 

Mean Body Weights and Survival of Female Mice in the 2-Year Feed Study of Primidone 

Weeks 0 ppm 300 ppm 600 ppm 1,300 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 (g) Survivors (g) controls) Survivors (g) controls) Survivors (g) controls) Survivors 

1 18.1 50 18.1 100 50 18.1 100 50 18.0 99 50 

2 19.0 50 19.0 100 50 18.8 99 50 19.0 100 50 

3 19.9 50 20.5 103 50 20.4 103 50 20.2 102 50 

4 21.4 50 22.0 103 50 21.5 101 50 21.6 101 50 

5 22.8 50 23.3 102 50 23.0 101 50 23.3 102 50 

6 24.0 50 24.4 102 50 24.3 101 50 24.2 101 50 

7 25.4 50 26.7 105 50 26.0 102 50 25.9 102 50 

8 25.9 50 27.8 107 50 27.0 104 50 26.5 102 50 

9 27.8 50 29.1 105 50 28.6 103 50 28.2 101 50 

10 29.0 50 29.5 102 50 29.5 102 50 28.9 100 50 

11 28.3 50 31.5 111 50 30.3 107 50 29.5 104 50 

12 31.5 50 33.5 106 50 31.8 101 50 31.0 98 50 

13 32.9 50 34.3 104 50 33.0 100 50 32.2 98 50 

17 37.7 50 39.9 106 50 37.9 101 50 37.1 98 50 

21 40.7 50 42.7 105 50 40.2 99 50 39.4 97 50 

25 43.3 50 45.5 105 50 42.5 98 50 41.8 97 50 

29 43.4 50 47.2 109 50 43.9 101 50 43.0 99 50 

33 45.2 50 48.3 107 50 44.8 99 50 43.1 95 50 

36 46.7 50 49.6 106 50 46.0 99 50 44.5 95 50 

41 49.1 50 51.5 105 50 48.2 98 50 46.3 94 50 

45 51.1 50 53.4 105 50 49.8 98 50 47.7 93 50 

49 53.4 50 55.3 104 50 52.4 98 50 49.7 93 50 

53 54.7 50 55.7 102 50 53.2 97 50 50.4 92 50 

57 55.6 50 57.2 103 50 54.5 98 50 51.7 93 50 

61 55.8 49 57.1 102 50 54.8 98 50 52.2 94 50 

65 56.2 49 57.5 102 50 54.8 98 50 51.8 92 50 

69 57.4 49 58.8 102 50 55.8 97 50 53.1 93 50 

73 59.0 49 60.0 102 50 57.0 97 49 54.5 92 50 

77 58.1 48 59.8 103 50 56.2 97 49 53.5 92 50 

81 59.3 48 61.2 103 50 57.8 98 48 53.9 91 50 

84 60.6 48 62.7 104 50 59.3 98 48 54.1 89 49 

88 60.3 46 63.1 105 49 59.7 99 48 52.2 87 48 

93 60.7 44 63.7 105 49 58.7 97 48 49.0 81 46 

97 61.6 43 64.7 105 48 58.7 95 48 47.6 77 46 

101 61.3 41 61.6 101 47 57.6 94 44 46.0 75 40 

Mean for weeks 

1-13 25.1 26.1 104 25.6 102 25.3 101 

14-52 45.6 48.2 106 45.1 99 43.6 96 

53-101 58.5 60.2 103 56.8 97 51.5 88 
 

56 Primidone, NTP TR 476 

Figure 5
 

Growth Curves for Male and Female Mice
 

Exposed to Primidone in Feed for 2 Years
 
 

Primidone, NTP TR 476 57 

Pathology and Statistical Analyses 

This section describes the statistically significant or 

biologically noteworthy changes in the incidences 

of neoplasms and/or nonneoplastic lesions of the liver, 

thyroid gland, spleen, and pancreatic islets. Summaries of the incidences of neoplasms and nonneoplastic lesions, individual animal tumor diagnoses, 

statistical analyses of primary neoplasms that occurred 

with an incidence of at least 5% in at least one animal 

group, and historical incidences for the neoplasms 

mentioned in this section are presented in Appendix C 

for male mice and Appendix D for female mice. 

Liver: The liver was a target organ in male and 

female mice. An increase in the incidences of proliferative lesions as well as nonproliferative changes 

occurred in males and females. The incidences of 

hepatocellular neoplasms (hepatocellular adenoma, 

hepatocellular carcinoma, and hepatoblastoma) in all 

exposed groups (except hepatoblastoma in females) 

were significantly greater than those in the controls 

(Tables 20, C3, and D3). The incidences of hepatocellular adenoma or carcinoma (combined) and hepatocellular adenoma, hepatocellular carcinoma, or 

hepatoblastoma (combined) in all exposed groups 

exceeded the ranges in historical controls in 2-year 

NTP feed studies (Tables 20, C4a, and D4). Hepatocellular adenomas were generally round, compressive 

masses of well-differentiated, uniform-appearing 

hepatocytes. In contrast to foci of cellular alteration, 

adenomas lacked normal anatomic features of the liver 

lobule, such as bile ducts, ductlets, and portal triad 

areas. In other respects, adenomas resembled foci of 

cellular alteration. Hepatocellular carcinomas were 

less differentiated than adenomas and generally had a 

distinct trabecular or glandular pattern, at least in a 

portion of the mass. Areas of necrosis or loss of cells 

were common. The staining quality of cells typically 

varied from one area to another, as did the size and 

shape of the cells. Many hepatocellular carcinomas 

metastasized to the lung. Hepatoblastomas were 

distinguished from hepatocellular carcinomas by their 

elongated, basophilic cells that were often arranged 

radially around small blood vessels. These liver neoplasms also metastasized to the lung in several 

instances. Hepatoblastomas occurred in conjunction 

with adenomas and/or carcinomas in almost every 

instance. 

The incidences of centrilobular hypertrophy of the 

hepatocytes were increased in exposed groups of 

males and females, and the severities increased with 

increasing exposure concentration (Tables 20, C5, and 

D5). The incidences of cytoplasmic vacuolization 

were increased in all exposed groups of females and 

in 300 ppm males. In 1,300 ppm mice, especially 

males, cytoplasmic and nuclear enlargement involved 

over half the distance from the central vein to the 

portal triad area in the most severely affected hepatic 

lobules. Incidences of eosinophilic focus in all 

exposed groups of females were significantly greater 

than those in the controls. 
58 Primidone, NTP TR 476 

TABLE 20 

Incidences of Neoplasms and Nonneoplastic Lesions of the Liver in Mice 

in the 2-Year Feed Study of Primidone 

0 ppm 300 ppm 600 ppm 1,300 ppm 

Male 

Number Examined Microscopically 50 50 50 50 

Hepatocyte, Centrilobular Hypertrophya 3 (1.7)b 30** (2.0) 21** (2.0) 18** (2.1) 

Hepatocyte, Vacuolization Cytoplasmic 1 (2.0) 8* (2.9) 3 (2.0) 2 (3.0) 

Hepatocellular Adenoma 

Overall ratec 22/50 (44%) 41/50 (82%) 39/50 (78%) 32/50 (64%) 

Adjusted rated 59.2% 93.1% 92.6% 87.3% 

Terminal ratee 20/35 (57%) 31/34 (91%) 28/31 (90%) 15/19 (79%) 

First incidence (days) 551 603 566 368 

Life table analysisf P<0.001 P<0.001 P<0.001 P<0.001 

Logistic regression testf P=0.040 P<0.001 P<0.001 P=0.013 

Hepatocellular Carcinoma 

Overall rate 12/50 (24%) 31/50 (62%) 35/50 (70%) 38/50 (76%) 

Adjusted rate 29.1% 71.6% 81.1% 100.0% 

Terminal rate 7/35 (20%) 22/34 (65%) 23/31 (74%) 19/19 (100%) 

First incidence (days) 526 614 575 470 

Life table analysis P<0.001 P<0.001 P<0.001 P<0.001 

Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001 

Hepatoblastoma 

Overall rate 0/50 (0%) 17/50 (34%) 26/50 (52%) 7/50 (14%) 

Adjusted rate 0.0% 39.9% 56.9% 23.4% 

Terminal rate 0/35 (0%) 9/34 (26%) 12/31 (39%) 1/19 (5%) 

First incidence (days) —g 617 422 551 

Life table analysis P<0.043 P<0.001 P<0.001 P<0.003 

Logistic regression test P=0.271 P<0.001 P<0.001 P=0.009 

Hepatocellular Adenoma, Hepatocellular Carcinoma, or Hepatoblastomah 

Overall rate 31/50 (62%) 49/50 (98%) 49/50 (98%) 46/50 (92%) 

Adjusted rate 73.5% 100.0% 100.0% 100.0% 

Terminal rate 24/35 (69%) 34/34 (100%) 31/31 (100%) 19/19 (100%) 

First incidence (days) 526 603 422 368 

Life table analysis P<0.001 P<0.001 P<0.001 P<0.001 

Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001 
 

Primidone, NTP TR 476 59 

TABLE 20 

Incidences of Neoplasms and Nonneoplastic Lesions of the Liver in Mice 

in the 2-Year Feed Study of Primidone 

0 ppm 300 ppm 600 ppm 1,300 ppm 

Female 

Number Examined Microscopically 50 50 49 50 

Eosinophilic Focus 8 23** 24** 17* 

Hepatocyte, Centrilobular Hypertrophy 1 (1.0) 11** (1.5) 11** (1.9) 21** (2.0) 

Hepatocyte, Vacuolization Cytoplasmic 3 (2.3) 35** (2.9) 39** (2.9) 28** (2.6) 

Hepatocellular Adenoma 

Overall rate 15/50 (30%) 42/50 (84%) 45/49 (92%) 47/50 (94%) 

Adjusted rate 34.9% 91.3% 97.8% 97.9% 

Terminal rate 13/41 (32%) 38/42 (90%) 43/44 (98%) 38/39 (97%) 

First incidence (days) 673 701 680 583 

Life table analysis P<0.001 P<0.001 P<0.001 P<0.001 

Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001 

Hepatocellular Carcinoma 

Overall rate 3/50 (6%) 11/50 (22%) 19/49 (39%) 38/50 (76%) 

Adjusted rate 7.1% 24.9% 42.2% 84.4% 

Terminal rate 2/41 (5%) 9/42 (21%) 18/44 (41%) 32/39 (82%) 

First incidence (days) 694 701 699 638 

Life table analysis P<0.001 P<0.029 P<0.001 P<0.001 

Logistic regression test P<0.001 P=0.032 P<0.001 P<0.001 

Hepatocellular Adenoma or Carcinoma 

Overall rate 16/50 (32%) 42/50 (84%) 45/49 (92%) 50/50 (100%) 

Adjusted rate 37.2% 91.3% 97.8% 100.0% 

Terminal rate 14/41 (34%) 38/42 (90%) 43/44 (98%) 39/39 (100%) 

First incidence (days) 673 701 680 583 

Life table analysis P<0.001 P<0.001 P<0.001 P<0.001 

Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001 

Hepatoblastoma	 1 4 4 4 

Hepatocellular Adenoma, Hepatocellular Carcinoma, or Hepatoblastomai 

Overall rate 16/50 (32%) 42/50 (84%) 46/49 (94%) 50/50 (100%) 

Adjusted rate 37.2% 91.3% 97.9% 100.0% 

Terminal rate 14/41 (34%) 38/42 (90%) 43/44 (98%) 39/39 (100%) 

First incidence (days) 673 701 677 583 

Life table analysis P<0.001 P<0.001 P<0.001 P<0.001 

Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001 

* Significantly different (P#0.05) from the control group by the logistic regression test 

** P#0.01 a Number of animals with lesion 

b Average severity of lesions in affected animals: 1=minimal; 2=mild; 3=moderate; 4=marked c Number of animals with neoplasm per number of animals with liver examined microscopically d Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality e Observed incidence in animals surviving until the end of the study f	 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 neoplasms in animals dying prior 

to terminal kill as nonfatal. 

g	 Not applicable; no neoplasms in animal group h	 Historical incidence for 2-year NTP feed studies with untreated controls (mean ± standard deviation): 596/1,465 (40.7% ± 14.5%); range, 

10%-68% 

i	 Historical incidence: 313/1,464 (21.4% ± 13.0%); range, 3%-56% 
 

 

 

60 

c 

Primidone, NTP TR 476 

Thyroid Gland: Proliferative changes occurred in the 

thyroid gland in an exposure-related manner in male 

and female mice. Incidences of follicular cell hyperplasia were increased in all exposed groups of males 

and in 600 and 1,300 ppm females, but incidences of 

follicular cell adenomas were increased only in male 

mice (Tables 21, C3, C5, and D5). No thyroid gland 

carcinomas were observed. Hyperplastic follicles 

were enlarged and were lined by an increased number 

of cuboidal to columnar epithelial cells that were 

larger than normal. In some instances, the epithelial 

cells formed small projections toward the center of 

the follicle. Adenomas were considered to be on a 

continuum with focal hyperplasia, but adenomas were 

larger, showed cellular atypia, and compressed the 

surrounding thyroid parenchyma. 

TABLE 21 

Incidences of Neoplasms and Nonneoplastic Lesions of the Thyroid Gland in Mice 

in the 2-Year Feed Study of Primidone 

0 ppm 300 ppm 600 ppm 1,300 ppm 

Male 

Number Examined Microscopically 

Follicular Cell, Hyperplasiaa 49 8 (1.1)b 

48 

20** (1.5) 

50 

31** (1.7) 

50 

42** (2.5) 

Follicular Cell Adenomac 

Overall rated 0/49 (0%) 3/48 (6%) 3/50 (6%) 6/50 (12%) 

Adjusted ratee 

Terminal ratef 

First incidence (days) 

0.0% 

0/35 (0%) 

—h 

9.1% 

3/33 (9%) 

726 (T) 

9.7% 

3/31 (10%) 

726 (T) 

25.3% 

3/19 (16%) 

674 

Logistic regression testg P=0.003 P=0.110 P=0.100 P=0.008 

Female 

Number Examined Microscopically 50 48 48 50 

Follicular Cell, Hyperplasia 13 (1.2) 12 (1.7) 28** (1.8) 49** (2.3) 

Follicular Cell Adenoma 1 1 0 2 

** Significantly different (P#0.01) from the control group by the logistic regression test 

(T)Terminal sacrifice 

a	 Number of animals with lesion 

b	 Average severity of lesions in affected animals: 1=minimal; 2=mild; 3=moderate; 4=marked 

Historical incidence in 2-year NTP feed studies with untreated controls (mean ± standard deviation): 22/1,455 (1.5% ± 1.5%); 

range, 0%-4% d	 Number of animals with neoplasm per number of animals with thyroid gland 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 

g	 In the control column is the P value 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 neoplasms in animals dying prior 

to terminal kill as nonfatal. 

h	 Not applicable; no neoplasms in animal group 
Primidone, NTP TR 476 61 

Other Organs: In the spleen, the incidences of 

hematopoietic cell proliferation were increased in 

600 ppm males and 1,300 ppm males and females 

compared to the controls (males: 0 ppm, 14/48; 

300 ppm, 15/49; 600 ppm, 26/50; 1,300 ppm, 37/49; 

females: 14/50, 13/48, 13/49, 23/50; Tables C5 and 

D5). Incidences of hyperplasia of the pancreatic 

islets were significantly decreased in 600 and 

1,300 ppm males and females (males: 32/45, 34/48, 

18/49, 4/47; females: 15/50, 17/47, 6/49, 5/50). 

GENETIC TOXICOLOGY 

Primidone (33 to 10,000 μg/plate) induced mutations 

in Salmonella typhimurium strain TA1535 in trials 

conducted in the absence of exogenous metabolic activation (S9); no mutagenic response was detected in 

TA1535 with S9 (Mortelmans et al., 1986; 

Table E1). Negative results were obtained in the 

S. typhimurium assay with strains TA98, TA100, and 

TA1537, with and without S9. No induction of sister 

chromatid exchanges (Table E2) or chromosomal 

aberrations (Table E3) was noted in cultured Chinese 

hamster ovary cells treated with concentrations of 

primidone ranging from 125 to 1,250 μg/mL, with or 

without S9. In vivo, no significant increase was 

observed in the frequency of micronucleated polychromatic erythrocytes in bone marrow of male mice 

treated with 87.5 to 350 mg primidone/kg body 

weight three times at 24-hour intervals in either of 

two trials (Table E4). 

In summary, primidone induced gene mutations in 

S. typhimurium in the absence of S9 activation, but 

did not induce chromosomal damage in mammalian 

cells, in vitro or in vivo, even at doses associated with 

marked toxicity. 
62 Primidone, NTP TR 476 
 

 

 

 

 

63 

DISCUSSION AND CONCLUSIONS
 

Primidone was nominated for toxicity and carcinogenicity studies by the National Cancer Institute because 

of its high production and its use in the treatment of 

epilepsy in both adults and children. The International Agency for Research on Cancer had recommended to the National Cancer Institute that 

primidone be a high-priority chemical for carcinogenicity studies. 

The recommended maintenance dose of primidone in 

the treatment of epilepsy is 10 to 25 mg/kg per day 

(given in divided doses). Clinically effective serum 

concentrations for primidone are between 5 and 

12 μg/mL (PDR, 1996). A primary metabolite of 

primidone is phenobarbital, which is also used in 

the treatment of epilepsy. Effective clinical plasma 

concentrations for phenobarbital are between 5 and 

40 μg/mL (PDR, 1996). 

In the 14-day studies, concentrations of 1,250 to 

20,000 ppm delivered approximately 120 to 1,100 mg 

primidone/kg body weight per day to rats and doses 

of 625 to 5,000 ppm delivered approximately 100 to 

900 mg/kg to mice. In the 14-week studies, doses of 

300 to 5,000 ppm delivered approximately 20 to 

400 mg/kg to rats and 50 to 1,000 mg/kg to mice. In 

the 2-year studies, rats received 600 to 2,500 ppm and 

mice received 300 to 1,300 ppm in feed. These doses 

delivered approximately 25 to 150 mg/kg per day. At 

2 years, the plasma concentrations of primidone 

(female rats) were generally within 1 to 8 μg/mL, and 

the plasma concentrations for phenobarbital (female 

rats and mice) were generally within 2 to 20 μg/mL. 

Peak concentrations of primidone and phenobarbital 

increased with increasing exposure concentration. 

In the 14-day and 14-week studies, the principal 

chemical-related findings were toxicity to the kidney 

of rats and the liver of rats and mice. Exposurerelated increases in liver weights occurred in rats 

and mice in the 14-week studies. In the 14-week 

studies, microscopic examination showed hepatocellular centrilobular hypertrophy in male rats exposed to 

600 ppm or greater and in female rats exposed to 

1,300, 2,500, or 5,000 ppm and chronic nephropathy 

in 2,500 and 5,000 ppm male rats. Based on these 

findings, the highest exposure concentration used in 

the 2-year study in rats was 2,500 ppm. An increased 

incidence of centrilobular hypertrophy was observed 

in all exposed groups of male and female mice. The 

morphologic appearance of the hepatocellular hypertrophy was typical of that seen in animals exposed to 

phenobarbital-type substances. Male mice in the 

2,500 and 5,000 ppm groups and 5,000 ppm females 

also showed adrenal cortical cytoplasmic alteration 

and splenic hematopoietic cell proliferation. The 

highest exposure concentration was set at 1,300 ppm 

for the 2-year mouse study. At the end of the 

14-week rat and mouse studies, results of the NTP 

sperm motility and/or vaginal cytology evaluations did 

not indicate any exposure-related changes in rats or 

male mice. There was an exposure-related increase in 

the estrous cycle length of female mice, but the 

estrous cycle was not affected in female rats. In the 

continuous breeding study in Swiss (CD-1®) mice, 

primidone was administered in feed at concentrations 

up to 2,000 ppm (approximately 350 mg/kg per day). 

Fertility and reproduction in F0 animals were not 

affected, and mating, pregnancy, and fertility indices 

in F1 mice were similar to those in the controls. 

Estrous cycle length was increased by approximately 7% in F1 females exposed to 1,500 ppm 

(Appendix O). 

It has been shown that rats treated with 100 mg/kg 

primidone, per os, twice daily, for up to 8 weeks, 

developed a folate deficiency (Carl et al., 1987b). 

Additionally, a macrocytic, normochromatic anemia 

with leukopenia has been experimentally induced in 

folate deficient rats (Kodicek and Carpenter, 1950). 

In the 14-week study, 5,000 ppm primidone resulted 

in an average daily dose of 400 mg/kg in rats; thus, 

development of an anemia related to a primidoneinduced folate deficiency could be hypothesized 

for rats in the 5,000 ppm group. There was, however, only minimal evidence (a minimal decrease of 

hemoglobin concentration) of an anemic tendency 

and no evidence of a macrocytosis. The lack of 

evidence indicating an anemia could be that, under the 

conditions of this 14-week study, there was little or 

no primidone-induced folate deficiency resulting in a 
 

 

 

 

 

 

 

 

 

 

 

64 Primidone, NTP TR 476 

minimal to no anemic response. Additionally, dehydration could have masked a minimal anemia. In the 

14-week study, rats in the 2,500 and 5,000 ppm 

groups had decreased mean body weight gains, suggesting decreased feed or water consumption or illness. There were minimal decreases of alkaline phosphatase activity in exposed groups of rats that would 

be consistent with decreased feed consumption. Circulating alkaline phosphatase in a normal rat is primarily of intestinal or bone origin (Righetti and 

Kaplan, 1971), and fasting or feed restriction causes 

decreases in serum alkaline phosphatase activity 

(Jenkins and Robinson, 1975). If feed consumption 

by rats was decreased, decreases in alkaline phosphatase activity might be related to loss of the normally circulating intestinal fraction. Also, mechanical 

difficulties with the automatic watering system caused 

inadequate water delivery to the 2,500 and 5,000 ppm 

male rats and resulted in dehydrated animals. There 

were increases in total protein and albumin concentrations in the exposed groups that would be consistent with dehydration (Kaneko, 1989). A relative 

erythrocytosis related to dehydration and hemoconcentration can occur in animals with a decreased 

plasma volume due to inadequate water intake or 

excessive water loss (e.g., diarrhea); thus, a relative 

erythrocytosis could mask the presence and/or the 

severity of an existing anemia. 

In the 14-week study, thrombocytosis occurred in all 

exposed groups of male rats and in 1,300, 2,500, and 

5,000 ppm female rats. These findings could be consistent with either physiologic thrombocytosis or a 

reactive thrombocytosis (Jain, 1986). 

In the 2-year studies, survival was similar among 

exposed and control groups of female rats and mice. 

Male rats exposed to 1,300 or 2,500 ppm began to die 

near the end of the study, and this was attributed to 

kidney toxicity. Some early deaths were also seen in 

1,300 ppm male mice; this was attributed to the toxic 

and carcinogenic effects in the liver. Mean body 

weights of 1,300 and 2,500 ppm male and female rats 

and 1,300 ppm male and female mice were lower than 

those of the controls during the second year of the 

study, but there was no evidence that these body 

weight effects masked the ability of the bioassay to 

detect a carcinogenic response. 

In the 2-year rat study, exposure-related toxicity in 

the liver included cytoplasmic vacuolization and 

centrilobular hepatocyte hypertrophy. While these 

studies did not determine whether the exposure-related 

effects were due to primidone or its metabolites, the 

liver toxicity observed in rats exposed to primidone 

was similar to that previously observed in F344/N rats 

exposed to phenobarbital. For example, Butler (1978) 

gave 1,000 ppm sodium phenobarbital in drinking 

water to male F344/N rats for 103 weeks and found 

liver toxicity in treated rats but no evidence of 

treatment-induced neoplasms in the liver. The liver 

lesions were described as marked centrilobular cytomegaly of the parenchymal cells. Near the end of the 

study, focal nodules of hepatic parenchymal cells 

were seen, as well as areas of focal fatty degeneration 

and some evidence of cell necrosis. Biliary proliferation was prominent in all animals. 

In the current studies there was no evidence for a 

treatment-related carcinogenic response in the liver of 

F344/N rats exposed to primidone. No hepatic neoplasms were seen in Sprague-Dawley rats receiving 

intraperitoneal injections of 2 mg/kg phenobarbital for 

life (Schmal and Habs, 1976). Liver neoplasms were 

seen in Wistar rats given 500 ppm phenobarbital in 

drinking water for their lifetime (Rossi et al., 1977). 

At 2 years, there was a marginal increase in the incidence of thyroid gland follicular cell adenoma or 

carcinoma (combined) in the 1,300 ppm male rats, 

and the incidences of these neoplasms were outside 

the historical range for 2-year NTP feed studies in all 

exposed groups of male rats. However, because this 

increase was not significant by the trend analysis and 

the incidence of follicular cell hyperplasia was not 

increased, this was considered equivocal evidence of 

carcinogenic activity. There was no increase in the 

incidence of follicular cell neoplasms in female rats. 

Thyroid gland proliferative effects have been related 

to exposure to phenobarbital (McClain et al., 1988, 

1989), but in the current study there was no evidence 

of thyroid gland follicular cell hyperplasia in exposed 

rats. Long-term changes in thyroid hormone levels 

are likely to predispose the rat to a higher incidence 

of proliferative lesions in the thyroid gland. The male 

rat has a higher circulating level of thyroid-stimulating 

hormone (TSH) than the female rat (Capen, 1996), 

and this might be one explanation why the thyroid 

gland neoplasms were seen in male but not female rats 

in the present study. 
 

 

 

 

 

 

Primidone, NTP TR 476 65 

Phenobarbital is a prototype agent for induction of a 

variety of biotransformation systems including glutathione S-epoxide transferases, UDP-glucuronyl transferases, aldehyde dehydrogenases, and cytochrome 

P450 monoxygenases (Waxman and Azaroff, 1992; 

Ramsden et al., 1993). UDP-glucuronosyl transferase inducers have been shown to lower plasma concentrations of tetraiodothyronine (T4) by increasing its 

glucuronidation and elimination by the liver (Liu 

et al., 1995). Plasma concentrations of triiodothyronine (T3) may also be decreased (de Sandro et al., 

1991). Many xenobiotics that increase the incidence 

of thyroid gland neoplasms in rodents do so by 

increasing the peripheral metabolism of thyroid hormones through an induction of hepatic microsomal 

enzymes as has been reported for phenobarbital 

(Capen, 1994). Lowering thyroid hormone levels 

results in a compensatory increase in secretion of 

TSH, increased incidences of follicular cell hypertrophy and hyperplasia, and an increased incidence of 

follicular cell neoplasms in rodents (Barter and 

Klaassen, 1994; Capen, 1994). 

The rodent may be more sensitive than other 

mammals to chemically induced changes in the 

thyroid gland because the rodent lacks a serum protein 

known as thyroxine-binding globulin that binds to 

thyroid hormones. Because rodents lack this protein, 

they quickly excrete thyroid hormones. In contrast, 

where thyroid globulin protein is present (in the 

human, monkey, and dog) (Döhler et al., 1979; 

Capen, 1996), the protein would serve to modulate 

the effects of a chemical (e.g., phenobarbital) that 

induces enzymes that cause conjugation and excretion 

of the hormone. However, there is some evidence 

that long-term treatment of humans with phenobarbital 

(greater than 6 years) may result in altered serum T3 

and T4 levels (Tanaka et al., 1987). 

Exposure to primidone for 2 years resulted in a 

marked increase in the severity of chronic nephropathy in male rats. In addition, increased incidences of 

parathyroid gland hyperplasia, mineralization of the 

forestomach and glandular stomach, fibrous osteodystrophy, and lung interstitium mineralization were seen 

in exposed male rats; these lesions were secondary to 

chronic nephropathy. The chemical-related increase 

in the severity of nephropathy in exposed males was 

a principal cause of decreased survival in these 

animals. The incidence of parathyroid gland hyperplasia as well as the mineralization observed in other 

organ systems are consistent with renal secondary 

hyperparathyroidism. Hyperparathyroidism accompanies severe nephropathy in rats because the progressive loss of renal function disrupts calcium and 

phosphorus homeostasis, leading to prolonged parathyroid gland stimulation. This has been observed in 

other NTP studies (e.g., coumarin; NTP, 1993a). 

The NTP has found that examination of the entire 

kidney, by step-sectioning through residual tissue, 

may enable a more precise evaluation of the potential chemical-related induction of renal proliferative 

lesions than observations made from single sections, 

particularly when the proliferative lesions are small 

and identified only by microscopic examination 

(Eustis et al., 1994). For primidone, this extended 

evaluation of the male rat kidney showed a marginal 

increase in neoplasms in the 2,500 ppm group when 

the results of the original and step-section evaluations 

were combined. The increased incidences of focal 

hyperplasia and the marginal increase in renal tubule 

neoplasms in the 2,500 ppm group were considered 

to be equivocal evidence of a carcinogenic effect. 

Chronic nephropathy may influence the induction, 

development, or progression of renal neoplasms in 

several ways, including a reduction in target cell 

population and/or increased number of cells in the 

replicative cycle due to chronic inflammation and 

continued degeneration and necrosis, alterations in 

vascularity as a result of fibrosis, or other alterations 

in microenvironment. 

Kidney changes consistent with nephropathy were 

observed in both the 14-week and 2-year rat studies. 

The nephropathy in the 14-week study was much less 

severe than in the 2-year study. In the 14-week study, 

a “minimal” lesion was considered to be only a few 

regenerative tubules, while a grade of “mild” 

nephropathy (equivalent to grade 2) was four or more 

regenerative tubules, and “moderate” nephropathy 

included the presence of protein casts and/or glomerular lesions. These lesions progressed with continued 

exposure to primidone. At 2 years, the lesions were 

quite advanced, far exceeding the severity of the 

nephropathy in the 14-week study and resulting in 

compromised renal function, especially in the 1,300 

and 2,500 ppm groups. In the 2-year study, nephropathy represented an entire spectrum of microscopic 

lesions including interstitial fibrosis, tubule degeneration and regeneration, protein casts, cyst formation, 

glomerular sclerosis, and eventual loss of nephrons. 
 

 

 

 

 

 

 

66 Primidone, NTP TR 476 

These studies could not differentiate between the 

effects of primidone or its metabolites in the kidney. 

Phenobarbital, a metabolite of primidone, can induce 

hepatic, intestinal, and renal UDP-glucuronosyltransferase and cytochrome P450 enzyme activities 

(Koster et al., 1986). Induction of these enzyme 

systems in the kidney may result in the accumulation 

of toxic metabolites and subsequent toxicity to the 

kidney (Nakagawa and Tayama, 1988; Imaoka et al., 

1989). Phenobarbital treatment has been shown to 

cause cell proliferation in the rat kidney, as measured 

by bromodeoxyuridine immunocytochemistry (Jones 

and Clarke, 1993); a phenobarbital-induced increase 

in cell proliferation may have contributed to the 

observed toxicity in the male rat kidney in this study. 

In the two long-term phenobarbital studies in male 

Wistar rats (Rossi et al., 1977) or male F344/N rats 

(Butler, 1978), an increase in severity of nephropathy 

was not reported. However, the extent to which the 

kidney was examined for nonneoplastic lesions in 

these studies was not reported. Generally, female rats 

exhibit less renal toxicity than male rats after phenobarbital/chemical treatment. Age-dependent DNA 

modifications induced by endogenous electrophiles 

can be derived during normal metabolism of nutrients. 

DNA damage due to aging is sex, species, and tissue 

specific (Li et al., 1992). 

In male and female mice, there was clear evidence for 

carcinogenic activity in the liver, where the incidences 

of hepatocellular adenoma, hepatocellular carcinoma, 

and hepatoblastomas were increased (except hepatoblastoma in females). The incidence and multiplicity 

of hepatocellular neoplasms increased in an exposurerelated manner. In addition, centrilobular hypertrophy and hepatocyte cytoplasmic vacuolization were 

increased. Thyroid gland follicular cell hyperplasia 

was seen in male and female mice. The incidence of 

thyroid gland follicular cell adenomas was increased 

in 1,300 ppm male mice. These effects may be due to 

phenobarbital-induced liver enzymes which can result 

in decreases in circulating T3 and T4 levels, followed 

by increases in TSH stimulation, but this was not 

measured in the current studies. 

These studies could not determine the extent to which 

the mouse liver carcinogenic effects were due to the 

parent compound, the metabolites, or a combination 

of exposures to these chemicals. However, there is 

considerable literature on the carcinogenic effects 

of phenobarbital in mice (reviewed by IARC, 1976) 

since its introduction in 1912 as a sedative-hypnotic 

and anticonvulsant (McClain, 1990). Phenobarbital 

has been shown to cause liver neoplasms in several 

strains of mice including CF1 mice (Thorpe and 

Walker, 1973; Ponomarkov et al., 1976) and 

C3H mice (Peraino et al., 1973a). Those species or 

strains with high incidences of spontaneous liver 

neoplasms appear to be more sensitive than species 

with low neoplasm incidences (McClain, 1990); most 

rat strains have relatively low incidences of spontaneous liver neoplasms, and it has been shown that 

rats, as compared to mice, are relatively resistant to 

phenobarbital-induced liver neoplasms. 

Hepatocellular adenomas, hepatocellular carcinomas, 

and hepatoblastomas represent a biological and morphological continuum in progression of proliferative 

lesions. It is probable that hepatoblastomas are composed of cells that are more primitive, rather than 

representing further progression to a more malignant 

state. Hepatoblastomas are considered to represent a 

phenotypic (and possibly genotypic) variant of a 

malignant liver neoplasm. Because the malignant 

potential of hepatoblastomas and hepatocellular carcinomas appears similar and hepatoblastomas are generally observed within hepatocellular neoplasms (mostly 

carcinomas), it is appropriate to combine the incidences of hepatoblastoma with those of adenoma and 

carcinoma when interpreting the carcinogenic 

potential of a chemical. Hepatoblastomas are rare 

and seen in relatively high numbers only after 

chemical administration [e.g., with oxazepam (NTP, 

1993b, 1998), o-nitroanisole (NTP, 1993c), benzofuran (NTP, 1989), ethylene thiourea (NTP, 1992), 

1-amino 2,4-dibromoanthraquinone (NTP, 1996), 

methylphenidate hydrochloride (NTP, 1995), and 

coumarin (NTP, 1993a)]. Liver cancer accounts for 

approximately 2% to 3% of all cancer deaths in the 

United States (Parker et al., 1996). In children, hepatoblastomas account for approximately 70% of all 

liver cancers (Ding et al., 1994). 

To date, liver disease associated with Helicobacter 

hepaticus infection has been identified in male mice 

from nine 2-year bioassays. Using a polymerase 

chain reaction-based assay (Malarkey et al., 1997); 

H. hepaticus was not identified in the livers of 

20 mice evaluated from this study. Furthermore, 

histologic lesions consistent with those described for 

H. hepaticus were not identified in livers of mice in 

this study. 
 

 

 

Primidone, NTP TR 476 67 

In NTP genetic toxicity studies, primidone did not 

induce chromosomal effects in a number of mammalian cell test systems (Appendix E). Primidone was 

mutagenic in Salmonella in the absence of S9, but 

no mutagenic activity was detected in the presence of 

S9. The metabolite phenobarbital has a longer halflife than the parent compound primidone, suggesting 

that the metabolite may have contributed to the carcinogenic effects observed, but these studies could not 

determine the extent to which the carcinogenic effects 

were due to parent compound, metabolite, or a combination of these exposures. 

Phenobarbital has been used clinically for more than 

80 years, and there is no conclusive evidence that it 

causes neoplasms in the clinical setting (Brodie and 

Dichter, 1996). The IARC has reviewed the data on 

phenobarbital and has found insufficient evidence 

to draw a conclusion that it is carcinogenic in humans, 

although the evidence for carcinogenicity in animals 

is clear (IARC, 1987). A recent review on the side 

effects of primidone did not report any evidence for 

cancer in humans for this or any other antiepileptic 

drug (Brodie and Dichter, 1996). 

CONCLUSIONS 

Under the conditions of these 2-year feed studies, 

there was equivocal evidence of carcinogenic activity* 

of primidone in male F344/N rats based on a marginal 

increase in thyroid gland follicular cell neoplasms, 

primarily adenomas, and a marginal increase in renal 

tubule neoplasms. There was no evidence of carcinogenic activity of primidone in female F344/N rats 

exposed to 600, 1,300, or 2,500 ppm. There was 

clear evidence of carcinogenic activity of primidone in 

male B6C3F1 mice based on the increased incidences 

of hepatocellular neoplasms, and the increased incidence of thyroid gland follicular cell adenomas was 

also considered to be chemical related. There was 

clear evidence of carcinogenic activity of primidone 

in female B6C3F1 mice based on the increased incidences of hepatocellular neoplasms. 

Exposure of rats to primidone resulted in increased 

incidences of hepatocyte cytoplasmic vacuolization 

and centrilobular hypertrophy in males and females 

and eosinophilic foci in females. The increased severity of nephropathy and increased incidence of renal 

tubule hyperplasia in male rats were related to 

primidone exposure. Exposure of male mice to 

primidone resulted in hepatocyte centrilobular hypertrophy and thyroid gland follicular cell hyperplasia. 

Exposure of female mice to primidone resulted in 

hepatocyte centrilobular hypertrophy and cytoplasmic 

vacuolization, eosinophilic focus, and thyroid gland 

follicular cell hyperplasia. 

* Explanation of Levels of Evidence of Carcinogenic Activity is on page 11. A summary of the Technical Reports Review Subcommittee 

comments and the public discussion on this Technical Report appears on page 13. 
68 Primidone, NTP TR 476
 
 

 

 

 

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81 

APPENDIX A 

SUMMARY OF LESIONS IN MALE RATS 

IN THE 2-YEAR FEED STUDY 

OF PRIMIDONE 

TABLE A1 

TABLE A2 

TABLE A3 

TABLE A4 

TABLE A5 

Summary of the Incidence of Neoplasms in Male Rats 

in the 2-Year Feed Study of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

Individual Animal Tumor Pathology of Male Rats 

in the 2-Year Feed Study of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

Statistical Analysis of Primary Neoplasms in Male Rats 

in the 2-Year Feed Study of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

Historical Incidence of Thyroid Gland Follicular Cell Neoplasms 

in Untreated Male F344/N Rats ...................................... 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats 

in the 2-Year Feed Study of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

82 

86 

104 

110 

111 
 

 

 

 

 

 

 

 

 

 

82 Primidone, NTP TR 476 

TABLE A1 

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study of Primidonea 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

Disposition Summary 

Animals initially in study 50 50 50 50 

Early deaths 

Moribund 33 33 39 36 

Natural deaths 4 10 7 14 

Survivors 

Terminal sacrifice 13 7 4 

Animals examined microscopically 50 50 50 50 

Alimentary System 

Intestine large, colon (50) (50) (49) (46) 

Polyp adenomatous 1 (2%) 

Intestine small, duodenum (50) (50) (50) (47) 

Intestine small, jejunum (50) (50) (49) (45) 

Carcinoma 1 (2%) 

Leiomyosarcoma 1 (2%) 

Intestine small, ileum (50) (50) (49) (46) 

Leiomyosarcoma 1 (2%) 

Liver (50) (50) (50) (50) 

Fibrosarcoma, metastatic, spleen 1 (2%) 

Hepatocellular adenoma 1 (2%) 1 (2%) 

Hepatocellular adenoma, multiple 1 (2%) 1 (2%) 

Mesentery (11) (6) (9) (3) 

Fat, fibrosarcoma, metastatic, spleen 1 (9%) 

Oral mucosa (1) 

Pharyngeal, squamous cell carcinoma 1 (100%) 

Pancreas (50) (50) (50) (49) 

Salivary glands (50) (50) (49) (49) 

Stomach, forestomach (50) (50) (50) (49) 

Squamous cell papilloma 1 (2%) 

Stomach, glandular (50) (50) (50) (49) 

Tongue (1) 

Squamous cell papilloma 1 (100%) 

Tooth (1) 

Cardiovascular System 

Heart (50) (50) (50) (50) 

Endocrine System 

Adrenal cortex (50) (50) (50) (50) 

Adrenal medulla (50) (50) (50) (49) 

Pheochromocytoma malignant 1 (2%) 1 (2%) 1 (2%) 

Pheochromocytoma benign 16 (32%) 19 (38%) 19 (38%) 7 (14%) 

Bilateral, pheochromocytoma benign 7 (14%) 9 (18%) 5 (10%) 5 (10%) 

Islets, pancreatic (50) (50) (50) (49) 

Adenoma 1 (2%) 

Carcinoma 1 (2%) 

Parathyroid gland (45) (41) (46) (49) 

Pituitary gland (49) (50) (48) (49) 

Pars distalis, adenoma 17 (35%) 12 (24%) 11 (23%) 4 (8%) 

Pars intermedia, adenoma 1 (2%) 1 (2%) 1 (2%) 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 83 

TABLE A1 

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study of Primidone 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

Endocrine System (continued) 

Thyroid gland (50) (50) (49) (49) 

Bilateral, C-cell, adenoma 1 (2%) 

C-cell, adenoma 7 (14%) 2 (4%) 7 (14%) 1 (2%) 

C-cell, carcinoma 1 (2%) 1 (2%) 

Follicular cell, adenoma 1 (2%) 1 (2%) 6 (12%) 3 (6%) 

Follicular cell, carcinoma 1 (2%) 3 (6%) 1 (2%) 1 (2%) 

General Body System 

Peritoneum (1) (1) (1) 

Tissue NOS (1) 

Genital System 

Coagulating gland (2) (1) (1) 

Epididymis (50) (50) (50) (48) 

Preputial gland (50) (50) (50) (49) 

Adenoma 2 (4%) 1 (2%) 2 (4%) 

Carcinoma 1 (2%) 

Prostate (50) (50) (50) (48) 

Seminal vesicle (50) (50) (50) (48) 

Testes (50) (50) (50) (48) 

Bilateral, interstitial cell, adenoma 33 (66%) 33 (66%) 35 (70%) 34 (71%) 

Interstitial cell, adenoma 8 (16%) 11 (22%) 7 (14%) 9 (19%) 

Hematopoietic System 

Bone marrow (50) (50) (50) (50) 

Lymph node (24) (23) (16) (10) 

Lymph node, mandibular (50) (50) (48) (49) 

Lymph node, mesenteric (50) (50) (50) (48) 

Spleen (50) (50) (50) (49) 

Fibrosarcoma 3 (6%) 1 (2%) 

Thymus (49) (47) (44) (46) 

Thymoma malignant 1 (2%) 

Integumentary System 

Mammary gland (48) (49) (50) (47) 

Adenoma 1 (2%) 

Carcinoma 2 (4%) 

Fibroadenoma 3 (6%) 3 (6%) 3 (6%) 

Skin (50) (50) (50) (50) 

Basal cell adenoma 1 (2%) 

Basal cell carcinoma 1 (2%) 

Basosquamous tumor benign 1 (2%) 

Keratoacanthoma 1 (2%) 1 (2%) 

Trichoepithelioma 1 (2%) 

Pinna, melanoma malignant 1 (2%) 

Pinna, squamous cell papilloma 1 (2%) 1 (2%) 1 (2%) 

Subcutaneous tissue, fibroma 3 (6%) 3 (6%) 3 (6%) 

Subcutaneous tissue, fibrosarcoma 2 (4%) 

Subcutaneous tissue, hemangiosarcoma 1 (2%) 

Subcutaneous tissue, pinna, melanoma benign 1 (2%) 1 (2%) 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

84 Primidone, NTP TR 476 

TABLE A1 

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study of Primidone 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

Musculoskeletal System 

Bone 

Chordoma

Mandible, osteosarcoma

Tibia, osteosarcoma

Skeletal muscle

(50) 

 1 (2%) 

 1 (2%) 

 (1) 

(50) 

 1 (2%) 

(50) (50) 

Nervous System 

Brain 

Astrocytoma malignant

Peripheral nerve

Spinal, schwannoma malignant

(50) 

 (1)

 1 (100%) 

(50) 

 (1) 

(49) (49) 

 2 (4%) 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma

Alveolar/bronchiolar carcinoma

Chordoma, metastatic, bone

Fibrosarcoma, metastatic, spleen

Hemangiosarcoma, metastatic, skin

Osteosarcoma, metastatic, bone

Squamous cell carcinoma

Nose 

(50) 

 1 (2%)

 1 (2%) 

 1 (2%) 

 1 (2%) 

(50) 

(50) 

 1 (2%) 

(50) 

(50) 

 1 (2%)

 1 (2%) 

 1 (2%) 

(50) 

(50) 

 1 (2%) 

(50) 

Special Senses System 

Zymbal’s gland

Carcinoma

 (1) 

 1 (100%) 

Urinary System 

Kidney 

Fibrosarcoma, metastatic, spleen

Lipoma

Osteosarcoma, metastatic, bone

Renal tubule, adenoma

Renal tubule, carcinoma

Urinary bladder 

Papilloma

(50) 

 1 (2%) 

 1 (2%) 

 2 (4%)

(50) 

(50) 

 1 (2%)

(50) 

(50) 

 1 (2%)

(50) 

 1 (2%) 

(50) 

 1 (2%) 

 1 (2%) 

 1 (2%) 

(48) 

Systemic Lesions 

Multiple organsb 

Leukemia mononuclear

Lymphoma malignant

Mesothelioma malignant

(50) 

 35 (70%)

 2 (4%)

(50) 

 30 (60%)

 3 (6%)

(50) 

 24 (48%)

 1 (2%) 

 2 (4%)

(50) 

 12 (24%) 

 1 (2%) 
 

85 

c 

Primidone, NTP TR 476 

TABLE A1 

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study of Primidone 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

Neoplasm Summary 

Total animals with primary neoplasmsc 50 49 50 47 

Total primary neoplasms 160 144 143 87 

Total animals with benign neoplasms 49 49 49 44 

Total benign neoplasms 105 103 107 69 

Total animals with malignant neoplasms 41 35 29 15 

Total malignant neoplasms 55 41 36 18 

Total animals with metastatic neoplasms 3 1 

Total metastatic neoplasms 7 1 

a Number of animals examined microscopically at the site and the number of animals with neoplasm b Number of animals with any tissue examined microscopically 

Primary neoplasms: all neoplasms except metastatic neoplasms 
 

 

 

 

 

86 Primidone, NTP TR 476 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Primidone: 0 ppm 

2344555555555566666666666 

Number of Days on Study 8056002667788912444555666 

1573596084535197156255279 

0000000000000000000000000 

Carcass ID Number 1441103112200230124534103 

4433754104079224592088616 

Alimentary System 

Esophagus +++++++++++++++++++++++++ 

Intestine large, colon +++++++++++++++++++++++++ 

Intestine large, rectum +++++++++++++++++++++++++ 

Intestine large, cecum +++++++++++++++++++++++++ 

Intestine small, duodenum +++++++++++++++++++++++++ 

Intestine small, jejunum +++++++++++++++++++++++++ 

Carcinoma X 

Intestine small, ileum +++++++++++++++++++++++++ 

Leiomyosarcoma X 

Liver +++++++++++++++++++++++++ 

Fibrosarcoma, metastatic, spleen X 

Hepatocellular adenoma 

Mesentery + ++ + + 

Fat, fibrosarcoma, metastatic, spleen X 

Oral mucosa + 

Pharyngeal, squamous cell carcinoma X 

Pancreas +++++++++++++++++++++++++ 

Salivary glands +++++++++++++++++++++++++ 

Stomach, forestomach +++++++++++++++++++++++++ 

Stomach, glandular +++++++++++++++++++++++++ 

Cardiovascular System 

Blood vessel 

Heart 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

Endocrine System 

Adrenal cortex +++++++++++++++++++++++++ 

Adrenal medulla +++++++++++++++++++++++++ 

Pheochromocytoma malignant 

Pheochromocytoma benign X X X X X 

Bilateral, pheochromocytoma benign X X X 

Islets, pancreatic +++++++++++++++++++++++++ 

Adenoma 

Carcinoma 

Parathyroid gland +M+M+++++++++M+++++++++++ 

Pituitary gland ++++++++++M++++++++++++++ 

Pars distalis, adenoma X X X XX X X 

Thyroid gland +++++++++++++++++++++++++ 

C-cell, adenoma X 

C-cell, carcinoma X 

Follicular cell, adenoma 

Follicular cell, carcinoma 

General Body System 

None 

+: Tissue examined microscopically M: Missing tissue X: Lesion present 

A: Autolysis precludes examination I: Insufficient tissue Blank: Not examined 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 87 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Primidone: 0 ppm 

Number of Days on Study 

6666666777777777777777777 

6778999011112222222222222 

9463278400593333333333333 

Carcass ID Number 

0000000000000000000000000 

3334010133440012222223444 

7316386909052821356785179 

Total 

Tissues/ 

Tumors 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Carcinoma 

Intestine small, ileum 

Leiomyosarcoma 

Liver 

Fibrosarcoma, metastatic, spleen 

Hepatocellular adenoma

Mesentery

Fat, fibrosarcoma, metastatic, spleen 

Oral mucosa 

Pharyngeal, squamous cell carcinoma 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

X 

 ++ + ++ + 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

50 

50 

50 

50 

50 

1 

50 

1 

50 

1 

1 

11 

1 

1 

1 

50 

50 

50 

50 

Cardiovascular System 

Blood vessel + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Pheochromocytoma malignant X 1 

Pheochromocytoma benign XX XXXX X X X X X 16 

Bilateral, pheochromocytoma benign X X X X 7 

Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Adenoma X 1 

Carcinoma X 1 

Parathyroid gland M + + + + + + + + + + M + + + + + + + + + + + + + 45 

Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Pars distalis, adenoma X X X X X X X X X X 17 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 50 

C-cell, adenoma X X X X X X 7 

C-cell, carcinoma 1 

Follicular cell, adenoma X 1 

Follicular cell, carcinoma X 1 

General Body System 

None 
 

 

 

 

 

 

 

 

 

88 Primidone, NTP TR 476 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Primidone: 0 ppm 

2344555555555566666666666 

Number of Days on Study 8056002667788912444555666 

1573596084535197156255279 

0000000000000000000000000 

Carcass ID Number 1441103112200230124534103 

4433754104079224592088616 

Genital System 

Coagulating gland + 

Epididymis +++++++++++++++++++++++++ 

Preputial gland +++++++++++++++++++++++++ 

Adenoma X 

Carcinoma X 

Prostate +++++++++++++++++++++++++ 

Seminal vesicle +++++++++++++++++++++++++ 

Testes +++++++++++++++++++++++++ 

Bilateral, interstitial cell, adenoma X XXXXX XX XXXX X 

Interstitial cell, adenoma X X X X 

Hematopoietic System 

Bone marrow +++++++++++++++++++++++++ 

Lymph node + +++ +++++ + +++ 

Lymph node, mandibular +++++++++++++++++++++++++ 

Lymph node, mesenteric +++++++++++++++++++++++++ 

Spleen +++++++++++++++++++++++++ 

Fibrosarcoma X X 

Thymus +++++++++++++++++++++++++ 

Thymoma malignant X 

Integumentary System 

Mammary gland +++++++++++++++++++M++M++ 

Carcinoma 

Fibroadenoma X 

Skin +++++++++++++++++++++++++ 

Basal cell adenoma 

Basal cell carcinoma 

Keratoacanthoma 

Subcutaneous tissue, fibroma X X X 

Subcutaneous tissue, pinna, melanoma benign X 

Musculoskeletal System 

Bone +++++++++++++++++++++++++ 

Chordoma X 

Mandible, osteosarcoma 

Skeletal muscle + 

Nervous System 

Brain +++++++++++++++++++++++++ 

Peripheral nerve + 

Spinal, schwannoma malignant X 

Spinal cord + 

Respiratory System 

Lung +++++++++++++++++++++++++ 

Alveolar/bronchiolar adenoma X 

Chordoma, metastatic, bone X 

Fibrosarcoma, metastatic, spleen X 

Osteosarcoma, metastatic, bone 

Nose +++++++++++++++++++++++++ 

Trachea +++++++++++++++++++++++++ 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 89 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Primidone: 0 ppm 

6666666777777777777777777 

Number of Days on Study 6778999011112222222222222 

9463278400593333333333333 

0000000000000000000000000 Total 

Carcass ID Number 3334010133440012222223444 Tissues/ 

7316386909052821356785179 Tumors 

Genital System 

Coagulating gland + 2 

Epididymis +++++++++++++++++++++++++ 50 

Preputial gland +++++++++++++++++++++++++ 50 

Adenoma X 2 

Carcinoma 1 

Prostate +++++++++++++++++++++++++ 50 

Seminal vesicle +++++++++++++++++++++++++ 50 

Testes +++++++++++++++++++++++++ 50 

Bilateral, interstitial cell, adenoma X XXXXXXX X XXXXXX XXXXX 33 

Interstitial cell, adenoma X X X X 8 

Hematopoietic System 

Bone marrow +++++++++++++++++++++++++ 50 

Lymph node +++++ +++++ + 24 

Lymph node, mandibular +++++++++++++++++++++++++ 50 

Lymph node, mesenteric +++++++++++++++++++++++++ 50 

Spleen +++++++++++++++++++++++++ 50 

Fibrosarcoma X 3 

Thymus ++++++++M++++++++++++++++ 49 

Thymoma malignant 1 

Integumentary System 

Mammary gland +++++++++++++++++++++++++ 48 

Carcinoma X X 2 

Fibroadenoma X X 3 

Skin +++++++++++++++++++++++++ 50 

Basal cell adenoma X 1 

Basal cell carcinoma X 1 

Keratoacanthoma X 1 

Subcutaneous tissue, fibroma 3 

Subcutaneous tissue, pinna, melanoma benign 1 

Musculoskeletal System 

Bone +++++++++++++++++++++++++ 50 

Chordoma 1 

Mandible, osteosarcoma X 1 

Skeletal muscle 1 

Nervous System 

Brain +++++++++++++++++++++++++ 50 

Peripheral nerve 1 

Spinal, schwannoma malignant 1 

Spinal cord 1 

Respiratory System 

Lung +++++++++++++++++++++++++ 50 

Alveolar/bronchiolar adenoma 1 

Chordoma, metastatic, bone 1 

Fibrosarcoma, metastatic, spleen 1 

Osteosarcoma, metastatic, bone X 1 

Nose +++++++++++++++++++++++++ 50 

Trachea +++++++++++++++++++++++++ 50 
 

 

90 Primidone, NTP TR 476 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Primidone: 0 ppm 

2344555555555566666666666 

Number of Days on Study 8056002667788912444555666 

1573596084535197156255279 

0000000000000000000000000 

Carcass ID Number 1441103112200230124534103 

4433754104079224592088616 

Special Senses System 

None 

Urinary System 

Kidney +++++++++++++++++++++++++ 

Fibrosarcoma, metastatic, spleen X 

Osteosarcoma, metastatic, bone 

Renal tubule, adenoma X 

Urinary bladder +++++++++++++++++++++++++ 

Systemic Lesions 

Multiple organs +++++++++++++++++++++++++ 

Leukemia mononuclear X XXXXXXXXXXXX XXXXX 

Mesothelioma malignant 
 

 

 

 

Primidone, NTP TR 476 91 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Primidone: 0 ppm 

6666666777777777777777777 

Number of Days on Study 6778999011112222222222222 

9463278400593333333333333 

0000000000000000000000000 Total 

Carcass ID Number 3 3 3 4 0 1 0 1 3 3 4 4 0 0 1 2 2 2 2 2 2 3 4 4 4 Tissues/ 

7316386909052821356785179 Tumors 

Special Senses System 

None 

Urinary System 

Kidney +++++++++++++++++++++++++ 50 

Fibrosarcoma, metastatic, spleen 1 

Osteosarcoma, metastatic, bone X 1 

Renal tubule, adenoma X 2 

Urinary bladder +++++++++++++++++++++++++ 50 

Systemic Lesions 

Multiple organs +++++++++++++++++++++++++ 50 

Leukemia mononuclear XXXX XXXXXX XX X XX XX 35 

Mesothelioma malignant XX 2 
 

 

 

 

 

 

 

92 Primidone, NTP TR 476 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Primidone: 600 ppm 

3555555555666666666666666 

Number of Days on Study 3000244678011123334445566 

0223624293909974551122419 

0000000000000000000000000 

Carcass ID Number 7589998659766886785566875 

9619034097463891367897011 

Alimentary System 

Esophagus +++++++++++++++++++++++++ 

Intestine large, colon +++++++++++++++++++++++++ 

Intestine large, rectum +++++++++++++++++++++++++ 

Intestine large, cecum +++++++++++++++++++++++++ 

Intestine small, duodenum +++++++++++++++++++++++++ 

Intestine small, jejunum +++++++++++++++++++++++++ 

Leiomyosarcoma 

Intestine small, ileum +++++++++++++++++++++++++ 

Liver +++++++++++++++++++++++++ 

Hepatocellular adenoma, multiple 

Mesentery ++ + 

Pancreas +++++++++++++++++++++++++ 

Salivary glands +++++++++++++++++++++++++ 

Stomach, forestomach +++++++++++++++++++++++++ 

Stomach, glandular +++++++++++++++++++++++++ 

Tooth + 

Cardiovascular System 

Blood vessel +++++++++++++++++++++++++ 

Heart +++++++++++++++++++++++++ 

Endocrine System 

Adrenal cortex +++++++++++++++++++++++++ 

Adrenal medulla +++++++++++++++++++++++++ 

Pheochromocytoma malignant X 

Pheochromocytoma benign X X XXX X XX X X 

Bilateral, pheochromocytoma benign X X 

Islets, pancreatic +++++++++++++++++++++++++ 

Parathyroid gland +++MMM+++M++++++++MM+++++ 

Pituitary gland +++++++++++++++++++++++++ 

Pars distalis, adenoma X XX X XX X 

Pars intermedia, adenoma X 

Thyroid gland +++++++++++++++++++++++++ 

Bilateral, C-cell, adenoma X 

C-cell, adenoma 

Follicular cell, adenoma 

Follicular cell, carcinoma X 

General Body System 

Peritoneum + 

Tissue NOS + 

Genital System 

Coagulating gland + 

Epididymis +++++++++++++++++++++++++ 

Preputial gland +++++++++++++++++++++++++ 

Adenoma 

Prostate +++++++++++++++++++++++++ 

Seminal vesicle +++++++++++++++++++++++++ 

Testes +++++++++++++++++++++++++ 

Bilateral, interstitial cell, adenoma X X XXXX XX XXX 

Interstitial cell, adenoma XXX X X XX XX 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 93 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Primidone: 600 ppm 

6666666666677777777777777 

Number of Days on Study 6677778899900011112222222 

9903443347739902253333333 

0000000000000000000000001 Total 

Carcass ID Number 5779996967896885975567780 Tissues/ 

5255685480512734262347820 Tumors 

Alimentary System 

Esophagus +++++++++++++++++++++++++ 50 

Intestine large, colon +++++++++++++++++++++++++ 50 

Intestine large, rectum +++++++++++++++++++++++++ 50 

Intestine large, cecum +++++++++++++++++++++++++ 50 

Intestine small, duodenum +++++++++++++++++++++++++ 50 

Intestine small, jejunum +++++++++++++++++++++++++ 50 

Leiomyosarcoma X 1 

Intestine small, ileum +++++++++++++++++++++++++ 50 

Liver +++++++++++++++++++++++++ 50 

Hepatocellular adenoma, multiple X 1 

Mesentery + + + 6 

Pancreas +++++++++++++++++++++++++ 50 

Salivary glands +++++++++++++++++++++++++ 50 

Stomach, forestomach +++++++++++++++++++++++++ 50 

Stomach, glandular +++++++++++++++++++++++++ 50 

Tooth 1 

Cardiovascular System 

Blood vessel +++++++++++++++++++++++++ 50 

Heart +++++++++++++++++++++++++ 50 

Endocrine System 

Adrenal cortex +++++++++++++++++++++++++ 50 

Adrenal medulla +++++++++++++++++++++++++ 50 

Pheochromocytoma malignant 1 

Pheochromocytoma benign XX X X X XXX X 19 

Bilateral, pheochromocytoma benign X X X X X X X 9 

Islets, pancreatic +++++++++++++++++++++++++ 50 

Parathyroid gland +++++++M+++++++++++++++MM 41 

Pituitary gland +++++++++++++++++++++++++ 50 

Pars distalis, adenoma X X X X X 12 

Pars intermedia, adenoma 1 

Thyroid gland +++++++++++++++++++++++++ 50 

Bilateral, C-cell, adenoma 1 

C-cell, adenoma X X 2 

Follicular cell, adenoma X 1 

Follicular cell, carcinoma X X 3 

General Body System 

Peritoneum 1 

Tissue NOS 1 

Genital System 

Coagulating gland 1 

Epididymis +++++++++++++++++++++++++ 50 

Preputial gland +++++++++++++++++++++++++ 50 

Adenoma X 1 

Prostate +++++++++++++++++++++++++ 50 

Seminal vesicle +++++++++++++++++++++++++ 50 

Testes +++++++++++++++++++++++++ 50 

Bilateral, interstitial cell, adenoma XX XXXXXXXXXXXXXXXXXXXX 33 

Interstitial cell, adenoma X X 11 
 

 

 

94 Primidone, NTP TR 476 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Primidone: 600 ppm 

Number of Days on Study 

3555555555666666666666666 

3000244678011123334445566 

0223624293909974551122419 

Carcass ID Number 

0000000000000000000000000 

7589998659766886785566875 

9619034097463891367897011 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Fibrosarcoma

Thymus 

+++++++++++++++++++++++++ 

+++ + +++++ +++ + + 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

X 

+++++++++++++++++++++++M+ 

Integumentary System 

Mammary gland 

Adenoma

Fibroadenoma 

Skin 

Basosquamous tumor benign 

Keratoacanthoma 

Pinna, squamous cell papilloma

Subcutaneous tissue, fibroma

+M+++++++++++++++++++++++ 

X 

+++++++++++++++++++++++++ 

X 

X X X 

Musculoskeletal System 

Bone 

Tibia, osteosarcoma 

+++++++++++++++++++++++++ 

Nervous System 

Brain 

Peripheral nerve

Spinal cord

+++++++++++++++++++++++++ 

+ 

+ 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma

Nose 

Trachea 

+++++++++++++++++++++++++ 

X 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

Special Senses System 

Eye

Zymbal’s gland

Carcinoma

 + 

+ 

X 

Urinary System 

Kidney 

Renal tubule, adenoma

Urinary bladder 

+++++++++++++++++++++++++ 

X 

+++++++++++++++++++++++++ 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear 

Mesothelioma malignant

+++++++++++++++++++++++++ 

XXX X XXXXX XXX XX X X 

X X 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 95 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Primidone: 600 ppm 

6666666666677777777777777 

Number of Days on Study 6677778899900011112222222 

9903443347739902253333333 

0000000000000000000000001 Total 

Carcass ID Number 5779996967896885975567780 

5255685480512734262347820 

Tissues/ 

Tumors 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Fibrosarcoma 

Thymus 

+++++++++++++++++++++++++ 

+ + + + ++++ + 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

++M++++++M+++++++++++++++ 

50 

23 

50 

50 

50 

1 

47 

Integumentary System 

Mammary gland 

Adenoma 

Fibroadenoma

Skin 

Basosquamous tumor benign

Keratoacanthoma 

Pinna, squamous cell papilloma 

Subcutaneous tissue, fibroma 

+++++++++++++++++++++++++ 

X X X 

+++++++++++++++++++++++++ 

X 

X 

49 

1 

3 

50 

1 

1 

1 

3 

Musculoskeletal System 

Bone 

Tibia, osteosarcoma

+++++++++++++++++++++++++ 

X 

50 

1 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

+++++++++++++++++++++++++ 50 

1 

1 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Nose 

Trachea 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

1 

50 

50 

Special Senses System 

Eye 

Zymbal’s gland 

Carcinoma 

1 

1 

1 

Urinary System 

Kidney 

Renal tubule, adenoma 

Urinary bladder 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

1 

50 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear 

+++++++++++++++++++++++++ 

X X XXXX X XXXX X X X 

50 

30 

Mesothelioma malignant X 3 
 

 

 

 

 

96 Primidone, NTP TR 476 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Primidone: 1,300 ppm 

3455555555555566666666666 

Number of Days on Study 5325566778888911112233334 

4791812263599923397712551 

1111111111111111111111111 

Carcass ID Number 2401141203103221203331230 

5916483043522279650471193 

Alimentary System 

Esophagus +++++++++++++++++++++++++ 

Intestine large, colon ++++++++++++++++++++A ++++ 

Polyp adenomatous X 

Intestine large, rectum ++++++++++++++++++++A ++++ 

Intestine large, cecum + + + + + + + + + + + + + + + + + + + + A + + + + 

Intestine small, duodenum +++++++++++++++++++++++++ 

Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + A + + + + 

Intestine small, ileum ++++++++++++++++++++M++++ 

Liver +++++++++++++++++++++++++ 

Hepatocellular adenoma, multiple 

Mesentery + + + ++ 

Pancreas +++++++++++++++++++++++++ 

Salivary glands +++++++++++++++++++++++++ 

Stomach, forestomach +++++++++++++++++++++++++ 

Squamous cell papilloma 

Stomach, glandular +++++++++++++++++++++++++ 

Tongue 

Squamous cell papilloma 

Cardiovascular System 

Blood vessel +++++++++++++++++++++++++ 

Heart +++++++++++++++++++++++++ 

Endocrine System 

Adrenal cortex +++++++++++++++++++++++++ 

Adrenal medulla +++++++++++++++++++++++++ 

Pheochromocytoma malignant X 

Pheochromocytoma benign X X X X XXXXX 

Bilateral, pheochromocytoma benign X X 

Islets, pancreatic +++++++++++++++++++++++++ 

Parathyroid gland +++++M+++++++++++++M+++++ 

Pituitary gland ++++++++++++M++++++++++++ 

Pars distalis, adenoma X X X X 

Pars intermedia, adenoma X 

Thyroid gland +++++++++++++++++++++++++ 

C-cell, adenoma X X 

C-cell, carcinoma X 

Follicular cell, adenoma X X X X 

Follicular cell, carcinoma 

General Body System 

Peritoneum + 

Genital System 

Epididymis +++++++++++++++++++++++++ 

Preputial gland +++++++++++++++++++++++++ 

Adenoma X 

Prostate +++++++++++++++++++++++++ 

Seminal vesicle +++++++++++++++++++++++++ 

Testes +++++++++++++++++++++++++ 

Bilateral, interstitial cell, adenoma XXX XX XXXX XX XXX X 

Interstitial cell, adenoma X X X X 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 97 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Primidone: 1,300 ppm 

6666666666666666666677777 

Number of Days on Study 4445556666677888899912222 

1384594778934333817823333 

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 

Carcass ID Number 1 4 2 2 1 4 3 0 4 0 4 3 3 0 1 4 2 0 4 5 2 1 3 4 4 Tissues/ 

2 2 3 9 8 5 5 8 4 9 0 8 6 7 7 3 8 6 1 0 4 0 1 6 7 Tumors 

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Polyp adenomatous 1 

Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Liver + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Hepatocellular adenoma, multiple X 1 

Mesentery + + + + 9 

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Salivary glands + M + + + + + + + + + + + + + + + + + + + + + + + 49 

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Squamous cell papilloma X 1 

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Tongue + 1 

Squamous cell papilloma X 1 

Cardiovascular System 

Blood vessel + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Pheochromocytoma malignant 1 

Pheochromocytoma benign XXX X X X XX X X 19 

Bilateral, pheochromocytoma benign X X X 5 

Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Parathyroid gland + M + + + + + + + + + + + + + + M + + + + + + + + 46 

Pituitary gland + M + + + + + + + + + + + + + + + + + + + + + + + 48 

Pars distalis, adenoma XXXX X X X 11 

Pars intermedia, adenoma 1 

Thyroid gland + M + + + + + + + + + + + + + + + + + + + + + + + 49 

C-cell, adenoma X X XXX 7 

C-cell, carcinoma 1 

Follicular cell, adenoma X X 6 

Follicular cell, carcinoma X 1 

General Body System 

Peritoneum 1 

Genital System 

Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Preputial gland + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Adenoma X 2 

Prostate + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Testes + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Bilateral, interstitial cell, adenoma X X X X X X X X X X X X X X X X X X X X 35 

Interstitial cell, adenoma X X X 7 
 

 

 

 

 

 

 

 

98 Primidone, NTP TR 476 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Primidone: 1,300 ppm 

3455555555555566666666666 

Number of Days on Study 5325566778888911112233334 

4791812263599923397712551 

1111111111111111111111111 

Carcass ID Number 2401141203103221203331230 

5916483043522279650471193 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

+++++++++++++++++++++++++ 

+ +++ ++++ + + 

++++++++++++M++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++M++++M++++ 

Integumentary System 

Mammary gland 

Fibroadenoma 

Skin 

Pinna, melanoma malignant 

Pinna, squamous cell papilloma 

Subcutaneous tissue, fibroma

Subcutaneous tissue, fibrosarcoma

Subcutaneous tissue, hemangiosarcoma 

Subcutaneous tissue, pinna, melanoma benign 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

X X 

X X 

X 

X 

Musculoskeletal System 

Bone +++++++++++++++++++++++++ 

Nervous System 

Brain +++++++++++++++++++++++++ 

Respiratory System 

Lung 

Alveolar/bronchiolar carcinoma

Hemangiosarcoma, metastatic, skin 

Squamous cell carcinoma

Nose 

Trachea 

+++++++++++++++++++++++++ 

X 

X 

X 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

Special Senses System 

Eye + 

Urinary System 

Kidney 

Renal tubule, adenoma

Urinary bladder 

Papilloma 

+++++++++++++++++++++++++ 

X 

+++++++++++++++++++++++++ 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear 

Lymphoma malignant 

Mesothelioma malignant

+++++++++++++++++++++++++ 

X XXXX XXX X XX XX 

X 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 99 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Primidone: 1,300 ppm 

6666666666666666666677777 

Number of Days on Study 4445556666677888899912222 

1384594778934333817823333 

1111111111111111111111111 Total 

Carcass ID Number 1422143040433014204521344 

2239855849086773861040167 

Tissues/ 

Tumors 

Hematopoietic System 

Bone marrow 

Lymph node

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

+++++++++++++++++++++++++ 

+ + + + + + 

+ M +++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++M+++M+++++M+++M+++++++ 

50 

16 

48 

50 

50 

44 

Integumentary System 

Mammary gland 

Fibroadenoma

Skin 

Pinna, melanoma malignant

Pinna, squamous cell papilloma

Subcutaneous tissue, fibroma

Subcutaneous tissue, fibrosarcoma 

Subcutaneous tissue, hemangiosarcoma 

Subcutaneous tissue, pinna, melanoma benign 

+++++++++++++++++++++++++ 

X X X 

+++++++++++++++++++++++++ 

X 

X 

X 

50 

3 

50 

1 

1 

3 

2 

1 

1 

Musculoskeletal System 

Bone +++++++++++++++++++++++++ 50 

Nervous System 

Brain +M+++++++++++++++++++++++ 49 

Respiratory System 

Lung 

Alveolar/bronchiolar carcinoma 

Hemangiosarcoma, metastatic, skin 

Squamous cell carcinoma 

Nose 

Trachea 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

1 

1 

1 

50 

50 

Special Senses System 

Eye + 2 

Urinary System 

Kidney 

Renal tubule, adenoma 

Urinary bladder 

Papilloma

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

X 

50 

1 

50 

1 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear

Lymphoma malignant

Mesothelioma malignant

+++++++++++++++++++++++++ 

X X X X X X X XXXX 

X 

X 

50 

24 

1 

2 
 

 

 

100 Primidone, NTP TR 476 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Primidone: 2,500 ppm 

2344455555555555555555555 

Number of Days on Study 5833301223333444556666677 

4067757292378248690112923 

1111121111111111111111111 

Carcass ID Number 6966808996685897998587955 

8424108506371676692440125 

Alimentary System 

Esophagus +++++++++++++++++++++++M+ 

Intestine large, colon A A ++++++++++M++M+++++++++ 

Intestine large, rectum +A ++++++++++A ++M+++++++++ 

Intestine large, cecum A A + + + + + + + + + + A + + M + + + + + + + + + 

Intestine small, duodenum + A + + + + + + + + + + A + + M + + + + + + + + + 

Intestine small, jejunum A A + + + + + + + + + + A + + M + + + + + + + + + 

Intestine small, ileum A A ++++++++++M++M+++++++++ 

Liver +++++++++++++++++++++++++ 

Hepatocellular adenoma 

Mesentery + + + 

Pancreas +++++++++++++++M+++++++++ 

Salivary glands +++++++++++++++++++++++M+ 

Stomach, forestomach +++++++++++++++M+++++++++ 

Stomach, glandular +++++++++++++++M+++++++++ 

Cardiovascular System 

Blood vessel +++++++++++++++++++++++++ 

Heart +++++++++++++++++++++++++ 

Endocrine System 

Adrenal cortex +++++++++++++++++++++++++ 

Adrenal medulla M++++++++++++++++++++++++ 

Pheochromocytoma benign X X 

Bilateral, pheochromocytoma benign X X 

Islets, pancreatic +++++++++++++++M+++++++++ 

Parathyroid gland +++++++++++++++++++++++M+ 

Pituitary gland +++++++++++++++++++++++M+ 

Pars distalis, adenoma X X 

Pars intermedia, adenoma 

Thyroid gland +++++++++++++++++++++++M+ 

C-cell, adenoma 

Follicular cell, adenoma X 

Follicular cell, carcinoma 

General Body System 

Peritoneum + 

Genital System 

Coagulating gland 

Epididymis ++++++++++++M++M+++++++++ 

Preputial gland +++++++++++++++M+++++++++ 

Prostate ++++++++++++M++M+++++++++ 

Seminal vesicle ++++++++++++M++M+++++++++ 

Testes ++++++++++++M++M+++++++++ 

Bilateral, interstitial cell, adenoma XXXXXXX XX XX XXXXX 

Interstitial cell, adenoma X X X 
 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 101 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Primidone: 2,500 ppm 

5555555555555555566666666 

Number of Days on Study 8888888888888899901112347 

1335558999999969930996214 

1111111111111111111111111 Total 

Carcass ID Number 8785776566668977775899575 Tissues/ 

3499120315790873596532887 Tumors 

Alimentary System 

Esophagus +++++++++++++++++++++++++ 49 

Intestine large, colon +++++++++++++++++++++++++ 46 

Intestine large, rectum +++++++++++++++++++++++++ 47 

Intestine large, cecum +++++++++++++++++++++++++ 46 

Intestine small, duodenum +++++++++++++++++++++++++ 47 

Intestine small, jejunum + + + + + A + + + + + + + + + + + + + + + + + + + 45 

Intestine small, ileum +++++++++++++++++++++++++ 46 

Liver +++++++++++++++++++++++++ 50 

Hepatocellular adenoma X 1 

Mesentery 3 

Pancreas +++++++++++++++++++++++++ 49 

Salivary glands +++++++++++++++++++++++++ 49 

Stomach, forestomach +++++++++++++++++++++++++ 49 

Stomach, glandular +++++++++++++++++++++++++ 49 

Cardiovascular System 

Blood vessel +++++++++++++++++++++++++ 50 

Heart +++++++++++++++++++++++++ 50 

Endocrine System 

Adrenal cortex +++++++++++++++++++++++++ 50 

Adrenal medulla +++++++++++++++++++++++++ 49 

Pheochromocytoma benign X X X X X 7 

Bilateral, pheochromocytoma benign X X X 5 

Islets, pancreatic +++++++++++++++++++++++++ 49 

Parathyroid gland +++++++++++++++++++++++++ 49 

Pituitary gland +++++++++++++++++++++++++ 49 

Pars distalis, adenoma X X 4 

Pars intermedia, adenoma X 1 

Thyroid gland +++++++++++++++++++++++++ 49 

C-cell, adenoma X 1 

Follicular cell, adenoma X X 3 

Follicular cell, carcinoma X 1 

General Body System 

Peritoneum 1 

Genital System 

Coagulating gland + 1 

Epididymis +++++++++++++++++++++++++ 48 

Preputial gland +++++++++++++++++++++++++ 49 

Prostate +++++++++++++++++++++++++ 48 

Seminal vesicle +++++++++++++++++++++++++ 48 

Testes +++++++++++++++++++++++++ 48 

Bilateral, interstitial cell, adenoma XX XXX XXX XXX X XXXXXX 34 

Interstitial cell, adenoma X X X X X X 9 
 

102 Primidone, NTP TR 476 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Primidone: 2,500 ppm 

Number of Days on Study 

2344455555555555555555555 

5833301223333444556666677 

4067757292378248690112923 

Carcass ID Number 

1111121111111111111111111 

6966808996685897998587955 

8424108506371676692440125 

Hematopoietic System 

Bone marrow 

Lymph node

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

+++++++++++++++++++++++++ 

+ + + + + + 

+++++++++++++++++++++++M+ 

++++++++++++M++M+++++++++ 

+++++++++++++++M+++++++++ 

+++++++++++++++++++++++M+ 

Integumentary System 

Mammary gland 

Skin 

Trichoepithelioma

Pinna, squamous cell papilloma

++++M+++M++++++M+++++++++ 

+++++++++++++++++++++++++ 

X 

X 

Musculoskeletal System 

Bone +++++++++++++++++++++++++ 

Nervous System 

Brain 

Astrocytoma malignant 

+++++++++++++++++++++++M+ 

Respiratory System 

Lung 

Alveolar/bronchiolar carcinoma 

Nose 

Trachea 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++M+ 

Special Senses System 

None 

Urinary System 

Kidney 

Lipoma

Renal tubule, adenoma

Renal tubule, carcinoma 

Urinary bladder 

+++++++++++++++++++++++++ 

X 

X 

++++++++++++M++M+++++++++ 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear

Mesothelioma malignant

+++++++++++++++++++++++++ 

 X X X X X 

X 
 

 

 

 

 

 

 

 

Primidone, NTP TR 476 103 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Primidone: 2,500 ppm 

Number of Days on Study 

5555555555555555566666666 

8888888888888899901112347 

1335558999999969930996214 

Carcass ID Number 

1111111111111111111111111 

8785776566668977775899575 

3499120315790873596532887 

Total 

Tissues/ 

Tumors 

Hematopoietic System 

Bone marrow 

Lymph node

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

+++++++++++++++++++++++++ 

+ + + + 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++M++++M++++++++++++++M+ 

50 

10 

49 

48 

49 

46 

Integumentary System 

Mammary gland 

Skin 

Trichoepithelioma 

Pinna, squamous cell papilloma 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

47 

50 

1 

1 

Musculoskeletal System 

Bone +++++++++++++++++++++++++ 50 

Nervous System 

Brain 

Astrocytoma malignant

+++++++++++++++++++++++++ 

X X 

49 

2 

Respiratory System 

Lung 

Alveolar/bronchiolar carcinoma

Nose 

Trachea 

+++++++++++++++++++++++++ 

X 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

1 

50 

49 

Special Senses System 

None 

Urinary System 

Kidney 

Lipoma 

Renal tubule, adenoma 

Renal tubule, carcinoma

Urinary bladder 

+++++++++++++++++++++++++ 

X 

+++++++++++++++++++++++++ 

50 

1 

1 

1 

48 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear

Mesothelioma malignant 

+++++++++++++++++++++++++ 

X X X X X X X 

50 

12 

1 
104 Primidone, NTP TR 476 

TABLE A3 

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Feed Study of Primidone 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

Adrenal Medulla: Benign Pheochromocytoma 

Overall ratea 23/50 (46%) 28/50 (56%) 24/50 (48%) 12/49 (24%) 

Adjusted rateb 79.6% 83.3% 87.0% 85.0% 

Terminal ratec 8/13 (62%) 3/7 (43%) 2/4 (50%) 0/0 (0%) 

First incidence (days) 305 562 529 522 

Life table testd P<0.001 P=0.066 P=0.017 P<0.001 

Logistic regression testd P=0.231N P=0.213 P=0.413 P=0.367N 

Cochran-Armitage testd P=0.006N 

Fisher exact testd P=0.212 P=0.500 P=0.021N 

Adrenal Medulla: Benign or Malignant Pheochromocytoma 

Overall rate 23/50 (46%) 28/50 (56%) 24/50 (48%) 12/49 (24%) 

Adjusted rate 79.6% 83.3% 87.0% 85.0% 

Terminal rate 8/13 (62%) 3/7 (43%) 2/4 (50%) 0/0 (0%) 

First incidence (days) 305 562 529 522 

Life table test P<0.001 P=0.066 P=0.017 P<0.001 

Logistic regression test P=0.231N P=0.213 P=0.413 P=0.367N 

Cochran-Armitage test P=0.006N 

Fisher exact test P=0.212 P=0.500 P=0.021N 

Kidney (Renal Tubule): Adenoma (Step Sections) 

Overall rate 2/50 (4%) 1/50 (2%) 4/50 (8%) 5/50 (10%) 

Adjusted rate 13.8% 4.5% 20.9% 54.9% 

Terminal rate 1/13 (8%) 0/7 (0%) 0/4 (0%) 0/0 (0%) 

First incidence (days) 715 673 613 585 

Life table test P<0.001 P=0.650N P=0.196 P<0.001 

Logistic regression test P=0.012 P=0.557N P=0.264 P=0.030 

Cochran-Armitage test P=0.076 

Fisher exact test P=0.500N P=0.500 P=0.218 

Kidney (Renal Tubule): Adenoma (Single and Step Sections) 

Overall rate 4/50 (8%) 2/50 (4%) 4/50 (8%) 6/50 (12%) 

Adjusted rate 23.2% 8.0% 20.9% 56.0% 

Terminal rate 2/13 (15%) 0/7 (0%) 0/4 (0%) 0/0 (0%) 

First incidence (days) 619 654 613 529 

Life table test P<0.001 P=0.492N P=0.304 P<0.001 

Logistic regression test P=0.056 P=0.355N P=0.563 P=0.098 

Cochran-Armitage test P=0.185 

Fisher exact test P=0.339N P=0.643N P=0.370 

Kidney (Renal Tubule): Adenoma or Carcinoma (Step Sections) 

Overall rate 2/50 (4%) 1/50 (2%) 4/50 (8%) 5/50 (10%) 

Adjusted rate 13.8% 4.5% 20.9% 54.9% 

Terminal rate 1/13 (8%) 0/7 (0%) 0/4 (0%) 0/0 (0%) 

First incidence (days) 715 673 613 585 

Life table test P<0.001 P=0.650N P=0.196 P<0.001 

Logistic regression test P=0.010 P=0.557N P=0.264 P=0.030 

Cochran-Armitage test P=0.076 

Fisher exact test P=0.500N P=0.500 P=0.218 
Primidone, NTP TR 476 105 

TABLE A3 

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Feed Study of Primidone 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

Kidney (Renal Tubule): Adenoma or Carcinoma (Single and Step Sections) 

Overall rate 4/50 (8%) 2/50 (4%) 4/50 (8%) 7/50 (14%) 

Adjusted rate 23.2% 8.0% 20.9% 62.3% 

Terminal rate 2/13 (15%) 0/7 (0%) 0/4 (0%) 0/0 (0%) 

First incidence (days) 619 654 613 529 

Life table test P<0.001 P=0.492N P=0.304 P<0.001 

Logistic regression test P=0.025 P=0.355N P=0.563 P=0.050 

Cochran-Armitage test P=0.105 

Fisher exact test P=0.339N P=0.643N P=0.262 

Mammary Gland: Fibroadenoma 

Overall rate 3/50 (6%) 3/50 (6%) 3/50 (6%) 0/50 (0%) 

Adjusted rate 13.8% 25.4% 35.8% 0.0% 

Terminal rate 1/13 (8%) 1/7 (14%) 1/4 (25%) 0/0 (0%) e First incidence (days) 627 697 669 —

Life table test P=0.296 P=0.530 P=0.339 P=0.878N 

Logistic regression test P=0.632N P=0.634 P=0.537 P=0.373N 

Cochran-Armitage test P=0.093N 

Fisher exact test P=0.661N P=0.661N P=0.121N 

Mammary Gland: Fibroadenoma or Adenoma 

Overall rate 3/50 (6%) 4/50 (8%) 3/50 (6%) 0/50 (0%)
 

Adjusted rate 13.8% 27.2% 35.8% 0.0%
 

Terminal rate 1/13 (8%) 1/7 (14%) 1/4 (25%) 0/0 (0%)
 

First incidence (days) 627 583 669 —
 

Life table test P=0.396 P=0.379 P=0.339 P=0.878N
 

Logistic regression test P=0.427N P=0.492 P=0.537 P=0.373N
 

Cochran-Armitage test P=0.076N
 

Fisher exact test P=0.500 P=0.661N P=0.121N
 

Mammary Gland: Fibroadenoma, Adenoma, or Carcinoma 

Overall rate 5/50 (10%) 4/50 (8%) 3/50 (6%) 0/50 (0%)
 

Adjusted rate 25.4% 27.2% 35.8% 0.0%
 

Terminal rate 2/13 (15%) 1/7 (14%) 1/4 (25%) 0/0 (0%)
 

First incidence (days) 627 583 669 —
 

Life table test P=0.573 P=0.601 P=0.495 P=0.878N
 

Logistic regression test P=0.261N P=0.524N P=0.576N P=0.362N
 

Cochran-Armitage test P=0.022N
 

Fisher exact test P=0.500N P=0.357N P=0.028N
 

Pituitary Gland (Pars Distalis): Adenoma 

Overall rate 17/49 (35%) 12/50 (24%) 11/48 (23%) 4/49 (8%) 

Adjusted rate 61.1% 37.7% 73.5% 22.9% 

Terminal rate 5/13 (38%) 0/7 (0%) 2/4 (50%) 0/0 (0%) 

First incidence (days) 281 526 437 436 

Life table test P=0.318 P=0.391N P=0.434 P=0.445 

Logistic regression test P=0.003N P=0.172N P=0.152N P=0.003N 

Cochran-Armitage test P=0.001N 

Fisher exact test P=0.172N P=0.146N P=0.001N 
106 Primidone, NTP TR 476 

TABLE A3 

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Feed Study of Primidone 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

Preputial Gland: Adenoma or Carcinoma 

Overall rate 3/50 (6%) 1/50 (2%) 2/50 (4%) 0/49 (0%)
 

Adjusted rate 12.8% 4.5% 8.0% 0.0%
 

Terminal rate 1/13 (8%) 0/7 (0%) 0/4 (0%) 0/0 (0%)
 

First incidence (days) 526 673 613 —
 

Life table test P=0.468N P=0.376N P=0.653N P=0.484N
 

Logistic regression test P=0.134N P=0.304N P=0.501N P=0.156N
 

Cochran-Armitage test P=0.109N
 

Fisher exact test P=0.309N P=0.500N P=0.125N
 

Skin: Squamous Cell Papilloma, Keratoacanthoma, Trichoepithelioma, Benign Basosquamous Tumor, Basal Cell Adenoma, 

or Basal Cell Carcinoma 

Overall rate 3/50 (6%) 3/50 (6%) 1/50 (2%) 2/50 (4%) 

Adjusted rate 23.1% 13.3% 14.3% 6.1% 

Terminal rate 3/13 (23%) 0/7 (0%) 0/4 (0%) 0/0 (0%) 

First incidence (days) 723 (T) 562 697 544 

Life table test P=0.158 P=0.496 P=0.716 P=0.182 

Logistic regression test P=0.605 P=0.645 P=0.704N P=0.517 

Cochran-Armitage test P=0.330N 

Fisher exact test P=0.661N P=0.309N P=0.500N 

Skin (Subcutaneous Tissue): Fibroma 

Overall rate 3/50 (6%) 3/50 (6%) 3/50 (6%) 0/50 (0%)
 

Adjusted rate 8.8% 8.8% 19.9% 0.0%
 

Terminal rate 0/13 (0%) 0/7 (0%) 0/4 (0%) 0/0 (0%)
 

First incidence (days) 619 610 627 —
 

Life table test P=0.610 P=0.645 P=0.504 P=0.723N
 

Logistic regression test P=0.083N P=0.661 P=0.655 P=0.163N
 

Cochran-Armitage test P=0.093N
 

Fisher exact test P=0.661N P=0.661N P=0.121N
 

Skin (Subcutaneous Tissue): Fibroma or Fibrosarcoma 

Overall rate 3/50 (6%) 3/50 (6%) 5/50 (10%) 0/50 (0%)
 

Adjusted rate 8.8% 8.8% 24.7% 0.0%
 

Terminal rate 0/13 (0%) 0/7 (0%) 0/4 (0%) 0/0 (0%)
 

First incidence (days) 619 610 576 —
 

Life table test P=0.400 P=0.645 P=0.231 P=0.723N
 

Logistic regression test P=0.079N P=0.661 P=0.356 P=0.163N
 

Cochran-Armitage test P=0.143N
 

Fisher exact test P=0.661N P=0.357 P=0.121N
 

Spleen: Fibrosarcoma 

Overall rate 3/50 (6%) 1/50 (2%) 0/50 (0%) 0/49 (0%)
 

Adjusted rate 12.9% 3.7% 0.0% 0.0%
 

Terminal rate 1/13 (8%) 0/7 (0%) 0/4 (0%) 0/0 (0%)
 

First incidence (days) 575 661 — —
 

Life table test P=0.147N P=0.367N P=0.210N P=0.565N
 

Logistic regression test P=0.045N P=0.304N P=0.124N P=0.210N
 

Cochran-Armitage test P=0.042N
 

Fisher exact test P=0.309N P=0.121N P=0.125N
 
Primidone, NTP TR 476 107 

TABLE A3 

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Feed Study of Primidone 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

Testes: Adenoma 

Overall rate 41/50 (82%) 44/50 (88%) 42/50 (84%) 43/48 (90%) 

Adjusted rate 100.0% 100.0% 100.0% 100.0% 

Terminal rate 13/13 (100%) 7/7 (100%) 4/4 (100%) 0/0 (0%) 

First incidence (days) 463 502 551 437 

Life table test P<0.001 P=0.070 P=0.003 P<0.001 

Logistic regression test P=0.009 P=0.387 P=0.559 P=0.009 

Cochran-Armitage test P=0.233 

Fisher exact test P=0.288 P=0.500 P=0.217 

Thyroid Gland (C-cell): Adenoma 

Overall rate 7/50 (14%) 3/50 (6%) 7/49 (14%) 1/49 (2%) 

Adjusted rate 43.3% 30.3% 46.7% 16.7% 

Terminal rate 5/13 (38%) 2/7 (29%) 0/4 (0%) 0/0 (0%) 

First incidence (days) 505 583 529 619 

Life table test P=0.171 P=0.396N P=0.114 P=0.589 

Logistic regression test P=0.365N P=0.173N P=0.510 P=0.546N 

Cochran-Armitage test P=0.066N 

Fisher exact test P=0.159N P=0.597 P=0.032N 

Thyroid Gland (C-cell): Adenoma or Carcinoma 

Overall rate 8/50 (16%) 3/50 (6%) 8/49 (16%) 1/49 (2%) 

Adjusted rate 45.4% 30.3% 48.5% 16.7% 

Terminal rate 5/13 (38%) 2/7 (29%) 0/4 (0%) 0/0 (0%) 

First incidence (days) 505 583 529 619 

Life table test P=0.177 P=0.279N P=0.118 P=0.607 

Logistic regression test P=0.298N P=0.108N P=0.509 P=0.415N 

Cochran-Armitage test P=0.049N 

Fisher exact test P=0.100N P=0.590 P=0.017N 

Thyroid Gland (Follicular Cell): Adenoma 

Overall rate 1/50 (2%) 1/50 (2%) 6/49 (12%) 3/49 (6%) 

Adjusted rate 5.0% 4.5% 21.7% 10.7% 

Terminal rate 0/13 (0%) 0/7 (0%) 0/4 (0%) 0/0 (0%) 

First incidence (days) 697 673 354 529 

Life table test P=0.003 P=0.726 P=0.026 P=0.069 

Logistic regression test P=0.227 P=0.754 P=0.047 P=0.280 

Cochran-Armitage test P=0.136 

Fisher exact test P=0.753N P=0.053 P=0.301 

Thyroid Gland (Follicular Cell): Carcinoma 

Overall rate 1/50 (2%) 3/50 (6%) 1/49 (2%) 1/49 (2%) 

Adjusted rate 7.7% 20.6% 5.6% 5.6% 

Terminal rate 1/13 (8%) 1/7 (14%) 0/4 (0%) 0/0 (0%) 

First incidence (days) 723 (T) 641 667 589 

Life table test P=0.135 P=0.196 P=0.583 P=0.356 

Logistic regression test P=0.461 P=0.281 P=0.662 P=0.583 

Cochran-Armitage test P=0.438N 

Fisher exact test P=0.309 P=0.747 P=0.747 
108 Primidone, NTP TR 476 

TABLE A3 

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Feed Study of Primidone 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

Thyroid Gland (Follicular Cell): Adenoma or Carcinoma 

Overall rate 2/50 (4%) 4/50 (8%) 7/49 (14%) 4/49 (8%) 

Adjusted rate 12.3% 24.2% 26.1% 15.7% 

Terminal rate 1/13 (8%) 1/7 (14%) 0/4 (0%) 0/0 (0%) 

First incidence (days) 697 641 354 529 

Life table test P<0.001 P=0.225 P=0.024 P=0.023 

Logistic regression test P=0.266 P=0.309 P=0.076 P=0.207 

Cochran-Armitage test P=0.262 

Fisher exact test P=0.339 P=0.075 P=0.329 

All Organs: Mononuclear Cell Leukemia 

Overall rate 35/50 (70%) 30/50 (60%) 24/50 (48%) 12/50 (24%) 

Adjusted rate 84.4% 79.3% 100.0% 100.0% 

Terminal rate 7/13 (54%) 2/7 (29%) 4/4 (100%) 0/0 (0%) 

First incidence (days) 505 502 354 380 

Life table test P=0.043 P=0.529 P=0.414 P=0.039 

Logistic regression test P<0.001N P=0.059N P=0.023N P<0.001N 

Cochran-Armitage test P<0.001N 

Fisher exact test P=0.201N P=0.021N P<0.001N 

All Organs: Malignant Mesothelioma 

Overall rate 2/50 (4%) 3/50 (6%) 2/50 (4%) 1/50 (2%) 

Adjusted rate 15.4% 18.4% 13.9% 2.5% 

Terminal rate 2/13 (15%) 1/7 (14%) 0/4 (0%) 0/0 (0%) 

First incidence (days) 723 (T) 544 627 533 

Life table test P=0.261 P=0.341 P=0.389 P=0.486 

Logistic regression test P=0.472N P=0.499 P=0.555 P=0.739 

Cochran-Armitage test P=0.307N 

Fisher exact test P=0.500 P=0.691N P=0.500N 

All Organs: Benign Neoplasms 

Overall rate 49/50 (98%) 49/50 (98%) 49/50 (98%) 44/50 (88%) 

Adjusted rate 100.0% 100.0% 100.0% 100.0% 

Terminal rate 13/13 (100%) 7/7 (100%) 4/4 (100%) 0/0 (0%) 

First incidence (days) 281 502 354 436 

Life table test P<0.001 P=0.158 P=0.008 P<0.001 

Logistic regression test P=0.043N P=0.666N P=0.761N P=0.127N 

Cochran-Armitage test P=0.009N 

Fisher exact test P=0.753N P=0.753N P=0.056N 

All Organs: Malignant Neoplasms 

Overall rate 41/50 (82%) 35/50 (70%) 29/50 (58%) 15/50 (30%) 

Adjusted rate 90.6% 88.8% 100.0% 100.0% 

Terminal rate 9/13 (69%) 4/7 (57%) 4/4 (100%) 0/0 (0%) 

First incidence (days) 305 502 354 380 

Life table test P=0.026 P=0.518 P=0.392 P=0.032 

Logistic regression test P<0.001N P=0.061N P=0.008N P<0.001N 

Cochran-Armitage test P<0.001N 

Fisher exact test P=0.121N P=0.008N P<0.001N 
109 

c 

Primidone, NTP TR 476 

TABLE A3 

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Feed Study of Primidone 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

All Organs: Benign or Malignant Neoplasms 

Overall rate 50/50 (100%) 49/50 (98%) 50/50 (100%) 47/50 (94%) 

Adjusted rate 100.0% 100.0% 100.0% 100.0% 

Terminal rate 13/13 (100%) 7/7 (100%) 4/4 (100%) 0/0 (0%) 

First incidence (days) 281 502 354 380 

Life table test P<0.001 P=0.189 P=0.008 P<0.001 

Logistic regression test P=0.091N P=0.248N —f P=0.172N 

Cochran-Armitage test P=0.046N 

Fisher exact test P=0.500N P=1.000N P=0.121N 

(T)Terminal sacrifice 

a Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for adrenal gland, 

kidney, pituitary gland, preputial gland, spleen, testes, and thyroid gland; for other tissues, denominator is number of animals necropsied. b Kaplan-Meier estimated neoplasm incidence after adjustment for intercurrent mortality 

Observed incidence at terminal kill 

d	 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 f Value of statistic cannot be computed. 
110 Primidone, NTP TR 476 

TABLE A4 

Historical Incidence of Thyroid Gland Follicular Cell Neoplasms in Untreated Male F344/N Ratsa 

Incidence in Controls 

Study Adenoma Carcinoma Adenoma or Carcinoma 

Historical Incidence at Battelle Columbus Laboratories 

4,4'-Thiobis(6-t-butyl-m-cresol) 0/50 1/50 1/50 

5,5-Diphenylhydantoin 0/49 1/49 1/49 

Ethylene Thiourea 0/49 1/49 1/49 

Polybrominated Biphenyls (Firemaster FF-1®) 1/49 0/49 1/49 

Manganese (II) Sulfate Monohydrate 1/52 0/52 1/52 

Triamterene 1/50 0/50 1/50 

Tricresyl Phosphate 0/51 0/51 0/51 

Overall Historical Incidence 

Total 11/1,295 (0.9%) 11/1,295 (0.9%) 22/1,295 (1.7%) 

Standard deviation 1.2% 1.2% 1.6% 

Range 0%-4% 0%-4% 0%-6% 

a Data as of 12 May 1995 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 111 

TABLE A5 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Study of Primidonea 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

Disposition Summary 

Animals initially in study 50 50 50 50 

Early deaths 

Moribund 33 33 39 36 

Natural deaths 4 10 7 14 

Survivors 

Terminal sacrifice 13 7 4 

Animals examined microscopically 50 50 50 50 

Alimentary System 

Esophagus (50) (50) (50) (49) 

Inflammation, chronic active 1 (2%) 

Ulcer 1 (2%) 

Intestine large, colon (50) (50) (49) (46) 

Mineralization 1 (2%) 2 (4%) 

Parasite metazoan 2 (4%) 1 (2%) 

Muscularis, mineralization 1 (2%) 

Intestine large, rectum (50) (50) (49) (47) 

Mineralization 1 (2%) 

Parasite metazoan 3 (6%) 3 (6%) 1 (2%) 2 (4%) 

Intestine small, duodenum (50) (50) (50) (47) 

Erosion 2 (4%) 

Inflammation, suppurative 2 (4%) 

Mineralization 1 (2%) 

Intestine small, jejunum (50) (50) (49) (45) 

Ulcer 1 (2%) 

Liver (50) (50) (50) (50) 

Basophilic focus 14 (28%) 9 (18%) 8 (16%) 6 (12%) 

Clear cell focus 2 (4%) 1 (2%) 

Eosinophilic focus 1 (2%) 6 (12%) 5 (10%) 2 (4%) 

Hepatodiaphragmatic nodule 3 (6%) 2 (4%) 5 (10%) 3 (6%) 

Inflammation, chronic 18 (36%) 19 (38%) 22 (44%) 24 (48%) 

Mixed cell focus 2 (4%) 1 (2%) 

Pigmentation, hemosiderin 19 (38%) 18 (36%) 15 (30%) 19 (38%) 

Bile duct, hyperplasia 47 (94%) 50 (100%) 50 (100%) 44 (88%) 

Hepatocyte, degeneration, cystic 16 (32%) 27 (54%) 27 (54%) 18 (36%) 

Hepatocyte, necrosis 10 (20%) 12 (24%) 6 (12%) 10 (20%) 

Hepatocyte, vacuolization cytoplasmic 26 (52%) 28 (56%) 33 (66%) 43 (86%) 

Hepatocyte, centrilobular, degeneration 1 (2%) 

Hepatocyte, centrilobular, hypertrophy 14 (28%) 33 (66%) 40 (80%) 

Portal vein, thrombosis 1 (2%) 

Mesentery (11) (6) (9) (3) 

Angiectasis 1 (33%) 

Hemorrhage 1 (11%) 

Inflammation, chronic active 1 (11%) 1 (33%) 

Artery, inflammation, chronic active 1 (33%) 

Artery, mineralization 1 (33%) 

Artery, necrosis, fibrinoid 1 (33%) 

Fat, inflammation, chronic active 1 (11%) 

Fat, mineralization 1 (11%) 

Fat, necrosis 6 (55%) 3 (50%) 4 (44%) 

Fat, pigmentation, hematoidin 1 (11%) 

Fat, pigmentation, hemosiderin 1 (9%) 1 (11%) 

a Number of animals examined microscopically at the site and the number of animals with lesion 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

112 Primidone, NTP TR 476 

TABLE A5 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Study of Primidone 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

Alimentary System (continued) 

Pancreas (50) (50) (50) (49) 

Inflammation, chronic active 1 (2%) 

Acinus, atrophy 25 (50%) 14 (28%) 12 (24%) 8 (16%) 

Acinus, hypertrophy 1 (2%) 

Artery, inflammation, chronic active 1 (2%) 

Stomach, forestomach (50) (50) (50) (49) 

Hyperplasia 1 (2%) 

Inflammation, chronic active 1 (2%) 

Mineralization 3 (6%) 3 (6%) 12 (24%) 

Perforation 2 (4%) 1 (2%) 

Epithelium, hyperplasia 4 (8%) 11 (22%) 19 (38%) 10 (20%) 

Epithelium, inflammation, chronic active 2 (4%) 10 (20%) 14 (28%) 9 (18%) 

Epithelium, ulcer 2 (4%) 7 (14%) 13 (26%) 8 (16%) 

Stomach, glandular (50) (50) (50) (49) 

Mineralization 6 (12%) 15 (30%) 36 (73%) 

Epithelium, erosion 2 (4%) 1 (2%) 

Epithelium, inflammation, chronic active 2 (4%) 1 (2%) 1 (2%) 

Epithelium, necrosis 1 (2%) 2 (4%) 1 (2%) 

Epithelium, ulcer 4 (8%) 1 (2%) 

Cardiovascular System 

Blood vessel (50) (50) (50) (50) 

Inflammation, chronic active 1 (2%) 1 (2%) 

Necrosis, fibrinoid 1 (2%) 

Aorta, mineralization 5 (10%) 9 (18%) 31 (62%) 

Heart (50) (50) (50) (50) 

Cardiomyopathy, chronic 42 (84%) 45 (90%) 43 (86%) 48 (96%) 

Mineralization 4 (8%) 8 (16%) 15 (30%) 

Pigmentation, hemosiderin 1 (2%) 

Atrium, thrombosis 2 (4%) 3 (6%) 2 (4%) 

Endocrine System 

Adrenal cortex (50) (50) (50) (50) 

Accessory adrenal cortical nodule 2 (4%) 1 (2%) 

Atrophy 1 (2%) 

Degeneration, fatty 26 (52%) 18 (36%) 19 (38%) 12 (24%) 

Hyperplasia 17 (34%) 4 (8%) 9 (18%) 6 (12%) 

Hypertrophy 3 (6%) 2 (4%) 

Inflammation, chronic active 1 (2%) 

Mineralization 1 (2%) 

Necrosis 1 (2%) 

Thrombosis 1 (2%) 

Adrenal medulla (50) (50) (50) (49) 

Hemorrhage 1 (2%) 

Hyperplasia 19 (38%) 22 (44%) 30 (60%) 29 (59%) 

Necrosis 1 (2%) 

Parathyroid gland (45) (41) (46) (49) 

Hyperplasia 2 (4%) 16 (39%) 26 (57%) 40 (82%) 

Pituitary gland (49) (50) (48) (49) 

Craniopharyngeal duct, cyst 1 (2%) 

Pars distalis, cyst 1 (2%) 7 (14%) 2 (4%) 4 (8%) 

Pars distalis, hyperplasia 17 (35%) 18 (36%) 16 (33%) 7 (14%) 

Pars intermedia, cyst 1 (2%) 2 (4%) 1 (2%) 1 (2%) 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 113 

TABLE A5 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Study of Primidone 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

Endocrine System (continued) 

Thyroid gland (50) (50) (49) (49) 

C-cell, hyperplasia 11 (22%) 11 (22%) 5 (10%) 3 (6%) 

Follicle, cyst 1 (2%) 3 (6%) 1 (2%) 6 (12%) 

Follicle, hyperplasia, cystic 1 (2%) 

Follicular cell, hyperplasia 1 (2%) 1 (2%) 

Follicular cell, hyperplasia, cystic 1 (2%) 1 (2%) 1 (2%) 

General Body System 

None 

Genital System 

Coagulating gland (2) (1) (1) 

Inflammation, chronic active 1 (50%) 1 (100%) 1 (100%) 

Epididymis (50) (50) (50) (48) 

Granuloma sperm 3 (6%) 3 (6%) 1 (2%) 

Mineralization 4 (8%) 

Necrosis 1 (2%) 

Preputial gland (50) (50) (50) (49) 

Cyst 2 (4%) 1 (2%) 3 (6%) 

Hyperplasia 5 (10%) 7 (14%) 3 (6%) 4 (8%) 

Inflammation, chronic active 47 (94%) 47 (94%) 40 (80%) 45 (92%) 

Prostate (50) (50) (50) (48) 

Cyst 1 (2%) 

Inflammation, chronic active 37 (74%) 34 (68%) 34 (68%) 30 (63%) 

Mineralization 2 (4%) 

Seminal vesicle (50) (50) (50) (48) 

Atrophy 2 (4%) 

Mineralization 1 (2%) 2 (4%) 8 (17%) 

Testes (50) (50) (50) (48) 

Necrosis 1 (2%) 

Germinal epithelium, degeneration 2 (4%) 2 (4%) 2 (4%) 3 (6%) 

Interstitial cell, hyperplasia 11 (22%) 11 (22%) 8 (16%) 11 (23%) 

Hematopoietic System 

Bone marrow (50) (50) (50) (50) 

Atrophy 1 (2%) 1 (2%) 1 (2%) 

Hyperplasia 38 (76%) 35 (70%) 28 (56%) 12 (24%) 

Inflammation, suppurative 1 (2%) 

Myelofibrosis 2 (4%) 1 (2%) 1 (2%) 

Lymph node (24) (23) (16) (10) 

Lumbar, hyperplasia, lymphoid 1 (6%) 

Mediastinal, hyperplasia, plasma cell 1 (6%) 

Renal, ectasia 1 (6%) 3 (30%) 

Renal, erythrophagocytosis 1 (10%) 

Renal, pigmentation, hemosiderin 2 (20%) 

Lymph node, mandibular (50) (50) (48) (49) 

Ectasia 3 (6%) 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

114 Primidone, NTP TR 476 

TABLE A5 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Study of Primidone 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

Hematopoietic System (continued) 

Lymph node, mesenteric (50) (50) (50) (48) 

Ectasia 2 (4%) 2 (4%) 4 (8%) 2 (4%) 

Fibrosis 1 (2%) 

Spleen (50) (50) (50) (49) 

Accessory spleen 1 (2%) 

Congestion 1 (2%) 1 (2%) 

Fibrosis 9 (18%) 19 (38%) 9 (18%) 12 (24%) 

Hematopoietic cell proliferation 2 (4%) 6 (12%) 1 (2%) 

Hyperplasia, lymphoid 1 (2%) 

Metaplasia, lipocyte 1 (2%) 

Necrosis 1 (2%) 1 (2%) 

Thymus (49) (47) (44) (46) 

Cyst 2 (4%) 1 (2%) 

Integumentary System 

Mammary gland (48) (49) (50) (47) 

Cyst 1 (2%) 1 (2%) 2 (4%) 

Hyperplasia, cystic 28 (58%) 25 (51%) 16 (32%) 6 (13%) 

Mineralization 1 (2%) 

Skin (50) (50) (50) (50) 

Hyperkeratosis 1 (2%) 

Hyperplasia, basal cell 1 (2%) 

Inflammation, chronic active 2 (4%) 1 (2%) 1 (2%) 

Inflammation, suppurative 1 (2%) 

Epidermis, hyperplasia 1 (2%) 1 (2%) 

Pinna, ulcer 1 (2%) 

Sebaceous gland, hyperplasia 1 (2%) 

Musculoskeletal System 

Bone (50) (50) (50) (50) 

Fibrous osteodystrophy 10 (20%) 26 (52%) 37 (74%) 

Necrosis 1 (2%) 

Osteopetrosis 2 (4%) 1 (2%) 

Nervous System 

Brain (50) (50) (49) (49) 

Hemorrhage 4 (8%) 2 (4%) 4 (8%) 1 (2%) 

Inflammation, chronic active 1 (2%) 

Spinal cord (1) (1) 

Demyelination 1 (100%) 

Respiratory System 

Lung (50) (50) (50) (50) 

Inflammation, chronic active 2 (4%) 3 (6%) 3 (6%) 

Metaplasia, osseous 1 (2%) 1 (2%) 2 (4%) 

Alveolar epithelium, hyperplasia 6 (12%) 1 (2%) 2 (4%) 4 (8%) 

Alveolus, hemorrhage 1 (2%) 

Alveolus, infiltration cellular, histiocyte 18 (36%) 18 (36%) 19 (38%) 15 (30%) 

Alveolus, inflammation, suppurative 1 (2%) 4 (8%) 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 115 

TABLE A5 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Study of Primidone 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

Respiratory System (continued) 

Lung (continued) (50) (50) (50) (50) 

Artery, thrombosis 1 (2%) 

Artery, vein, mineralization 46 (92%) 42 (84%) 43 (86%) 45 (90%)

 Interstitium, inflammation, chronic active 2 (4%) 

Interstitium, mineralization 3 (6%) 7 (14%) 25 (50%) 

Interstitium, alveolus, inflammation,

 suppurative 5 (10%) 

Perivascular, infiltration cellular,

 lymphocyte 1 (2%) 1 (2%) 1 (2%) 1 (2%) 

Nose (50) (50) (50) (50) 

Inflammation, chronic active 8 (16%) 12 (24%) 10 (20%) 9 (18%) 

Inflammation, suppurative 6 (12%) 7 (14%) 3 (6%) 9 (18%) 

Arteriole, venule, thrombosis 1 (2%) 2 (4%) 1 (2%) 

Nasolacrimal duct, inflammation, suppurative 2 (4%) 2 (4%) 

Respiratory epithelium, metaplasia, squamous 1 (2%) 

Special Senses System 

Eye (1) (2) 

Anterior chamber, inflammation, suppurative 1 (50%) 

Lens, cataract 1 (50%) 

Retina, degeneration 2 (100%) 

Urinary System 

Kidney (50) (50) (50) (50) 

Cyst 2 (4%) 2 (4%) 12 (24%) 9 (18%) 

Infarct 1 (2%) 

Inflammation, suppurative 1 (2%) 

Nephropathy, chronic 49 (98%) 48 (96%) 50 (100%) 50 (100%) 

Papilla, hyperplasia, atypical 1 (2%) 

Renal tubule, hyperplasia 1 (2%) 2 (4%) 4 (8%) 10 (20%) 

Urinary bladder (50) (50) (50) (48) 

Hemorrhage 1 (2%) 1 (2%) 

Inflammation, suppurative 1 (2%) 
116 Primidone, NTP TR 476
 
117 

APPENDIX B 

SUMMARY OF LESIONS IN FEMALE RATS 

IN THE 2-YEAR FEED STUDY 

OF PRIMIDONE 

TABLE B1 

TABLE B2 

TABLE B3 

TABLE B4 

TABLE B5 

Summary of the Incidence of Neoplasms in Female Rats 

in the 2-Year Feed Study of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

Individual Animal Tumor Pathology of Female Rats 

in the 2-Year Feed Study of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

Statistical Analysis of Primary Neoplasms in Female Rats 

in the 2-Year Feed Study of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

Historical Incidence of Mononuclear Cell Leukemia 

in Untreated Female F344/N Rats ..................................... 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats 

in the 2-Year Feed Study of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

119 

122 

138 

142 

143 
118 Primidone, NTP TR 476
 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 119 

TABLE B1 

Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Feed Study of Primidonea 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

Disposition Summary 

Animals initially in study 50 50 50 50 

Early deaths 

Moribund 21 18 14 19 

Natural deaths 5 5 5 3 

Survivors 

Died last week of study 1 

Terminal sacrifice 24 27 31 27 

Animals examined microscopically 50 50 50 50 

Alimentary System 

Intestine large, colon (50) (50) (50) (50) 

Intestine large, cecum (49) (50) (50) (49) 

Fibrosarcoma, metastatic, spleen 1 (2%) 

Intestine small, duodenum (49) (50) (50) (50) 

Intestine small, jejunum (49) (50) (50) (49) 

Fibrosarcoma, metastatic, spleen 1 (2%) 

Intestine small, ileum (50) (50) (50) (49) 

Fibrosarcoma, metastatic, spleen 1 (2%) 

Liver (50) (50) (50) (50) 

Fibrosarcoma, metastatic, spleen 1 (2%) 

Hepatocellular adenoma 1 (2%) 1 (2%) 

Mesentery (9) (10) (7) (7) 

Fibrosarcoma, metastatic, spleen 1 (14%) 

Oral mucosa (2) 

Gingival, squamous cell carcinoma 1 (50%) 

Pharyngeal, squamous cell carcinoma 1 (50%) 

Pancreas (49) (50) (50) (50) 

Fibrosarcoma, metastatic, spleen 1 (2%) 

Salivary glands (49) (50) (50) (50) 

Stomach, forestomach (49) (50) (50) (50) 

Stomach, glandular (49) (50) (50) (50) 

Carcinoid tumor benign 1 (2%) 

Carcinoid tumor malignant 1 (2%) 

Fibrosarcoma, metastatic, spleen 1 (2%) 

Cardiovascular System 

Blood vessel (49) (50) (50) (50) 

Heart (49) (50) (50) (50) 

Schwannoma malignant 1 (2%) 

Endocrine System 

Adrenal cortex (50) (50) (50) (50) 

Adenoma 1 (2%) 2 (4%) 1 (2%) 

Thymoma malignant, metastatic, thymus 1 (2%) 

Adrenal medulla (50) (50) (49) (50) 

Pheochromocytoma benign 2 (4%) 2 (4%) 3 (6%) 

Islets, pancreatic (50) (50) (50) (50) 

Adenoma 1 (2%) 

Parathyroid gland (38) (44) (42) (39) 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

120 Primidone, NTP TR 476 

TABLE B1 

Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Feed Study of Primidone 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

Endocrine System (continued) 

Pituitary gland (49) (50) (49) (50) 

Pars distalis, adenoma 16 (33%) 20 (40%) 13 (27%) 18 (36%) 

Pars distalis, adenoma, multiple 1 (2%) 1 (2%) 2 (4%) 

Pars distalis, carcinoma 1 (2%) 

Thyroid gland (49) (50) (50) (50) 

Bilateral, C-cell, adenoma 1 (2%) 

C-cell, adenoma 5 (10%) 2 (4%) 6 (12%) 4 (8%) 

Follicular cell, carcinoma 1 (2%) 

General Body System 

None 

Genital System 

Clitoral gland (49) (49) (50) (50) 

Adenoma 4 (8%) 1 (2%) 4 (8%) 1 (2%) 

Carcinoma 1 (2%) 1 (2%) 2 (4%) 

Ovary (50) (50) (50) (49) 

Granulosa-theca tumor malignant 1 (2%) 

Uterus (50) (50) (50) (49) 

Leiomyosarcoma 1 (2%) 

Polyp stromal 7 (14%) 7 (14%) 6 (12%) 7 (14%) 

Polyp stromal, multiple 1 (2%) 

Sarcoma stromal 3 (6%) 3 (6%) 

Vagina (1) (1) (2) 

Polyp 1 (100%) 2 (100%) 

Hematopoietic System 

Bone marrow (50) (50) (50) (50) 

Lymph node (11) (7) (8) (12) 

Thoracic, carcinoma, metastatic,

 mammary gland 1 (13%) 

Lymph node, mandibular (49) (50) (50) (50) 

Lymph node, mesenteric (50) (50) (50) (49) 

Spleen (49) (50) (50) (50) 

Fibrosarcoma 1 (2%) 

Thymus (49) (45) (47) (46) 

Thymoma benign 1 (2%) 

Thymoma malignant 1 (2%) 

Integumentary System 

Mammary gland (50) (50) (50) (50) 

Adenoma 2 (4%) 

Carcinoma 1 (2%) 2 (4%) 1 (2%) 

Fibroadenoma 13 (26%) 21 (42%) 14 (28%) 11 (22%) 

Fibroadenoma, multiple 9 (18%) 4 (8%) 2 (4%) 1 (2%) 

Skin (50) (50) (50) (50) 

Basal cell carcinoma 1 (2%) 

Pinna, squamous cell papilloma 1 (2%) 
 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 121 

TABLE B1 

Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Feed Study of Primidone 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

Integumentary System (continued) 

Skin 

 Subcutaneous tissue, fibroma

Subcutaneous tissue, fibrosarcoma

Subcutaneous tissue, liposarcoma

Subcutaneous tissue, sarcoma

(50) 

 1 (2%)

 1 (2%)

 1 (2%) 

(50) 

 1 (2%)

 1 (2%)

 1 (2%) 

(50) 

 1 (2%)

(50)

 1 (2%) 

 2 (4%) 

Musculoskeletal System 

Skeletal muscle (1) (2) 

Nervous System 

Brain 

Astrocytoma malignant

Carcinoma, metastatic, pituitary gland

Spinal cord

(50) 

 1 (2%)

(50) 

 (1)

(50) 

 1 (2%) 

(50) 

 1 (2%) 

 (2) 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma

Carcinoma, metastatic, mammary gland

Thymoma malignant, metastatic, thymus

Trachea 

(49) 

 1 (2%) 

(49) 

(50) 

(50) 

(50) 

 1 (2%)

 1 (2%) 

(50) 

(50) 

 2 (4%) 

(50) 

Special Senses System 

Zymbal’s gland

Carcinoma

 (1)

 1 (100%)

 (1) 

 1 (100%) 

Urinary System 

Kidney 

Urinary bladder 

(50) 

(50) 

(50) 

(50) 

(50) 

(50) 

(50) 

(49) 

Systemic Lesions 

Multiple organsb 

Leukemia mononuclear

(50) 

 13 (26%)

(50) 

 13 (26%)

(50) 

 13 (26%)

(50) 

 22 (44%) 

Neoplasm Summary 

Total animals with primary neoplasmsc 

Total primary neoplasms 

Total animals with benign neoplasms 

Total benign neoplasms 

Total animals with malignant neoplasms 

Total malignant neoplasms 

Total animals with metastatic neoplasms 

Total metastatic neoplasms 

47 

86 

39 

63 

21 

23 

1 

2 

44 

81 

35 

60 

20 

21 

41 

72 

35 

53 

17 

19 

1 

2 

46 

88 

33 

54 

28 

34 

2 

8 

a Number of animals examined microscopically at the site and the number of animals with neoplasm b Number of animals with any tissue examined microscopically 

Primary neoplasms: all neoplasms except metastatic neoplasms c 
 

 

 

 

 

122 Primidone, NTP TR 476 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Primidone: 0 ppm 

3455556666666666666667777 

Number of Days on Study 6134662233444466667890000 

7561166648011604885272222 

2222222222222222222222222 

Carcass ID Number 3031310344414301120102334 

7450428174845016953121681 

Alimentary System 

Esophagus + + M ++++++++++++++++++++++ 

Intestine large, colon +++++++++++++++++++++++++ 

Intestine large, rectum +++++A +++++++++++++++++++ 

Intestine large, cecum + + + + + A + + + + + + + + + + + + + + + + + + + 

Intestine small, duodenum + + + + + A + + + + + + + + + + + + + + + + + + + 

Intestine small, jejunum + + + + + A + + + + + + + + + + + + + + + + + + + 

Intestine small, ileum +++++++++++++++++++++++++ 

Liver +++++++++++++++++++++++++ 

Mesentery + ++ + + 

Pancreas ++M++++++++++++++++++++++ 

Salivary glands ++M++++++++++++++++++++++ 

Stomach, forestomach ++M++++++++++++++++++++++ 

Stomach, glandular + + M ++++++++++++++++++++++ 

Carcinoid tumor benign 

Carcinoid tumor malignant X 

Cardiovascular System 

Blood vessel 

Heart 

+ + M ++++++++++++++++++++++ 

++M++++++++++++++++++++++ 

Endocrine System 

Adrenal cortex +++++++++++++++++++++++++ 

Adenoma 

Thymoma malignant, metastatic, thymus X 

Adrenal medulla +++++++++++++++++++++++++ 

Pheochromocytoma benign X 

Islets, pancreatic +++++++++++++++++++++++++ 

Parathyroid gland ++MM+++MM+M+++++++M++++++ 

Pituitary gland +++++++++++++++++++++++++ 

Pars distalis, adenoma X X XXX X X X 

Thyroid gland ++M++++++++++++++++++++++ 

C-cell, adenoma X 

Follicular cell, carcinoma 

General Body System 

None 

Genital System 

Clitoral gland ++M++++++++++++++++++++++ 

Adenoma X 

Carcinoma X 

Ovary +++++++++++++++++++++++++ 

Uterus +++++++++++++++++++++++++ 

Polyp stromal X X X 

Polyp stromal, multiple X 

Vagina + 

Polyp X 

+: Tissue examined microscopically M: Missing tissue X: Lesion present 

A: Autolysis precludes examination I: Insufficient tissue Blank: Not examined 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 123 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Primidone: 0 ppm 

7777777777777777777777777 

Number of Days on Study 0222222222222222222222222 

3222333333333334444444444 

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Total 

Carcass ID Number 1 1 2 2 0 0 0 0 2 2 2 3 4 4 4 1 1 2 2 2 3 3 4 4 5 Tissues/ 

8 3 4 8 5 6 7 9 2 3 7 9 3 6 9 5 7 0 6 9 2 3 0 2 0 Tumors 

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Liver + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Mesentery + + + + 9 

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Carcinoid tumor benign X 1 

Carcinoid tumor malignant 1 

Cardiovascular System 

Blood vessel + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Adenoma X 1 

Thymoma malignant, metastatic, thymus 1 

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Pheochromocytoma benign X 2 

Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Parathyroid gland + + + + + + + M + M + M + M + + + M M + + + + + + 38 

Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + M + 49 

Pars distalis, adenoma XX X X XXX X 16 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 49 

C-cell, adenoma X X X X 5 

Follicular cell, carcinoma X 1 

General Body System 

None 

Genital System 

Clitoral gland + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Adenoma X X X 4 

Carcinoma 1 

Ovary + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Uterus + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Polyp stromal X X X X 7 

Polyp stromal, multiple 1 

Vagina 1 

Polyp 1 
 

 

 

 

124 Primidone, NTP TR 476 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Primidone: 0 ppm 

Number of Days on Study 

3455556666666666666667777 

6134662233444466667890000 

7561166648011604885272222 

Carcass ID Number 

2222222222222222222222222 

3031310344414301120102334 

7450428174845016953121681 

Hematopoietic System 

Bone marrow 

Lymph node

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Thymoma malignant

+++++++++++++++++++++++++ 

+ + + + + + 

+ + M ++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

++M++++++++++++++++++++++ 

++M++++++++++++++++++++++ 

X 

Integumentary System 

Mammary gland 

Adenoma

Carcinoma

Fibroadenoma

Fibroadenoma, multiple

Skin 

Basal cell carcinoma

Subcutaneous tissue, fibroma

Subcutaneous tissue, fibrosarcoma

Subcutaneous tissue, sarcoma 

+++++++++++++++++++++++++ 

X 

X 

X X X X X 

X X X X X 

+++++++++++++++++++++++++ 

X 

X 

X 

X 

Musculoskeletal System 

Bone +++++++++++++++++++++++++ 

Nervous System 

Brain 

Astrocytoma malignant

+++++++++++++++++++++++++ 

X 

Respiratory System 

Lung 

Thymoma malignant, metastatic, thymus

Nose 

Trachea 

++M++++++++++++++++++++++ 

X 

+++++++++++++++++++++++++ 

++M++++++++++++++++++++++ 

Special Senses System 

Eye

Zymbal’s gland

Carcinoma

 + 

+ 

X 

Urinary System 

Kidney 

Urinary bladder 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear

+++++++++++++++++++++++++ 

X X X X X X X 
 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 125 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Primidone: 0 ppm 

7777777777777777777777777 

Number of Days on Study 0222222222222222222222222 

3222333333333334444444444 

2222222222222222222222222 Total 

Carcass ID Number 1122000022234441122233445 

8348567923793695706923020 

Tissues/ 

Tumors 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Thymoma malignant 

+++++++++++++++++++++++++ 

+ + + + + 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

11 

49 

50 

49 

49 

1 

Integumentary System 

Mammary gland 

Adenoma

Carcinoma 

Fibroadenoma 

Fibroadenoma, multiple

Skin 

Basal cell carcinoma 

Subcutaneous tissue, fibroma 

Subcutaneous tissue, fibrosarcoma 

Subcutaneous tissue, sarcoma 

+++++++++++++++++++++++++ 

X 

X X X X X X X X 

X X X X 

+++++++++++++++++++++++++ 

50 

2 

1 

13 

9 

50 

1 

1 

1 

1 

Musculoskeletal System 

Bone +++++++++++++++++++++++++ 50 

Nervous System 

Brain 

Astrocytoma malignant 

+++++++++++++++++++++++++ 50 

1 

Respiratory System 

Lung 

Thymoma malignant, metastatic, thymus 

Nose 

Trachea 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

49 

1 

50 

49 

Special Senses System 

Eye 

Zymbal’s gland 

Carcinoma 

1 

1 

1 

Urinary System 

Kidney 

Urinary bladder 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

50 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear

+++++++++++++++++++++++++ 

 X X X X X X 

50 

13 
 

 

 

126 Primidone, NTP TR 476 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Primidone: 600 ppm 

3444444555556666666667777 

Number of Days on Study 6111256167780111477780022 

7059666178845288555522222 

2222222232222222222222222 

Carcass ID Number 9665687805588968978976955 

9427945801395517890048689 

Alimentary System 

Esophagus +++++++++++++++++++++++++ 

Intestine large, colon +++++++++++++++++++++++++ 

Intestine large, rectum +++++++++++++++++++++++++ 

Intestine large, cecum +++++++++++++++++++++++++ 

Intestine small, duodenum +++++++++++++++++++++++++ 

Intestine small, jejunum +++++++++++++++++++++++++ 

Intestine small, ileum +++++++++++++++++++++++++ 

Liver +++++++++++++++++++++++++ 

Hepatocellular adenoma X 

Mesentery ++ +++ + + + 

Pancreas +++++++++++++++++++++++++ 

Salivary glands +++++++++++++++++++++++++ 

Stomach, forestomach +++++++++++++++++++++++++ 

Stomach, glandular +++++++++++++++++++++++++ 

Cardiovascular System 

Blood vessel 

Heart 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

Endocrine System 

Adrenal cortex +++++++++++++++++++++++++ 

Adenoma 

Adrenal medulla +++++++++++++++++++++++++ 

Islets, pancreatic +++++++++++++++++++++++++ 

Parathyroid gland ++++M+++++M++++++++++++++ 

Pituitary gland +++++++++++++++++++++++++ 

Pars distalis, adenoma X X X XXXX X X 

Pars distalis, adenoma, multiple X 

Thyroid gland +++++++++++++++++++++++++ 

C-cell, adenoma X X 

General Body System 

None 

Genital System 

Clitoral gland ++++++++++++++++++++++M++ 

Adenoma 

Ovary +++++++++++++++++++++++++ 

Granulosa-theca tumor malignant 

Uterus +++++++++++++++++++++++++ 

Polyp stromal X X X X 

Sarcoma stromal X X 

Hematopoietic System 

Bone marrow +++++++++++++++++++++++++ 

Lymph node + + + + + + + 

Lymph node, mandibular +++++++++++++++++++++++++ 

Lymph node, mesenteric +++++++++++++++++++++++++ 

Spleen +++++++++++++++++++++++++ 

Thymus +++++++++++++++++++++++++ 
 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 127 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Primidone: 600 ppm 

Number of Days on Study 

7777777777777777777777777 

2222222222222222222222222 

2333333333333444444444444 

Carcass ID Number 

2222222222222222222222222 

9555667777899566677788899 

2456670128347203536712613 

Total 

Tissues/ 

Tumors 

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 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+ + 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

50 

50 

50 

50 

50 

50 

50 

1 

10 

50 

50 

50 

50 

Cardiovascular System 

Blood vessel + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Adenoma X X 2 

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Parathyroid gland + + M + + + + + + + + + M + + + + M + + + + + + M 44 

Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Pars distalis, adenoma X XX X X X XXXXX 20 

Pars distalis, adenoma, multiple 1 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 50 

C-cell, adenoma 2 

General Body System 

None 

Genital System 

Clitoral gland + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Adenoma X 1 

Ovary + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Granulosa-theca tumor malignant X 1 

Uterus + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Polyp stromal X X X 7 

Sarcoma stromal X 3 

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Lymph node 7 

Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Thymus M + M + + + M + + M + + + + M + + + + + + + + + + 45 
 

 

 

 

128 Primidone, NTP TR 476 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Primidone: 600 ppm 

Number of Days on Study 

3444444555556666666667777 

6111256167780111477780022 

7059666178845288555522222 

Carcass ID Number 

2222222232222222222222222 

9665687805588968978976955 

9427945801395517890048689 

Integumentary System 

Mammary gland 

Carcinoma 

Fibroadenoma

Fibroadenoma, multiple

Skin 

Subcutaneous tissue, fibroma 

Subcutaneous tissue, fibrosarcoma

Subcutaneous tissue, liposarcoma

+++++++++++++++++++++++++ 

X 

 X X X X X X X 

X X 

+++++++++++++++++++++++++ 

X 

X 

Musculoskeletal System 

Bone 

Skeletal muscle

+++++++++++++++++++++++++ 

+ 

Nervous System 

Brain 

Spinal cord

+++++++++++++++++++++++++ 

+ 

Respiratory System 

Lung 

Nose 

Trachea 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

Special Senses System 

Eye 

Urinary System 

Kidney 

Urinary bladder 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear 

+++++++++++++++++++++++++ 

X XXXX X XX X 
 

 

 

 

 

 

 

 

Primidone, NTP TR 476 129 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Primidone: 600 ppm 

Number of Days on Study 

7777777777777777777777777 

2222222222222222222222222 

2333333333333444444444444 

Carcass ID Number 

2222222222222222222222222 

9555667777899566677788899 

2456670128347203536712613 

Total 

Tissues/ 

Tumors 

Integumentary System 

Mammary gland 

Carcinoma

Fibroadenoma 

Fibroadenoma, multiple

Skin 

Subcutaneous tissue, fibroma

Subcutaneous tissue, fibrosarcoma 

Subcutaneous tissue, liposarcoma 

+++++++++++++++++++++++++ 

X 

XX XXX X XXXXX XX X 

X X 

+++++++++++++++++++++++++ 

X 

50 

2 

21 

4 

50 

1 

1 

1 

Musculoskeletal System 

Bone 

Skeletal muscle 

+++++++++++++++++++++++++ 50 

1 

Nervous System 

Brain 

Spinal cord 

+++++++++++++++++++++++++ 50 

1 

Respiratory System 

Lung 

Nose 

Trachea 

+++++++++++++++++++++++++ 

+++++M+++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

49 

50 

Special Senses System 

Eye + 1 

Urinary System 

Kidney 

Urinary bladder 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

50 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear

+++++++++++++++++++++++++ 

X X X X 

50 

13 
 

130 Primidone, NTP TR 476 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Primidone: 1,300 ppm 

3455566666666666777777777 

Number of Days on Study 8504501134689999000222222 

9613030844820356379222222 

3333333333333333333333333 

Carcass ID Number 2330313213201445014001123 

2067112997433960628280854 

Alimentary System 

Esophagus +++++++++++++++++++++++++ 

Intestine large, colon +++++++++++++++++++++++++ 

Intestine large, rectum +++++++++++++++++++++++++ 

Intestine large, cecum +++++++++++++++++++++++++ 

Intestine small, duodenum +++++++++++++++++++++++++ 

Intestine small, jejunum +++++++++++++++++++++++++ 

Intestine small, ileum +++++++++++++++++++++++++ 

Liver +++++++++++++++++++++++++ 

Mesentery + + + + 

Pancreas +++++++++++++++++++++++++ 

Salivary glands +++++++++++++++++++++++++ 

Stomach, forestomach +++++++++++++++++++++++++ 

Stomach, glandular +++++++++++++++++++++++++ 

Cardiovascular System 

Blood vessel 

Heart 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

Endocrine System 

Adrenal cortex +++++++++++++++++++++++++ 

Adrenal medulla ++++++++++++++M++++++++++ 

Pheochromocytoma benign X 

Islets, pancreatic +++++++++++++++++++++++++ 

Adenoma 

Parathyroid gland ++M++M+++++++M+++++++++++ 

Pituitary gland +++++++++++++++++++++++++ 

Pars distalis, adenoma XX X XX XX XX X 

Pars distalis, adenoma, multiple 

Thyroid gland +++++++++++++++++++++++++ 

Bilateral, C-cell, adenoma 

C-cell, adenoma X 

General Body System 

None 

Genital System 

Clitoral gland +++++++++++++++++++++++++ 

Adenoma X X 

Carcinoma X 

Ovary +++++++++++++++++++++++++ 

Uterus +++++++++++++++++++++++++ 

Leiomyosarcoma X 

Polyp stromal X X 

Vagina + 

Hematopoietic System 

Bone marrow +++++++++++++++++++++++++ 

Lymph node + + + + + + 

Thoracic, carcinoma, metastatic,

 mammary gland X 

Lymph node, mandibular +++++++++++++++++++++++++ 

Lymph node, mesenteric +++++++++++++++++++++++++ 
 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 131 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Primidone: 1,300 ppm 

Number of Days on Study 

7777777777777777777777777 

2222222222222222222222222 

2222333333333444444444444 

Carcass ID Number 

3333333333333333333333333 

3344000123444011122222344 

5825145679017945701368334 

Total 

Tissues/ 

Tumors 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Mesentery

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+ + + 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

50 

50 

50 

50 

50 

50 

50 

7 

50 

50 

50 

50 

Cardiovascular System 

Blood vessel + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Pheochromocytoma benign X 2 

Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Adenoma X 1 

Parathyroid gland + + + M + M M + + + M + + + + + + + + M + + + + + 42 

Pituitary gland + + + + + + + + M + + + + + + + + + + + + + + + + 49 

Pars distalis, adenoma X X X 13 

Pars distalis, adenoma, multiple X 1 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Bilateral, C-cell, adenoma X 1 

C-cell, adenoma X X X X X 6 

General Body System 

None 

Genital System 

Clitoral gland 

Adenoma

Carcinoma 

Ovary 

Uterus 

Leiomyosarcoma 

Polyp stromal

Vagina 

+++++++++++++++++++++++++ 

X X 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

X X X X 

50 

4 

1 

50 

50 

1 

6 

1 

Hematopoietic System 

Bone marrow 

Lymph node

Thoracic, carcinoma, metastatic, 

mammary gland 

Lymph node, mandibular 

Lymph node, mesenteric 

+++++++++++++++++++++++++ 

 + + 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

8 

1 

50 

50 
 

 

 

132 Primidone, NTP TR 476 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Primidone: 1,300 ppm 

Number of Days on Study 

3455566666666666777777777 

8504501134689999000222222 

9613030844820356379222222 

Carcass ID Number 

3333333333333333333333333 

2330313213201445014001123 

2067112997433960628280854 

Hematopoietic System (continued)

 Spleen 

Thymus 

Thymoma benign

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

X 

Integumentary System 

Mammary gland 

Carcinoma 

Fibroadenoma

Fibroadenoma, multiple

Skin 

Pinna, squamous cell papilloma 

Subcutaneous tissue, fibrosarcoma

+++++++++++++++++++++++++ 

X 

X XXX X X X 

X 

+++++++++++++++++++++++++ 

X 

Musculoskeletal System 

Bone +++++++++++++++++++++++++ 

Nervous System 

Brain 

Astrocytoma malignant 

+++++++++++++++++++++++++ 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma

Carcinoma, metastatic, mammary gland 

Nose 

Trachea 

+++++++++++++++++++++++++ 

X 

X 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

Special Senses System 

Eye 

Harderian gland 

Zymbal’s gland 

Carcinoma 

Urinary System 

Kidney 

Urinary bladder 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear

+++++++++++++++++++++++++ 

X XXXX X X X X 
 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 133 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Primidone: 1,300 ppm 

Number of Days on Study 

7777777777777777777777777 

2222222222222222222222222 

2222333333333444444444444 

Carcass ID Number 

3333333333333333333333333 

3344000123444011122222344 

5825145679017945701368334 

Total 

Tissues/ 

Tumors 

Hematopoietic System (continued)

 Spleen 

Thymus 

Thymoma benign 

+++++++++++++++++++++++++ 

+++++M+++++++M++++++++M++ 

50 

47 

1 

Integumentary System 

Mammary gland 

Carcinoma 

Fibroadenoma 

Fibroadenoma, multiple

Skin 

Pinna, squamous cell papilloma

Subcutaneous tissue, fibrosarcoma 

+++++++++++++++++++++++++ 

X X X X X X X 

X 

+++++++++++++++++++++++++ 

X 

50 

1 

14 

2 

50 

1 

1 

Musculoskeletal System 

Bone +++++++++++++++++++++++++ 50 

Nervous System 

Brain 

Astrocytoma malignant

+++++++++++++++++++++++++ 

X 

50 

1 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Carcinoma, metastatic, mammary gland 

Nose 

Trachea 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

1 

1 

50 

50 

Special Senses System 

Eye

Harderian gland

Zymbal’s gland

Carcinoma

 + 

X 

 ++ 

+ 

2 

1 

1 

1 

Urinary System 

Kidney 

Urinary bladder 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

50 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear

+++++++++++++++++++++++++ 

 X X X X 

50 

13 
 

 

 

 

 

 

 

 

 

 

 

 

134 Primidone, NTP TR 476 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Primidone: 2,500 ppm 

Number of Days on Study 

4444555555666666677777777 

3789445699002777800001222 

3791380106556445222888222 

Carcass ID Number 

3333333333333333333333333 

5896956577796685689668677 

5093295708274766841692234 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Fibrosarcoma, metastatic, spleen

Intestine small, duodenum 

Intestine small, jejunum 

Fibrosarcoma, metastatic, spleen

Intestine small, ileum 

Fibrosarcoma, metastatic, spleen

Liver 

Fibrosarcoma, metastatic, spleen

Hepatocellular adenoma

Mesentery

Fibrosarcoma, metastatic, spleen

Oral mucosa 

Gingival, squamous cell carcinoma

Pharyngeal, squamous cell carcinoma 

Pancreas 

Fibrosarcoma, metastatic, spleen

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Fibrosarcoma, metastatic, spleen

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

++++++++++++++M++++++++++ 

X 

+++++++++++++++++++++++++ 

++++++++++++++M++++++++++ 

X 

++++++++++++++M++++++++++ 

X 

+++++++++++++++++++++++++ 

X 

X 

+ + + + 

X 

+ + 

X 

X 

+++++++++++++++++++++++++ 

X 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

X 

Cardiovascular System 

Blood vessel 

Heart 

Schwannoma malignant

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

X 

Endocrine System 

Adrenal cortex +++++++++++++++++++++++++ 

Adenoma 

Adrenal medulla +++++++++++++++++++++++++ 

Pheochromocytoma benign 

Islets, pancreatic +++++++++++++++++++++++++ 

Parathyroid gland +++M+++++M+M+M++++++++M++ 

Pituitary gland +++++++++++++++++++++++++ 

Pars distalis, adenoma X X XX XX XX X 

Pars distalis, adenoma, multiple X 

Pars distalis, carcinoma X 

Thyroid gland +++++++++++++++++++++++++ 

C-cell, adenoma X 

General Body System 

None 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 135 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Primidone: 2,500 ppm 

Number of Days on Study 

7777777777777777777777777 

2222222222222222222222222 

2333333333333344444444444 

Carcass ID Number 

4333333333333333333333333 

0555677888999955677788899 

0123069179046848115735835 

Total 

Tissues/ 

Tumors 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Fibrosarcoma, metastatic, spleen 

Intestine small, duodenum 

Intestine small, jejunum 

Fibrosarcoma, metastatic, spleen 

Intestine small, ileum 

Fibrosarcoma, metastatic, spleen 

Liver 

Fibrosarcoma, metastatic, spleen 

Hepatocellular adenoma 

Mesentery

Fibrosarcoma, metastatic, spleen 

Oral mucosa 

Gingival, squamous cell carcinoma 

Pharyngeal, squamous cell carcinoma 

Pancreas 

Fibrosarcoma, metastatic, spleen 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Fibrosarcoma, metastatic, spleen 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+ + + 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

50 

50 

49 

1 

50 

49 

1 

49 

1 

50 

1 

1 

7 

1 

2 

1 

1 

50 

1 

50 

50 

50 

1 

Cardiovascular System 

Blood vessel 

Heart 

Schwannoma malignant 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

50 

1 

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Adenoma X 1 

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Pheochromocytoma benign X X X 3 

Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Parathyroid gland ++M+++++M+ M M M + + + + + M + + + + + + 39 

Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Pars distalis, adenoma X X X XX XX X X 18 

Pars distalis, adenoma, multiple X 2 

Pars distalis, carcinoma 1 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 50 

C-cell, adenoma X X X 4 

General Body System 

None 
 

 

136 Primidone, NTP TR 476 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Primidone: 2,500 ppm 

Number of Days on Study 

4444555555666666677777777 

3789445699002777800001222 

3791380106556445222888222 

Carcass ID Number 

3333333333333333333333333 

5896956577796685689668677 

5093295708274766841692234 

Genital System 

Clitoral gland +++++++++++++++++++++++++ 

Adenoma X 

Carcinoma X 

Ovary ++++++++++++++M++++++++++ 

Uterus ++++++++++++++M++++++++++ 

Polyp stromal X X X X X 

Sarcoma stromal X X 

Vagina + 

Polyp X 

Hematopoietic System 

Bone marrow 

Lymph node

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Fibrosarcoma

Thymus 

+++++++++++++++++++++++++ 

+ + + + + + + 

+++++++++++++++++++++++++ 

++++++++++++++M++++++++++ 

+++++++++++++++++++++++++ 

X 

+++++++++++++M+++++++M+++ 

Integumentary System 

Mammary gland 

Fibroadenoma

Fibroadenoma, multiple 

Skin 

Subcutaneous tissue, fibroma 

Subcutaneous tissue, fibrosarcoma

+++++++++++++++++++++++++ 

X X X X X 

+++++++++++++++++++++++++ 

X 

Musculoskeletal System 

Bone 

Skeletal muscle

+++++++++++++++++++++++++ 

+ + 

Nervous System 

Brain 

Carcinoma, metastatic, pituitary gland

Peripheral nerve 

Spinal cord

+++++++++++++++++++++++++ 

X 

+ 

+ + 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Nose 

Trachea 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

Special Senses System 

Eye 

Urinary System 

Kidney 

Urinary bladder 

+++++++++++++++++++++++++ 

++++++++++++++M++++++++++ 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear

+++++++++++++++++++++++++ 

 X X X X X X X X X X X X X 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 137 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Primidone: 2,500 ppm 

7777777777777777777777777 

Number of Days on Study 2222222222222222222222222 

2333333333333344444444444 

4 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Total 

Carcass ID Number 0 5 5 5 6 7 7 8 8 8 9 9 9 9 5 5 6 7 7 7 8 8 8 9 9 Tissues/ 

0 1 2 3 0 6 9 1 7 9 0 4 6 8 4 8 1 1 5 7 3 5 8 3 5 Tumors 

Genital System 

Clitoral gland + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Adenoma 1 

Carcinoma X 2 

Ovary + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Uterus + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Polyp stromal X X 7 

Sarcoma stromal X 3 

Vagina + 2 

Polyp X 2 

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Lymph node + + + + + 12 

Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Fibrosarcoma 1 

Thymus + + + + + + M + + + + + + + + M + + + + + + + + + 46 

Integumentary System 

Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Fibroadenoma X X X X X X 11 

Fibroadenoma, multiple X 1 

Skin + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Subcutaneous tissue, fibroma X 1 

Subcutaneous tissue, fibrosarcoma X 2 

Musculoskeletal System 

Bone + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Skeletal muscle 2 

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Carcinoma, metastatic, pituitary gland 1 

Peripheral nerve 1 

Spinal cord 2 

Respiratory System 

Lung + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Alveolar/bronchiolar adenoma X X 2 

Nose + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Trachea + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Special Senses System 

Eye + + 2 

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Systemic Lesions 

Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Leukemia mononuclear X X X X X X X X X 22 
138 Primidone, NTP TR 476 

TABLE B3 

Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Feed Study of Primidone 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

Adrenal Medulla: Benign Pheochromocytoma 

Overall ratea 

Adjusted rateb 2/50 (4%) 

6.8% 

0/50 (0%) 

0.0% 

2/49 (4%) 

5.2% 

3/50 (6%) 

10.7% 

Terminal ratec 1/24 (4%) 0/27 (0%) 1/31 (3%) 3/28 (11%) 

First incidence (days) 

Life table testd 

660 

P=0.263 

—e 

P=0.236N 

389 

P=0.635N 

722 (T) 

P=0.559 

Logistic regression testd 

Cochran-Armitage testd 

Fisher exact testd 

P=0.226 

P=0.228 

P=0.251N 

P=0.247N 

P=0.666 

P=0.684 

P=0.519 

P=0.500 

Clitoral Gland: Adenoma 

Overall rate 4/49 (8%) 1/49 (2%) 4/50 (8%) 1/50 (2%) 

Adjusted rate 14.9% 3.7% 10.7% 2.5% 

Terminal rate 3/24 (13%) 1/27 (4%) 2/31 (6%) 0/28 (0%) 

First incidence (days) 660 722 (T) 501 605 

Life table test P=0.196N P=0.157N P=0.529N P=0.160N 

Logistic regression test P=0.221N P=0.199N P=0.631N P=0.175N 

Cochran-Armitage test P=0.224N 

Fisher exact test P=0.181N P=0.631N P=0.175N 

Clitoral Gland: Adenoma or Carcinoma 

Overall rate 5/49 (10%) 1/49 (2%) 5/50 (10%) 3/50 (6%) 

Adjusted rate 17.9% 3.7% 12.6% 8.7% 

Terminal rate 3/24 (13%) 1/27 (4%) 2/31 (6%) 1/28 (4%) 

First incidence (days) 660 722 (T) 456 605 

Life table test P=0.417N P=0.093N P=0.514N P=0.309N 

Logistic regression test P=0.464N P=0.118N P=0.630 P=0.349N 

Cochran-Armitage test P=0.463N 

Fisher exact test P=0.102N P=0.617N P=0.346N 

Mammary Gland: Fibroadenoma 

Overall rate 22/50 (44%) 25/50 (50%) 16/50 (32%) 12/50 (24%) 

Adjusted rate 61.7% 72.9% 40.9% 35.5% 

Terminal rate 11/24 (46%) 18/27 (67%) 9/31 (29%) 8/28 (29%) 

First incidence (days) 415 578 550 489 

Life table test P=0.003N P=0.450 P=0.057N P=0.017N 

Logistic regression test P=0.004N P=0.209 P=0.128N P=0.028N 

Cochran-Armitage test P=0.006N 

Fisher exact test P=0.344 P=0.151N P=0.028N 

Mammary Gland: Fibroadenoma or Adenoma 

Overall rate 24/50 (48%) 25/50 (50%) 16/50 (32%) 12/50 (24%) 

Adjusted rate 65.4% 72.9% 40.9% 35.5% 

Terminal rate 12/24 (50%) 18/27 (67%) 9/31 (29%) 8/28 (29%) 

First incidence (days) 415 578 550 489 

Life table test P=0.001N P=0.548N P=0.027N P=0.007N 

Logistic regression test P=0.001N P=0.350 P=0.065N P=0.011N 

Cochran-Armitage test P=0.002N 

Fisher exact test P=0.500 P=0.076N P=0.011N 
Primidone, NTP TR 476 139 

TABLE B3 

Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Feed Study of Primidone 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

Mammary Gland: Adenoma or Carcinoma 

Overall rate 3/50 (6%) 2/50 (4%) 1/50 (2%) 0/50 (0%)
 

Adjusted rate 9.5% 5.6% 2.0% 0.0%
 

Terminal rate 1/24 (4%) 1/27 (4%) 0/31 (0%) 0/28 (0%)
 

First incidence (days) 566 367 389 —
 

Life table test P=0.053N P=0.485N P=0.271N P=0.111N
 

Logistic regression test P=0.068N P=0.438N P=0.335N P=0.120N
 

Cochran-Armitage test P=0.059N
 

Fisher exact test P=0.500N P=0.309N P=0.121N
 

Mammary Gland: Fibroadenoma, Adenoma, or Carcinoma 

Overall rate 25/50 (50%) 26/50 (52%) 17/50 (34%) 12/50 (24%) 

Adjusted rate 66.7% 73.4% 42.0% 35.5% 

Terminal rate 12/24 (50%) 18/27 (67%) 9/31 (29%) 8/28 (29%) 

First incidence (days) 415 367 389 489 

Life table test P<0.001N P=0.546N P=0.030N P=0.005N 

Logistic regression test P<0.001N P=0.372 P=0.074N P=0.006N 

Cochran-Armitage test P=0.001N 

Fisher exact test P=0.500 P=0.078N P=0.006N 

Pituitary Gland (Pars Distalis): Adenoma 

Overall rate 16/49 (33%) 21/50 (42%) 14/49 (29%) 20/50 (40%) 

Adjusted rate 47.9% 56.7% 36.2% 54.5% 

Terminal rate 8/23 (35%) 12/27 (44%) 7/30 (23%) 12/28 (43%) 

First incidence (days) 541 410 543 491 

Life table test P=0.510N P=0.281 P=0.223N P=0.460 

Logistic regression test P=0.413 P=0.184 P=0.399N P=0.289 

Cochran-Armitage test P=0.389 

Fisher exact test P=0.226 P=0.413N P=0.291 

Pituitary Gland (Pars Distalis): Adenoma or Carcinoma 

Overall rate 16/49 (33%) 21/50 (42%) 14/49 (29%) 21/50 (42%) 

Adjusted rate 47.9% 56.7% 36.2% 56.0% 

Terminal rate 8/23 (35%) 12/27 (44%) 7/30 (23%) 12/28 (43%) 

First incidence (days) 541 410 543 491 

Life table test P=0.482 P=0.281 P=0.223N P=0.394 

Logistic regression test P=0.330 P=0.184 P=0.399N P=0.223 

Cochran-Armitage test P=0.307 

Fisher exact test P=0.226 P=0.413N P=0.226 

Skin (Subcutaneous Tissue): Fibroma, Fibrosarcoma, or Sarcoma 

Overall rate 3/50 (6%) 2/50 (4%) 1/50 (2%) 3/50 (6%) 

Adjusted rate 7.0% 6.6% 2.2% 10.0% 

Terminal rate 0/24 (0%) 1/27 (4%) 0/31 (0%) 2/28 (7%) 

First incidence (days) 367 675 603 702 

Life table test P=0.568N P=0.513N P=0.297N P=0.633N 

Logistic regression test P=0.560 P=0.432N P=0.339N P=0.661N 

Cochran-Armitage test P=0.573 

Fisher exact test P=0.500N P=0.309N P=0.661N 
140 Primidone, NTP TR 476 

TABLE B3 

Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Feed Study of Primidone 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

Thyroid Gland (C-cell): Adenoma 

Overall rate 5/49 (10%) 2/50 (4%) 7/50 (14%) 4/50 (8%) 

Adjusted rate 19.4% 5.1% 22.6% 12.6% 

Terminal rate 4/24 (17%) 0/27 (0%) 7/31 (23%) 3/28 (11%) 

First incidence (days) 682 578 722 (T) 491 

Life table test P=0.541N P=0.210N P=0.565 P=0.420N 

Logistic regression test P=0.535 P=0.227N P=0.536 P=0.493N 

Cochran-Armitage test P=0.521 

Fisher exact test P=0.210N P=0.394 P=0.487N 

Uterus: Stromal Polyp 

Overall rate 8/50 (16%) 7/50 (14%) 6/50 (12%) 7/50 (14%) 

Adjusted rate 24.9% 21.1% 19.4% 19.9% 

Terminal rate 4/24 (17%) 3/27 (11%) 6/31 (19%) 3/28 (11%) 

First incidence (days) 415 612 722 (T) 489 

Life table test P=0.356N P=0.488N P=0.245N P=0.433N 

Logistic regression test P=0.429N P=0.517N P=0.375N P=0.498N 

Cochran-Armitage test P=0.439N 

Fisher exact test P=0.500N P=0.387N P=0.500N 

Uterus: Stromal Sarcoma 

Overall rate 0/50 (0%) 3/50 (6%) 0/50 (0%) 3/50 (6%) 

Adjusted rate 0.0% 7.9% 0.0% 9.3% 

Terminal rate 0/24 (0%) 1/27 (4%) 0/31 (0%) 2/28 (7%) 

First incidence (days) — 415 — 561 

Life table test P=0.192 P=0.123 — P=0.137 

Logistic regression test P=0.149 P=0.178 — P=0.121 

Cochran-Armitage test P=0.175 

Fisher exact test P=0.121 — P=0.121 

Uterus: Stromal Polyp or Stromal Sarcoma 

Overall rate 8/50 (16%) 10/50 (20%) 6/50 (12%) 10/50 (20%) 

Adjusted rate 24.9% 27.7% 19.4% 28.1% 

Terminal rate 4/24 (17%) 4/27 (15%) 6/31 (19%) 5/28 (18%) 

First incidence (days) 415 415 722 (T) 489 

Life table test P=0.529 P=0.414 P=0.245N P=0.476 

Logistic regression test P=0.433 P=0.427 P=0.375N P=0.399 

Cochran-Armitage test P=0.436 

Fisher exact test P=0.398 P=0.387N P=0.398 

All Organs: Mononuclear Cell Leukemia 

Overall rate 13/50 (26%) 13/50 (26%) 13/50 (26%) 22/50 (44%) 

Adjusted rate 38.3% 31.7% 31.3% 53.0% 

Terminal rate 6/24 (25%) 4/27 (15%) 5/31 (16%) 10/28 (36%) 

First incidence (days) 561 367 501 477 

Life table test P=0.068 P=0.574 P=0.415N P=0.114 

Logistic regression test P=0.018 P=0.483N P=0.582 P=0.047 

Cochran-Armitage test P=0.024 

Fisher exact test P=0.590N P=0.590N P=0.046 
141 

c 

Primidone, NTP TR 476 

TABLE B3 

Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Feed Study of Primidone 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

All Organs: Benign Neoplasms 

Overall rate 39/50 (78%) 35/50 (70%) 35/50 (70%) 33/50 (66%) 

Adjusted rate 90.5% 87.2% 77.5% 79.8% 

Terminal rate 20/24 (83%) 22/27 (81%) 21/31 (68%) 20/28 (71%) 

First incidence (days) 415 410 389 489 

Life table test P=0.069N P=0.240N P=0.060N P=0.083N 

Logistic regression test P=0.102N P=0.439N P=0.216N P=0.134N 

Cochran-Armitage test P=0.134N 

Fisher exact test P=0.247N P=0.247N P=0.133N 

All Organs: Malignant Neoplasms 

Overall rate 21/50 (42%) 20/50 (40%) 17/50 (34%) 28/50 (56%) 

Adjusted rate 52.1% 46.7% 39.1% 63.6% 

Terminal rate 7/24 (29%) 7/27 (26%) 7/31 (23%) 13/28 (46%) 

First incidence (days) 367 367 389 433 

Life table test P=0.213 P=0.508N P=0.165N P=0.258 

Logistic regression test P=0.059 P=0.351N P=0.327N P=0.114 

Cochran-Armitage test P=0.086 

Fisher exact test P=0.500N P=0.268N P=0.115 

All Organs: Benign or Malignant Neoplasms 

Overall rate 47/50 (94%) 44/50 (88%) 41/50 (82%) 46/50 (92%) 

Adjusted rate 94.0% 89.7% 82.0% 92.0% 

Terminal rate 21/24 (88%) 22/27 (81%) 22/31 (71%) 24/28 (86%) 

First incidence (days) 367 367 389 433 

Life table test P=0.243N P=0.352N P=0.042N P=0.288N 

Logistic regression test P=0.551 P=0.237N P=0.129N P=0.586N 

Cochran-Armitage test P=0.452N 

Fisher exact test P=0.243N P=0.061N P=0.500N 

(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, pituitary gland, and thyroid gland; for other tissues, denominator is number of animals necropsied. b	 Kaplan-Meier estimated neoplasm incidence after adjustment for intercurrent mortality 

Observed incidence at terminal kill 

d	 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 
142 Primidone, NTP TR 476 

TABLE B4 

Historical Incidence of Mononuclear Cell Leukemia in Untreated Female F344/N Ratsa 

Study Incidence in Controls 

Historical Incidence at Battelle Columbus Laboratories 

4,4N-Thiobis(6-t-butyl-m-cresol) 18/50 

5,5-Diphenylhydantoin 13/50 

Ethylene Thiourea 18/50 

Polybrominated Biphenyls (Firemaster FF-1®) 14/50 

Manganese (II) Sulfate Monohydrate 19/50 

Triamterene 8/50 

Tricresyl Phosphate 8/51 

Overall Historical Incidence 

Total 355/1,301 (27.3%) 

Standard deviation 9.0% 

Range 14%-52% 

a Data as of 12 May 1995. Includes data for lymphocytic, monocytic, mononuclear, and undifferentiated cell type leukemias 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 143 

TABLE B5 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Feed Study of Primidonea 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

Disposition Summary 

Animals initially in study 50 50 50 50 

Early deaths 

Moribund 21 18 14 19 

Natural deaths 5 5 5 3 

Survivors 

Died last week of study 1 

Terminal sacrifice 24 27 31 27 

Animals examined microscopically 50 50 50 50 

Alimentary System 

Esophagus (49) (50) (50) (50) 

Cyst 1 (2%) 

Intestine large, colon (50) (50) (50) (50) 

Parasite metazoan 1 (2%) 

Intestine large, rectum (49) (50) (50) (50) 

Parasite metazoan 2 (4%) 2 (4%) 3 (6%) 

Intestine small, ileum (50) (50) (50) (49) 

Inflammation, chronic active 1 (2%) 

Liver (50) (50) (50) (50) 

Angiectasis 2 (4%) 1 (2%) 3 (6%) 

Basophilic focus 39 (78%) 35 (70%) 38 (76%) 10 (20%) 

Clear cell focus 6 (12%) 5 (10%) 

Eosinophilic focus 2 (4%) 1 (2%) 18 (36%) 

Hematopoietic cell proliferation 1 (2%) 

Hepatodiaphragmatic nodule 5 (10%) 13 (26%) 11 (22%) 5 (10%) 

Inflammation, chronic 38 (76%) 38 (76%) 44 (88%) 34 (68%) 

Mixed cell focus 5 (10%) 1 (2%) 6 (12%) 

Pigmentation, hemosiderin 25 (50%) 14 (28%) 12 (24%) 19 (38%) 

Bile duct, hyperplasia 30 (60%) 23 (46%) 35 (70%) 33 (66%) 

Hepatocyte, degeneration 1 (2%) 

Hepatocyte, degeneration, cystic 1 (2%) 1 (2%) 

Hepatocyte, necrosis 4 (8%) 1 (2%) 3 (6%) 4 (8%) 

Hepatocyte, vacuolization cytoplasmic 25 (50%) 44 (88%) 46 (92%) 44 (88%) 

Hepatocyte, centrilobular, degeneration 1 (2%) 1 (2%) 

Hepatocyte, centrilobular, hypertrophy 1 (2%) 36 (72%) 38 (76%) 35 (70%) 

Portal vein, thrombosis 1 (2%) 

Mesentery (9) (10) (7) (7) 

Accessory spleen 1 (11%) 

Fat, inflammation, chronic active 1 (10%) 

Fat, necrosis 7 (78%) 5 (50%) 5 (71%) 6 (86%) 

Pancreas (49) (50) (50) (50) 

Cyst 1 (2%) 

Inflammation, chronic active 1 (2%) 1 (2%) 

Acinus, atrophy 8 (16%) 7 (14%) 11 (22%) 11 (22%) 

Salivary glands (49) (50) (50) (50) 

Parotid gland, atrophy 1 (2%) 

Parotid gland, inflammation, chronic active 1 (2%) 

Stomach, forestomach (49) (50) (50) (50) 

Hyperplasia, basal cell 1 (2%) 

Epithelium, hyperplasia 4 (8%) 4 (8%) 3 (6%) 4 (8%) 

Epithelium, inflammation, chronic active 2 (4%) 2 (4%) 2 (4%) 2 (4%) 

Epithelium, ulcer 2 (4%) 2 (4%) 2 (4%) 2 (4%) 

a Number of animals examined microscopically at the site and the number of animals with lesion 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

144 Primidone, NTP TR 476 

TABLE B5 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Feed Study of Primidone 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

Alimentary System (continued) 

Stomach, glandular (49) (50) (50) (50) 

Epithelium, erosion 2 (4%) 1 (2%) 

Epithelium, inflammation, chronic active 1 (2%) 

Epithelium, ulcer 1 (2%) 1 (2%) 

Cardiovascular System 

Blood vessel (49) (50) (50) (50) 

Inflammation, chronic active 1 (2%) 

Heart (49) (50) (50) (50) 

Cardiomyopathy, chronic 21 (43%) 29 (58%) 27 (54%) 21 (42%) 

Atrium, thrombosis 1 (2%) 

Endocrine System 

Adrenal cortex (50) (50) (50) (50) 

Accessory adrenal cortical nodule 1 (2%) 

Angiectasis 1 (2%) 

Atrophy 1 (2%) 

Degeneration, fatty 13 (26%) 11 (22%) 8 (16%) 6 (12%) 

Hyperplasia 10 (20%) 8 (16%) 10 (20%) 8 (16%) 

Hypertrophy 1 (2%) 1 (2%) 2 (4%) 3 (6%) 

Inflammation, chronic active 1 (2%) 

Inflammation, granulomatous 1 (2%) 

Necrosis 1 (2%) 

Adrenal medulla (50) (50) (49) (50) 

Hyperplasia 6 (12%) 5 (10%) 9 (18%) 7 (14%) 

Pituitary gland (49) (50) (49) (50) 

Angiectasis 1 (2%) 

Pars distalis, cyst 24 (49%) 24 (48%) 36 (73%) 28 (56%) 

Pars distalis, hyperplasia 26 (53%) 25 (50%) 26 (53%) 21 (42%) 

Pars intermedia, cyst 1 (2%) 1 (2%) 1 (2%) 

Thyroid gland (49) (50) (50) (50) 

C-cell, hyperplasia 14 (29%) 15 (30%) 11 (22%) 5 (10%) 

Follicular cell, hyperplasia 1 (2%) 

General Body System 

None 

Genital System 

Clitoral gland (49) (49) (50) (50) 

Cyst 2 (4%) 1 (2%) 2 (4%) 

Hyperplasia 12 (24%) 5 (10%) 8 (16%) 10 (20%) 

Inflammation, chronic active 4 (8%) 10 (20%) 2 (4%) 3 (6%) 

Ovary (50) (50) (50) (49) 

Atrophy 1 (2%) 1 (2%) 

Cyst 3 (6%) 4 (8%) 2 (4%) 1 (2%) 

Inflammation, chronic active 1 (2%) 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 145 

TABLE B5 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Feed Study of Primidone 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

Genital System (continued) 

Uterus (50) (50) (50) (49) 

Angiectasis 1 (2%) 

Edema 1 (2%) 

Hemorrhage 1 (2%) 1 (2%) 

Hydrometra 2 (4%) 

Hyperplasia, cystic 2 (4%) 4 (8%) 3 (6%) 

Inflammation, chronic active 1 (2%) 

Vagina (1) (1) (2) 

Cyst 1 (100%) 

Hematopoietic System 

Bone marrow (50) (50) (50) (50) 

Atrophy 1 (2%) 

Hyperplasia 5 (10%) 10 (20%) 7 (14%) 7 (14%) 

Hyperplasia, histiocytic 3 (6%) 

Hyperplasia, mast cell 1 (2%) 

Myelofibrosis 4 (8%) 1 (2%) 1 (2%) 1 (2%) 

Lymph node (11) (7) (8) (12) 

Mediastinal, hyperplasia, histiocytic 1 (9%) 

Mediastinal, necrosis 1 (9%) 

Lymph node, mandibular (49) (50) (50) (50) 

Ectasia 1 (2%) 

Inflammation, chronic active 1 (2%) 

Lymph node, mesenteric (50) (50) (50) (49) 

Ectasia 1 (2%) 3 (6%) 

Spleen (49) (50) (50) (50) 

Accessory spleen 2 (4%) 

Fibrosis 1 (2%) 1 (2%) 1 (2%) 

Hematopoietic cell proliferation 2 (4%) 3 (6%) 1 (2%) 2 (4%) 

Hemorrhage 1 (2%) 

Necrosis 2 (4%) 1 (2%) 1 (2%) 

Thymus (49) (45) (47) (46) 

Cyst 1 (2%) 

Integumentary System 

Mammary gland (50) (50) (50) (50) 

Cyst 1 (2%) 1 (2%) 

Hyperplasia, cystic 37 (74%) 44 (88%) 43 (86%) 37 (74%) 

Inflammation, chronic active 1 (2%) 

Skin (50) (50) (50) (50) 

Hyperkeratosis 1 (2%) 1 (2%) 

Inflammation, chronic active 2 (4%) 

Ulcer 1 (2%) 

Epidermis, hyperplasia 1 (2%) 2 (4%) 

Subcutaneous tissue, hemorrhage, acute 1 (2%) 

Musculoskeletal System 

Bone (50) (50) (50) (50) 

Osteopetrosis 2 (4%) 5 (10%) 1 (2%) 

Skeletal muscle (1) (2) 

Hemorrhage, acute 1 (50%) 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

146 Primidone, NTP TR 476 

TABLE B5 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Feed Study of Primidone 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

Nervous System 

Brain (50) (50) (50) (50) 

Hemorrhage 1 (2%) 3 (6%) 3 (6%) 1 (2%) 

Hydrocephalus 1 (2%) 1 (2%) 

Spinal cord (1) (2) 

Hemorrhage 1 (50%) 

Respiratory System 

Lung (49) (50) (50) (50) 

Inflammation, chronic active 1 (2%) 4 (8%) 1 (2%) 3 (6%) 

Necrosis 1 (2%) 

Alveolar epithelium, hyperplasia 5 (10%) 5 (10%) 6 (12%) 2 (4%) 

Alveolus, infiltration cellular, histiocyte 36 (73%) 44 (88%) 45 (90%) 46 (92%) 

Artery, vein, mineralization 38 (78%) 39 (78%) 38 (76%) 44 (88%) 

Interstitium, inflammation, chronic active 1 (2%) 

Perivascular, infiltration cellular,

 lymphocyte 4 (8%) 1 (2%) 3 (6%) 5 (10%) 

Nose (50) (49) (50) (50) 

Inflammation, chronic active 2 (4%) 4 (8%) 5 (10%) 

Inflammation, suppurative 1 (2%) 1 (2%) 1 (2%) 

Nasolacrimal duct, inflammation, suppurative 2 (4%) 3 (6%) 3 (6%) 4 (8%) 

Special Senses System 

Eye (1) (1) (2) (2) 

Inflammation, chronic active 1 (50%) 

Anterior chamber, inflammation, suppurative 1 (50%) 

Lens, cataract 1 (100%) 1 (100%) 1 (50%) 1 (50%) 

Retina, degeneration 1 (100%) 1 (100%) 2 (100%) 1 (50%) 

Harderian gland (1) 

Inflammation, chronic active 1 (100%) 

Urinary System 

Kidney (50) (50) (50) (50) 

Cyst 1 (2%) 1 (2%) 

Hydronephrosis 1 (2%) 

Mineralization 12 (24%) 10 (20%) 5 (10%) 8 (16%) 

Nephropathy, chronic 43 (86%) 43 (86%) 44 (88%) 43 (86%) 

Urinary bladder (50) (50) (50) (49) 

Inflammation, chronic active 1 (2%) 
147 

APPENDIX C 

SUMMARY OF LESIONS IN MALE MICE 

IN THE 2-YEAR FEED STUDY 

OF PRIMIDONE 

TABLE C1 

TABLE C2 

TABLE C3 

TABLE C4a 

TABLE C4b 

TABLE C5 

Summary of the Incidence of Neoplasms in Male Mice 

in the 2-Year Feed Study of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

Individual Animal Tumor Pathology of Male Mice 

in the 2-Year Feed Study of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

Statistical Analysis of Primary Neoplasms in Male Mice 

in the 2-Year Feed Study of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

Historical Incidence of Liver Neoplasms in Untreated Male B6C3F1 Mice . . . . . . . . . . 

Historical Incidence of Thyroid Gland Follicular Cell Adenoma 

in Untreated Male B6C3F1 Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice 

in the 2-Year Feed Study of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

149 

152 

168 

172 

173 

174 
148 Primidone, NTP TR 476
 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 149 

TABLE C1 

Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Feed Study of Primidonea 

0 ppm 300 ppm 600 ppm 1,300 ppm 

Disposition Summary 

Animals initially in study 50 50 50 50 

Early deaths 

Moribund 6 7 6 17 

Natural deaths 9 9 13 14 

Survivors 

Died last week of study 1 

Terminal sacrifice 35 33 31 19 

Animals examined microscopically 50 50 50 50 

Alimentary System 

Intestine large, rectum (48) (49) (50) (47) 

Fibrous histiocytoma 1 (2%) 

Intestine small, jejunum (46) (49) (50) (47) 

Histiocytic sarcoma 1 (2%) 

Intestine small, ileum (42) (47) (49) (48) 

Histiocytic sarcoma 1 (2%) 

Liver (50) (50) (50) (50) 

Hemangiosarcoma 2 (4%) 3 (6%) 

Hepatoblastoma 15 (30%) 21 (42%) 6 (12%) 

Hepatoblastoma, multiple 2 (4%) 5 (10%) 1 (2%) 

Hepatocellular carcinoma 10 (20%) 21 (42%) 13 (26%) 19 (38%) 

Hepatocellular carcinoma, multiple 2 (4%) 10 (20%) 22 (44%) 19 (38%) 

Hepatocellular adenoma 16 (32%) 12 (24%) 5 (10%) 10 (20%) 

Hepatocellular adenoma, multiple 6 (12%) 29 (58%) 34 (68%) 22 (44%) 

Histiocytic sarcoma 4 (8%) 

Mast cell tumor malignant 1 (2%) 

Mesentery (1) (2) (1) (1) 

Fat, histiocytic sarcoma 1 (100%) 

Pancreas (46) (48) (50) (47) 

Hemangioma 1 (2%) 

Hepatoblastoma, metastatic, liver 1 (2%) 

Stomach, forestomach (50) (49) (50) (49) 

Squamous cell carcinoma 1 (2%) 

Squamous cell papilloma 1 (2%) 

Cardiovascular System 

None 

Endocrine System 

Adrenal cortex (50) (49) (50) (50) 

Histiocytic sarcoma 1 (2%) 

Capsule, adenoma 1 (2%) 1 (2%) 

Adrenal medulla (50) (49) (50) (50) 

Pheochromocytoma benign 1 (2%) 1 (2%) 

Islets, pancreatic (45) (48) (49) (47) 

Adenoma 1 (2%) 

Thyroid gland (49) (48) (50) (50) 

Follicular cell, adenoma 3 (6%) 3 (6%) 6 (12%) 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

150 Primidone, NTP TR 476 

TABLE C1 

Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Feed Study of Primidone 

0 ppm 300 ppm 600 ppm 1,300 ppm 

General Body System 

None 

Genital System 

Epididymis (50) (50) (50) (50) 

Hepatoblastoma, metastatic, liver 1 (2%) 

Prostate (49) (50) (50) (50) 

Hematopoietic System 

Bone marrow (50) (50) (50) (50) 

Mast cell tumor malignant 1 (2%) 

Lymph node (1) (4) (1) (3) 

Mediastinal, alveolar/bronchiolar carcinoma,

 metastatic, lung 1 (25%) 

Lymph node, mesenteric (48) (48) (49) (42) 

Histiocytic sarcoma 1 (2%) 

Spleen (48) (49) (50) (49) 

Hemangiosarcoma 2 (4%) 

Hepatoblastoma, metastatic, uncertain

 primary site 1 (2%) 

Histiocytic sarcoma 2 (4%) 

Mast cell tumor malignant 1 (2%) 

Thymus (42) (40) (42) (34) 

Integumentary System 

Skin (50) (50) (50) (50) 

Subcutaneous tissue, fibrous histiocytoma 1 (2%) 

Subcutaneous tissue, histiocytic sarcoma 1 (2%) 1 (2%) 

Subcutaneous tissue, mast cell tumor

 malignant 1 (2%) 

Musculoskeletal System 

Skeletal muscle (1) 

Hepatoblastoma, metastatic, liver 1 (100%) 

Nervous System 

None 

Respiratory System 

Lung (50) (50) (50) (50) 

Alveolar/bronchiolar adenoma 6 (12%) 7 (14%) 5 (10%) 5 (10%) 

Alveolar/bronchiolar adenoma, multiple 1 (2%) 1 (2%) 

Alveolar/bronchiolar carcinoma 4 (8%) 1 (2%) 1 (2%) 3 (6%) 

Alveolar/bronchiolar carcinoma, multiple 1 (2%) 

Carcinoma, metastatic, harderian gland 1 (2%) 

Hepatoblastoma, metastatic, liver 2 (4%) 11 (22%) 5 (10%) 

Hepatocellular carcinoma, metastatic, liver 5 (10%) 5 (10%) 4 (8%) 5 (10%) 
 

 

 

 

 

 

 

 

 

 

 

151 

c 

Primidone, NTP TR 476 

TABLE C1 

Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Feed Study of Primidone 

0 ppm 300 ppm 600 ppm 1,300 ppm 

Respiratory System (continued)

Lung 

Histiocytic sarcoma

Mast cell tumor malignant

Sarcoma, metastatic, uncertain primary site

Mediastinum, hepatoblastoma, metastatic, liver

 1 (2%) 

 1 (2%) 

 1 (2%) 

 1 (2%) 

Special Senses System 

Harderian gland

Adenoma

Carcinoma

 (2)

 2 (100%)

 (3)

 2 (67%)

 1 (33%) 

 (1)

 1 (100%)

 (2) 

 1 (50%) 

Urinary System 

Kidney 

Alveolar/bronchiolar carcinoma,

 metastatic, lung

Hepatoblastoma, metastatic, liver

Histiocytic sarcoma

Renal tubule, carcinoma

(50) 

 1 (2%)

(49) 

 1 (2%) 

(50) 

 1 (2%) 

(50) 

 1 (2%) 

 1 (2%) 

Systemic Lesions 

Multiple organsb 

Histiocytic sarcoma

Lymphoma malignant

(50) 

 1 (2%)

 7 (14%)

(50) 

 4 (8%)

(50) 

 2 (4%) 

(50) 

 5 (10%) 

Neoplasm Summary 

Total animals with primary neoplasmsc 

Total primary neoplasms 

Total animals with benign neoplasms 

Total benign neoplasms 

Total animals with malignant neoplasms 

Total malignant neoplasms 

Total animals with metastatic neoplasms 

Total metastatic neoplasms 

Total animals with malignant neoplasms

 of uncertain primary site 

40 

68 

26 

34 

22 

34 

5 

6 

50 

112 

42 

54 

42 

58 

9 

11 

1 

50 

115 

39 

50 

42 

65 

14 

20 

47 

101 

36 

46 

42 

55 

11 

11 

1 

a Number of animals examined microscopically at the site and the number of animals with neoplasm b Number of animals with any tissue examined microscopically 

Primary neoplasms: all neoplasms except metastatic neoplasms 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

152 Primidone, NTP TR 476 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Primidone: 0 ppm 

4555555556666777777777777 

Number of Days on Study 3125577783449002222222222 

2161236835298116666666666 

0000000000000000000000000 

Carcass ID Number 5212423330141010011122234 

0739859058718953712913614 

Alimentary System 

Esophagus +++++++++++++++++++++++++ 

Gallbladder +++++M+++++++++++M+++++++ 

Intestine large, colon ++++A +++A ++++++++++++++++ 

Intestine large, rectum ++++A +++A ++++++++++++++++ 

Fibrous histiocytoma X 

Intestine large, cecum + + + + A + + + A + + + + + + + + + + + + + + + + 

Intestine small, duodenum + + M + A + + M A + A A +++++++++++++ 

Intestine small, jejunum + + + + A + + + A + A A + + + + + + + + + + + + + 

Intestine small, ileum ++M+A +M+A +A A +++++++++++++ 

Liver +++++++++++++++++++++++++ 

Hemangiosarcoma X X 

Hepatocellular carcinoma X X X X X X X 

Hepatocellular carcinoma, multiple X 

Hepatocellular adenoma X X X X X 

Hepatocellular adenoma, multiple X 

Mesentery + 

Pancreas + + + + A + + + A + A A + + + + + + + + + + + + + 

Hemangioma 

Salivary glands +++++++++++++++++++++++++ 

Stomach, forestomach +++++++++++++++++++++++++ 

Stomach, glandular +++++++++++++++++++++++++ 

Cardiovascular System 

Blood vessel 

Heart 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

Endocrine System 

Adrenal cortex +++++++++++++++++++++++++ 

Capsule, adenoma 

Adrenal medulla +++++++++++++++++++++++++ 

Islets, pancreatic +++A A +++A +A A +++++++++++++ 

Adenoma 

Parathyroid gland +M++MM+M+MMMMMM++M++M+M+M 

Pituitary gland M+++M+++M+++++++++++++I ++ 

Thyroid gland ++++A ++++++++++++++++++++ 

General Body System 

None 

Genital System 

Epididymis +++++++++++++++++++++++++ 

Preputial gland +++++++++++++++++++++++++ 

Prostate ++++++++M++++++++++++++++ 

Seminal vesicle ++++++++++A ++++++++++++++ 

Testes +++++++++++++++++++++++++ 

+: Tissue examined microscopically M: Missing tissue X: Lesion present 

A: Autolysis precludes examination I: Insufficient tissue Blank: Not examined 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 153 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Primidone: 0 ppm 

7777777777777777777777777 

Number of Days on Study 2222222222222222222222222 

6777777777777777777777777 

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Total 

Carcass ID Number 4 0 0 0 0 0 1 1 1 2 2 2 2 3 3 3 3 3 3 4 4 4 4 4 4 Tissues/ 

5 1 2 4 5 6 0 4 6 0 2 4 8 2 3 4 6 7 8 0 2 3 6 7 9 Tumors 

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Gallbladder + + M + + + + + + + + + + + + + + + + + + + + + + 47 

Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + 48 

Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + 48 

Fibrous histiocytoma 1 

Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + 48 

Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + 44 

Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + 46 

Intestine small, ileum + + M + + + + + + + M + + + + + + + + + + + + + + 42 

Liver + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Hemangiosarcoma 2 

Hepatocellular carcinoma X X X 10 

Hepatocellular carcinoma, multiple X 2 

Hepatocellular adenoma X X X X X X X X X X X 16 

Hepatocellular adenoma, multiple X X X X X 6 

Mesentery 1 

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 46 

Hemangioma X 1 

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Cardiovascular System 

Blood vessel + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Capsule, adenoma X 1 

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + 45 

Adenoma X 1 

Parathyroid gland + + M + + + M + + + + + + + + + + + + M + M M + + 31 

Pituitary gland + + M + + + + + + + + + + + + + + + + + + + + + + 45 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 49 

General Body System 

None 

Genital System 

Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Preputial gland + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Prostate + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Testes + + + + + + + + + + + + + + + + + + + + + + + + + 50 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

154 Primidone, NTP TR 476 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Primidone: 0 ppm 

4555555556666777777777777 

Number of Days on Study 3125577783449002222222222 

2161236835298116666666666 

0000000000000000000000000 

Carcass ID Number 5212423330141010011122234 

0739859058718953712913614 

Hematopoietic System 

Bone marrow 

Mast cell tumor malignant

Lymph node

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Hemangiosarcoma

Mast cell tumor malignant

Thymus 

+++++++++++++++++++++++++ 

X 

+ 

M + MMM+++++++M++++++M+++++ 

++++M+M++++++++++++++++++ 

+ + + A A + + + + + + + + + + + + + + + + + + + + 

X X 

X 

++M+++++++++M++++++++++++ 

Integumentary System 

Mammary gland 

Skin 

Subcutaneous tissue, fibrous histiocytoma

Subcutaneous tissue, histiocytic sarcoma 

Subcutaneous tissue, mast cell tumor malignant

MMMMMMMMMMMMMMMMMMMMMMMMM 

+++++++++++++++++++++++++ 

X 

X 

Musculoskeletal System 

Bone +++++++++++++++++++++++++ 

Nervous System 

Brain +++++++++++++++++++++++++ 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma

Alveolar/bronchiolar adenoma, multiple 

Alveolar/bronchiolar carcinoma

Hepatocellular carcinoma, metastatic, liver

Mast cell tumor malignant

Nose 

Trachea 

+++++++++++++++++++++++++ 

X X X 

X 

X X X 

X 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

Special Senses System 

Harderian gland

Adenoma

 + 

X 

Urinary System 

Kidney 

Alveolar/bronchiolar carcinoma, 

metastatic, lung

Urinary bladder 

+++++++++++++++++++++++++ 

X 

++++++++++A ++++++++++++++ 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant 

+++++++++++++++++++++++++ 

X X X X 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 155 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Primidone: 0 ppm 

7777777777777777777777777 

Number of Days on Study 2222222222222222222222222 

6777777777777777777777777 

0000000000000000000000000 Total 

Carcass ID Number 4000001112222333333444444 

5124560460248234678023679 

Tissues/ 

Tumors 

Hematopoietic System 

Bone marrow 

Mast cell tumor malignant 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Hemangiosarcoma 

Mast cell tumor malignant 

Thymus 

+++++++++++++++++++++++++ 

++++++M++++M+++M+++MM++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

++MMM+M ++++++M++M++++++++ 

50 

1 

1 

39 

48 

48 

2 

1 

42 

Integumentary System 

Mammary gland 

Skin 

Subcutaneous tissue, fibrous histiocytoma 

Subcutaneous tissue, histiocytic sarcoma

Subcutaneous tissue, mast cell tumor malignant 

MMMMMMMMMMMMMMMMMMMMMMMMM 

+++++++++++++++++++++++++ 

X 

50 

1 

1 

1 

Musculoskeletal System 

Bone +++++++++++++++++++++++++ 50 

Nervous System 

Brain +++++++++++++++++++++++++ 50 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma

Alveolar/bronchiolar adenoma, multiple

Alveolar/bronchiolar carcinoma

Hepatocellular carcinoma, metastatic, liver

Mast cell tumor malignant 

Nose 

Trachea 

+++++++++++++++++++++++++ 

X X X 

X 

X X X 

X X 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

6 

1 

4 

5 

1 

50 

50 

Special Senses System 

Harderian gland

Adenoma

 + 

X 

2 

2 

Urinary System 

Kidney 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 

Urinary bladder 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

1 

49 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma

Lymphoma malignant

+++++++++++++++++++++++++ 

X 

X X X 

50 

1 

7 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

156 Primidone, NTP TR 476 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Primidone: 300 ppm 

6666666666666677777777777 

Number of Days on Study 0011113344477911222222222 

3347776923623502666666666 

0000000000000000000000000 

Carcass ID Number 5995697889585858556666677 

8016665107185379492378912 

Alimentary System 

Esophagus + + + + + + + + + A + + + + + + + + + + + + + + + 

Gallbladder + + M + + M + + M A + M M + + + + + + + + + + + + 

Intestine large, colon +++++++++A +++++++++++++++ 

Intestine large, rectum +++++++++A +++++++++++++++ 

Intestine large, cecum + + + + + + + + + A + + + + + + + + + + + + + + + 

Intestine small, duodenum M + + + + + + + + A + + + + + + + + + + + + + + + 

Intestine small, jejunum + + + + + + + + + A + + + + + + + + + + + + + + + 

Intestine small, ileum +M+++++++A +++++++++++++++ 

Liver +++++++++++++++++++++++++ 

Hemangiosarcoma 

Hepatoblastoma X X XX XXX XX 

Hepatoblastoma, multiple X 

Hepatocellular carcinoma X X XXX XXX X X 

Hepatocellular carcinoma, multiple X X X 

Hepatocellular adenoma X X X X X X X 

Hepatocellular adenoma, multiple XX X X X X XX XXX 

Mesentery + +
 

Pancreas + M + + + + + + + A + + + + + + + + + + + + + + + 
 

Salivary glands +++++++++++++++++++++++++ 
 

Stomach, forestomach +++++++++A +++++++++++++++ 
 

Stomach, glandular + + + + + + + + + A + + + + + + + + + + + + + + + 
 

Cardiovascular System 

Blood vessel 

Heart 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

Endocrine System 

Adrenal cortex +++++++++M+++++++++++++++ 

Adrenal medulla +++++++++M+++++++++++++++ 

Pheochromocytoma benign 

Islets, pancreatic +M+++++++A +++++++++++++++ 

Parathyroid gland +++++++++M+++++++++M+M+++ 

Pituitary gland +++++++++A +++++++++I ++M++ 

Thyroid gland +++++++++A +++++++++++++++ 

Follicular cell, adenoma X 

General Body System 

None 

Genital System 

Epididymis +++++++++++++++++++++++++ 

Preputial gland +++++++++++++++++++++++++ 

Prostate +++++++++++++++++++++++++ 

Seminal vesicle +++++++++A +++++++++++++++ 

Testes +++++++++++++++++++++++++ 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 157 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Primidone: 300 ppm 

7777777777777777777777777 

Number of Days on Study 2222222222222222222222222 

6666666667777777777777777 

0000000010000000000000000 Total 

Carcass ID Number 7788999905566667777788899 Tissues/ 

7827234502301450346945689 Tumors 

Alimentary System 

Esophagus +++++++++++++++++++++++++ 49 

Gallbladder +++++++++++++++++++++++++ 44 

Intestine large, colon +++++++++++++++++++++++++ 49 

Intestine large, rectum +++++++++++++++++++++++++ 49 

Intestine large, cecum +++++++++++++++++++++++++ 49 

Intestine small, duodenum +++++++++++++++++++++++++ 48 

Intestine small, jejunum +++++++++++++++++++++++++ 49 

Intestine small, ileum +++++++++++M+++++++++++++ 47 

Liver +++++++++++++++++++++++++ 50 

Hemangiosarcoma X X X 3 

Hepatoblastoma X X X X X X 15 

Hepatoblastoma, multiple X 2 

Hepatocellular carcinoma X X X X XXXX XX X 21 

Hepatocellular carcinoma, multiple XXXX X X X 10 

Hepatocellular adenoma X X X X X 12 

Hepatocellular adenoma, multiple X XX XXXX XX XXXX XXXXX 29 

Mesentery 2 

Pancreas +++++++++++++++++++++++++ 48 

Salivary glands +++++++++++++++++++++++++ 50 

Stomach, forestomach +++++++++++++++++++++++++ 49 

Stomach, glandular +++++++++++++++++++++++++ 49 

Cardiovascular System 

Blood vessel 

Heart 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

50 

Endocrine System 

Adrenal cortex +++++++++++++++++++++++++ 49 

Adrenal medulla +++++++++++++++++++++++++ 49 

Pheochromocytoma benign X 1 

Islets, pancreatic +++++++++++++++++++++++++ 48 

Parathyroid gland ++++++++++M++M+++M+MM++++ 42 

Pituitary gland +++++++++++M+++++++++++++ 46 

Thyroid gland +++++++++M+++++++++++++++ 48 

Follicular cell, adenoma X X 3 

General Body System 

None 

Genital System 

Epididymis +++++++++++++++++++++++++ 50 

Preputial gland +++++++++++++++++++++++++ 50 

Prostate +++++++++++++++++++++++++ 50 

Seminal vesicle +++++++++++++++++++++++++ 49 

Testes +++++++++++++++++++++++++ 50 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

158 Primidone, NTP TR 476 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Primidone: 300 ppm 

6666666666666677777777777 

Number of Days on Study 0011113344477911222222222 

3347776923623502666666666 

0000000000000000000000000 

Carcass ID Number 5995697889585858556666677 

8016665107185379492378912 

Hematopoietic System 

Bone marrow +++++++++++++++++++++++++ 

Lymph node + + 

Mediastinal, alveolar/bronchiolar 

carcinoma, metastatic, lung 

Lymph node, mandibular + + + + + + + + + A + M + M + + + + + + + + + + + 

Lymph node, mesenteric + + + + + + + + + A + + + + + + + + + + + + + + + 

Spleen + + + + + + + + + A + + + + + + + + + + + + + + + 

Hepatoblastoma, metastatic,

 uncertain primary site X 

Thymus + + + + + M + + + A + + + + + + + + + M + + + + + 

Integumentary System 

Mammary gland MMMMMMMMMMMMMMMMMMMMMMMMM 

Skin +++++++++++++++++++++++++ 

Musculoskeletal System 

Bone +++++++++++++++++++++++++ 

Nervous System 

Brain +++++++++++++++++++++++++ 

Respiratory System 

Lung +++++++++++++++++++++++++ 

Alveolar/bronchiolar adenoma X X X 

Alveolar/bronchiolar carcinoma X 

Alveolar/bronchiolar carcinoma, multiple 

Carcinoma, metastatic, harderian gland X 

Hepatoblastoma, metastatic, liver X X 

Hepatocellular carcinoma, metastatic, liver X X 

Nose 

Trachea 

+++++++++++++++++++++++++ 

+ + + + + + + + + A + + + + + + + + + + + + + + + 

Special Senses System 

Harderian gland

Adenoma

Carcinoma

 +

 X 

+ 

X 

Urinary System 

Kidney 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 

Urinary bladder 

+ + + + + + + + + A + + + + + + + + + + + + + + + 

+++++++++A +++++++++++++++ 

Systemic Lesions 

Multiple organs 

Lymphoma malignant 

+++++++++++++++++++++++++ 

X 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 159 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Primidone: 300 ppm 

7777777777777777777777777 

Number of Days on Study 2222222222222222222222222 

6666666667777777777777777 

0000000010000000000000000 Total 

Carcass ID Number 7788999905566667777788899 

7827234502301450346945689 

Tissues/ 

Tumors 

Hematopoietic System 

Bone marrow 

Lymph node

Mediastinal, alveolar/bronchiolar 

carcinoma, metastatic, lung

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Hepatoblastoma, metastatic, 

uncertain primary site 

Thymus 

+++++++++++++++++++++++++ 

+ + 

X 

+++M+M+++M+++++M+++++++++ 

+++++++++++++++M+++++++++ 

+++++++++++++++++++++++++ 

++++ MMM+MM+M +++M+++++++++ 

50 

4 

1 

43 

48 

49 

1 

40 

Integumentary System 

Mammary gland 

Skin 

MMMMMMMMMMMMMMMMMMMMMMMMM 

+++++++++++++++++++++++++ 50 

Musculoskeletal System 

Bone +++++++++++++++++++++++++ 50 

Nervous System 

Brain +++++++++++++++++++++++++ 50 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma

Alveolar/bronchiolar carcinoma 

Alveolar/bronchiolar carcinoma, multiple

Carcinoma, metastatic, harderian gland 

Hepatoblastoma, metastatic, liver 

Hepatocellular carcinoma, metastatic, liver 

Nose 

Trachea 

+++++++++++++++++++++++++ 

X X X X 

X 

X X X 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

7 

1 

1 

1 

2 

5 

50 

49 

Special Senses System 

Harderian gland

Adenoma

Carcinoma 

+ 

X 

3 

2 

1 

Urinary System 

Kidney 

Alveolar/bronchiolar carcinoma, 

metastatic, lung

Urinary bladder 

+++++++++++++++++++++++++ 

X 

+++++++++++++++++++++++++ 

49 

1 

49 

Systemic Lesions 

Multiple organs 

Lymphoma malignant

+++++++++++++++++++++++++ 

 X X X 

50 

4 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

160 Primidone, NTP TR 476 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Primidone: 600 ppm 

4555666666666666667777777 

Number of Days on Study 2677011224445568890222222 

2658249153562871721666666 

1111111111111111111111111 

Carcass ID Number 0442301244032424113000001 

4520799138665471219125780 

Alimentary System 

Esophagus +++++++++++++++++++++++++ 

Gallbladder ++M++++++++++++M+M+++++++ 

Intestine large, colon +++++++++++++++++++++++++ 

Intestine large, rectum +++++++++++++++++++++++++ 

Intestine large, cecum +++++++++++++++++++++++++ 

Intestine small, duodenum + M + M +++++++++++++++++++++ 

Intestine small, jejunum +++++++++++++++++++++++++ 

Intestine small, ileum ++M++++++++++++++++++++++ 

Liver +++++++++++++++++++++++++ 

Hepatoblastoma XX XX XX X XXXXX X 

Hepatoblastoma, multiple X X X 

Hepatocellular carcinoma X X X X X X X 

Hepatocellular carcinoma, multiple X X X X XX XXXXX 

Hepatocellular adenoma X X X X 

Hepatocellular adenoma, multiple XX X XX XX XXX XX 

Mast cell tumor malignant X 

Mesentery + 

Pancreas +++++++++++++++++++++++++ 

Hepatoblastoma, metastatic, liver X 

Salivary glands +++++++++++++++++++++++++ 

Stomach, forestomach +++++++++++++++++++++++++ 

Stomach, glandular +++++++++++++++++++++++++ 

Cardiovascular System 

Blood vessel 

Heart 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

Endocrine System 

Adrenal cortex +++++++++++++++++++++++++ 

Capsule, adenoma 

Adrenal medulla +++++++++++++++++++++++++ 

Pheochromocytoma benign 

Islets, pancreatic +++++A +++++++++++++++++++ 

Parathyroid gland M+++M+++++M++M+++MM++++M+ 

Pituitary gland +++++++++++++++++++++++++ 

Thyroid gland +++++++++++++++++++++++++ 

Follicular cell, adenoma X 

General Body System 

None 

Genital System 

Epididymis +++++++++++++++++++++++++ 

Hepatoblastoma, metastatic, liver X 

Preputial gland ++M++++++++++++++M+++++++ 

Prostate +++++++++++++++++++++++++ 

Seminal vesicle +++++++++++++++++++++++++ 

Testes +++++++++++++++++++++++++ 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 161 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Primidone: 600 ppm 

7777777777777777777777777 

Number of Days on Study 2222222222222222222222222 

6666666666666677777777777 

1111111111111111111111111 Total 

Carcass ID Number 1111223333344501122223344 Tissues/ 

3567290124809034834683567 Tumors 

Alimentary System 

Esophagus +++++++++++++++++++++++++ 50 

Gallbladder ++++++++++++++++++++++++M 46 

Intestine large, colon +++++++++++++++++++++++++ 50 

Intestine large, rectum +++++++++++++++++++++++++ 50 

Intestine large, cecum +++++++++++++++++++++++++ 50 

Intestine small, duodenum +++++++++++++++++++++++++ 48 

Intestine small, jejunum +++++++++++++++++++++++++ 50 

Intestine small, ileum +++++++++++++++++++++++++ 49 

Liver +++++++++++++++++++++++++ 50 

Hepatoblastoma X XX XXX X X 21 

Hepatoblastoma, multiple X X 5 

Hepatocellular carcinoma X X X X X X 13 

Hepatocellular carcinoma, multiple X XXXX XX X XX X 22 

Hepatocellular adenoma X 5 

Hepatocellular adenoma, multiple X XXXXXXXXXXXXXXXXXX XXX 34 

Mast cell tumor malignant 1 

Mesentery 1 

Pancreas +++++++++++++++++++++++++ 50 

Hepatoblastoma, metastatic, liver 1 

Salivary glands +++++++++++++++++++++++++ 50 

Stomach, forestomach +++++++++++++++++++++++++ 50 

Stomach, glandular +++++++++++++++++++++++++ 50 

Cardiovascular System 

Blood vessel 

Heart 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

50 

Endocrine System 

Adrenal cortex +++++++++++++++++++++++++ 50 

Capsule, adenoma X 1 

Adrenal medulla +++++++++++++++++++++++++ 50 

Pheochromocytoma benign X 1 

Islets, pancreatic +++++++++++++++++++++++++ 49 

Parathyroid gland +++M+++++++M+++++M+++++++ 40 

Pituitary gland ++M++++++++++++M+++++++++ 48 

Thyroid gland +++++++++++++++++++++++++ 50 

Follicular cell, adenoma X X 3 

General Body System 

None 

Genital System 

Epididymis +++++++++++++++++++++++++ 50 

Hepatoblastoma, metastatic, liver 1 

Preputial gland +++++++++++++++++++++++++ 48 

Prostate +++++++++++++++++++++++++ 50 

Seminal vesicle +++++++++++++++++++++++++ 50 

Testes +++++++++++++++++++++++++ 50 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

162 Primidone, NTP TR 476 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Primidone: 600 ppm 

Number of Days on Study 

4555666666666666667777777 

2677011224445568890222222 

2658249153562871721666666 

Carcass ID Number 

1111111111111111111111111 

0442301244032424113000001 

4520799138665471219125780 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

+++++++++++++++++++++++++ 

+ M ++++M+++++++++++M++++++ 

+++++++++++++++M+++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++M++M+M++M+ 

Integumentary System 

Mammary gland 

Skin 

MMMMMMMMMMMMMMM+MMMMMMMMM 

+++++++++++++++++++++++++ 

Musculoskeletal System 

Bone 

Skeletal muscle

Hepatoblastoma, metastatic, liver

+++++++++++++++++++++++++ 

+ 

X 

Nervous System 

Brain +++++++++++++++++++++++++ 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma

Alveolar/bronchiolar carcinoma

Hepatoblastoma, metastatic, liver

Hepatocellular carcinoma, metastatic, liver

Mediastinum, hepatoblastoma, metastatic, liver

Nose 

Trachea 

+++++++++++++++++++++++++ 

X X 

X 

X X XX X X X 

X X 

X 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

Special Senses System 

Harderian gland

Adenoma

 + 

X 

Urinary System 

Kidney 

Hepatoblastoma, metastatic, liver

Urinary bladder 

+++++++++++++++++++++++++ 

X 

+++++++++++++++++++++++++ 

Systemic Lesions 

Multiple organs 

Lymphoma malignant

+++++++++++++++++++++++++ 

X 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 163 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Primidone: 600 ppm 

Number of Days on Study 

7777777777777777777777777 

2222222222222222222222222 

6666666666666677777777777 

Carcass ID Number 

1111111111111111111111111 

1111223333344501122223344 

3567290124809034834683567 

Total 

Tissues/ 

Tumors 

Hematopoietic System 

Bone marrow 

Lymph node

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

+++++++++++++++++++++++++ 

+ 

+++++++++++++++++M+++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

++M++++M++++++M+M++++++++ 

50 

1 

46 

49 

50 

42 

Integumentary System 

Mammary gland 

Skin 

MMMMMMMMMMMMMMMMMMMMMMMMM

+++++++++++++++++++++++++ 

1 

50 

Musculoskeletal System 

Bone 

Skeletal muscle 

Hepatoblastoma, metastatic, liver 

+++++++++++++++++++++++++ 50 

1 

1 

Nervous System 

Brain +++++++++++++++++++++++++ 50 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma

Alveolar/bronchiolar carcinoma 

Hepatoblastoma, metastatic, liver

Hepatocellular carcinoma, metastatic, liver

Mediastinum, hepatoblastoma, metastatic, liver 

Nose 

Trachea 

+++++++++++++++++++++++++ 

X X X 

XXX X 

X X 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

5 

1 

11 

4 

1 

50 

50 

Special Senses System 

Harderian gland 

Adenoma 

1 

1 

Urinary System 

Kidney 

Hepatoblastoma, metastatic, liver 

Urinary bladder 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

1 

50 

Systemic Lesions 

Multiple organs 

Lymphoma malignant

+++++++++++++++++++++++++ 

X 

50 

2 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

164 Primidone, NTP TR 476 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Primidone: 1,300 ppm 

Number of Days on Study 

0344555556666666666666666 

2679155660122233345577799 

2802619256755527866844478 

Carcass ID Number 

1111111111111111111111111 

9877986759956775588588976 

1524523342891657241306474 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Histiocytic sarcoma

Intestine small, ileum 

Histiocytic sarcoma

Liver 

Hepatoblastoma

Hepatoblastoma, multiple 

Hepatocellular carcinoma

Hepatocellular carcinoma, multiple

Hepatocellular adenoma

Hepatocellular adenoma, multiple

Histiocytic sarcoma

Mesentery

Fat, histiocytic sarcoma

Pancreas 

Salivary glands 

Stomach, forestomach 

Squamous cell carcinoma 

Squamous cell papilloma 

Stomach, glandular 

+++++++++++++++++++++++++ 

+++++M+++++++++++M++M++++ 

++++++++M++++++++++++++M+ 

++++++M+M++++++++++++++++ 

+++++M+++++++++++++++++++ 

M + M + + M + + + + + + + + + + + + + + + + + A + 

++++++M++++++++++++++++M+ 

X 

+++++++++++++++++++++++M+ 

X 

+++++++++++++++++++++++++ 

X X X X 

X XX X X X X X 

X X X X X X X 

 X X X X X 

XX X XX XXXX X 

X X 

+ 

X 

++M+++++M++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

Cardiovascular System 

Blood vessel +++++++++++++++++++++++++ 

Heart +++++++++++++++++++++++++ 

Endocrine System 

Adrenal cortex +++++++++++++++++++++++++ 

Histiocytic sarcoma X 

Adrenal medulla +++++++++++++++++++++++++ 

Islets, pancreatic ++M+++++M++++++++++++++++ 

Parathyroid gland M+++M+M++++++MM++M++ MMMMM 

Pituitary gland ++M++++++++++++++++++++I + 

Thyroid gland +++++++++++++++++++++++++ 

Follicular cell, adenoma X X 

General Body System 

None 

Genital System 

Epididymis +++++++++++++++++++++++++ 

Preputial gland +++++++++++++++++++++++++ 

Prostate +++++++++++++++++++++++++ 

Seminal vesicle +++++++++++++++++++++++++ 

Testes +++++++++++++++++++++++++ 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 165 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Primidone: 1,300 ppm 

Number of Days on Study 

6777777777777777777777777 

9001122222222222222222222 

9262606666666666667777777 

Carcass ID Number 

1111111111111111111111112 

8996595566667778895667890 

8679691825670893705081930 

Total 

Tissues/ 

Tumors 

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Gallbladder M + + + + + + + + + + + + M + + + + + + + + + + + 45 

Intestine large, colon A + + + + + + + + + + + + + + + + + + + + + + + + 47 

Intestine large, rectum A + + + + + + + + + + + + + + + + + + + + + + + + 47 

Intestine large, cecum A ++++++++++++++++++++++++ 48 

Intestine small, duodenum A ++++++++++++++++++++++++ 45 

Intestine small, jejunum A ++++++++++++++++++++++++ 47 

Histiocytic sarcoma 1 

Intestine small, ileum A + + + + + + + + + + + + + + + + + + + + + + + + 48 

Histiocytic sarcoma 1 

Liver + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Hepatoblastoma X X 6 

Hepatoblastoma, multiple X 1 

Hepatocellular carcinoma XXXX X X XX X X X 19 

Hepatocellular carcinoma, multiple X X X X X X X X X X X X 19 

Hepatocellular adenoma X X X X X 10 

Hepatocellular adenoma, multiple X X X X X X X X X X X X 22 

Histiocytic sarcoma X X 4 

Mesentery 1 

Fat, histiocytic sarcoma 1 

Pancreas A + + + + + + + + + + + + + + + + + + + + + + + + 47 

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Stomach, forestomach A + + + + + + + + + + + + + + + + + + + + + + + + 49 

Squamous cell carcinoma X 1 

Squamous cell papilloma X 1 

Stomach, glandular A ++++++++++++++++++++++++ 49 

Cardiovascular System 

Blood vessel 

Heart 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

50 

Endocrine System 

Adrenal cortex +++++++++++++++++++++++++ 50 

Histiocytic sarcoma 1 

Adrenal medulla +++++++++++++++++++++++++ 50 

Islets, pancreatic A ++++++++++++++++++++++++ 47 

Parathyroid gland M++M+++++++M+M++M++++++++ 34 

Pituitary gland I ++++++++++++++++++++++++ 47 

Thyroid gland +++++++++++++++++++++++++ 50 

Follicular cell, adenoma X X X X 6 

General Body System 

None 

Genital System 

Epididymis +++++++++++++++++++++++++ 50 

Preputial gland +++++++++++++++++++++++++ 50 

Prostate +++++++++++++++++++++++++ 50 

Seminal vesicle +++++++++++++++++++++++++ 50 

Testes +++++++++++++++++++++++++ 50 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

166 Primidone, NTP TR 476 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Primidone: 1,300 ppm 

0344555556666666666666666 

Number of Days on Study 2679155660122233345577799 

2802619256755527866844478 

1111111111111111111111111 

Carcass ID Number 9877986759956775588588976 

1524523342891657241306474 

Hematopoietic System 

Bone marrow 

Lymph node

Lymph node, mandibular 

Lymph node, mesenteric 

Histiocytic sarcoma 

Spleen 

Histiocytic sarcoma

Thymus 

+++++++++++++++++++++++++ 

+ + + 

M ++++M++M+++++++++M++++++ 

++++++M+M++++++++++++++M+ 

+++++++++++++++++++++++++ 

X 

++MM++M+MMM+++M++M++MM+M+ 

Integumentary System 

Mammary gland 

Skin 

Subcutaneous tissue, histiocytic sarcoma 

MMMMMMMMMMMMMMMMMMMMMMMMM 

+++++++++++++++++++++++++ 

Musculoskeletal System 

Bone +++++++++++++++++++++++++ 

Nervous System 

Brain +++++++++++++++++++++++++ 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma

Alveolar/bronchiolar adenoma, multiple 

Alveolar/bronchiolar carcinoma

Hepatoblastoma, metastatic, liver

Hepatocellular carcinoma, metastatic, liver

Histiocytic sarcoma

Sarcoma, metastatic, uncertain primary site

Nose 

Trachea 

+++++++++++++++++++++++++ 

X X X 

 X X 

XXX 

X X 

X 

X 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

Special Senses System 

Harderian gland

Adenoma 

+ 

Urinary System 

Kidney 

Histiocytic sarcoma

Renal tubule, carcinoma 

Urinary bladder 

+++++++++++++++++++++++++ 

X 

+++++++++++++++++++++++++ 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma

+++++++++++++++++++++++++ 

X X 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 167 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Primidone: 1,300 ppm 

6777777777777777777777777 

Number of Days on Study 9001122222222222222222222 

9262606666666666667777777 

1111111111111111111111112 Total 

Carcass ID Number 8996595566667778895667890 

8679691825670893705081930 

Tissues/ 

Tumors 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Histiocytic sarcoma

Spleen 

Histiocytic sarcoma

Thymus 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

A + M ++++++++M+++MM++++++++ 

X 

A + + + + + + + + + + + + + + + + + + + + + + + + 

X 

+++MMM+++++++++++M++++M++ 

50 

3 

46 

42 

1 

49 

2 

34 

Integumentary System 

Mammary gland 

Skin 

Subcutaneous tissue, histiocytic sarcoma

MMMMMMMMMMMMMMMMMMMMMMMMM 

+++++++++++++++++++++++++ 

X 

50 

1 

Musculoskeletal System 

Bone +++++++++++++++++++++++++ 50 

Nervous System 

Brain +++++++++++++++++++++++++ 50 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma

Alveolar/bronchiolar adenoma, multiple

Alveolar/bronchiolar carcinoma

Hepatoblastoma, metastatic, liver

Hepatocellular carcinoma, metastatic, liver

Histiocytic sarcoma 

Sarcoma, metastatic, uncertain primary site 

Nose 

Trachea 

+++++++++++++++++++++++++ 

X X 

X 

X 

X X 

X X X 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

5 

1 

3 

5 

5 

1 

1 

50 

50 

Special Senses System 

Harderian gland

Adenoma

 + 

X 

2 

1 

Urinary System 

Kidney 

Histiocytic sarcoma 

Renal tubule, carcinoma

Urinary bladder 

+++++++++++++++++++++++++ 

X 

+++++++++++++++++++++++++ 

50 

1 

1 

50 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma

+++++++++++++++++++++++++ 

 X X X 

50 

5 
168 Primidone, NTP TR 476 

TABLE C3 

Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Feed Study of Primidone 

0 ppm 300 ppm 600 ppm 1,300 ppm 

Harderian Gland: Adenoma or Carcinoma 

Overall ratea 

Adjusted rateb 2/50 (4%) 

5.7% 

3/50 (6%) 

7.6% 

1/50 (2%) 

3.2% 

1/50 (2%) 

5.3% 

Terminal ratec 2/35 (6%) 1/34 (3%) 1/31 (3%) 1/19 (5%) 

First incidence (days) 

Life table testd 726 (T) 

P=0.456N 

639 

P=0.501 

726 (T) 

P=0.543N 

726 (T) 

P=0.708N 

Logistic regression testd 

Cochran-Armitage testd 

Fisher exact testd 

P=0.332N 

P=0.288N 

P=0.499 

P=0.500 

P=0.543N 

P=0.500N 

P=0.708N 

P=0.500N 

Liver: Hemangiosarcoma 

Overall rate 2/50 (4%) 3/50 (6%) 0/50 (0%) 0/50 (0%) 

Adjusted rate 4.4% 8.8% 0.0% 0.0% 

Terminal rate 0/35 (0%) 3/34 (9%) 0/31 (0%) 0/19 (0%) 

First incidence (days) 552 726 (T) —e — 

Life table test P=0.121N P=0.511 P=0.226N P=0.252N 

Logistic regression test P=0.062N P=0.417 P=0.266N P=0.152N 

Cochran-Armitage test P=0.079N 

Fisher exact test P=0.500 P=0.247N P=0.247N 

Liver: Hepatocellular Adenoma 

Overall rate 22/50 (44%) 41/50 (82%) 39/50 (78%) 32/50 (64%) 

Adjusted rate 59.2% 93.1% 92.6% 87.3% 

Terminal rate 20/35 (57%) 31/34 (91%) 28/31 (90%) 15/19 (79%) 

First incidence (days) 551 603 566 368 

Life table test P<0.001 P<0.001 P<0.001 P<0.001 

Logistic regression test P=0.040 P<0.001 P<0.001 P=0.013 

Cochran-Armitage test P=0.155 

Fisher exact test P<0.001 P<0.001 P=0.035 

Liver: Hepatocellular Carcinoma 

Overall rate 12/50 (24%) 31/50 (62%) 35/50 (70%) 38/50 (76%) 

Adjusted rate 29.1% 71.6% 81.1% 100.0% 

Terminal rate 7/35 (20%) 22/34 (65%) 23/31 (74%) 19/19 (100%) 

First incidence (days) 526 614 575 470 

Life table test P<0.001 P<0.001 P<0.001 P<0.001 

Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001 

Cochran-Armitage test P<0.001 

Fisher exact test P<0.001 P<0.001 P<0.001 

Liver: Hepatocellular Adenoma or Carcinoma 

Overall rate 31/50 (62%) 48/50 (96%) 47/50 (94%) 46/50 (92%) 

Adjusted rate 73.5% 100.0% 100.0% 100.0% 

Terminal rate 24/35 (69%) 34/34 (100%) 31/31 (100%) 19/19 (100%) 

First incidence (days) 526 603 566 368 

Life table test P<0.001 P=0.002 P<0.001 P<0.001 

Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001 

Cochran-Armitage test P<0.001 

Fisher exact test P<0.001 P<0.001 P<0.001 
Primidone, NTP TR 476 169 

TABLE C3 

Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Feed Study of Primidone 

0 ppm 300 ppm 600 ppm 1,300 ppm 

Liver: Hepatoblastoma 

Overall rate 0/50 (0%) 17/50 (34%) 26/50 (52%) 7/50 (14%) 

Adjusted rate 0.0% 39.9% 56.9% 23.4% 

Terminal rate 0/35 (0%) 9/34 (26%) 12/31 (39%) 1/19 (5%) 

First incidence (days) — 617 422 551 

Life table test P=0.043 P<0.001 P<0.001 P=0.003 

Logistic regression test P=0.271 P<0.001 P<0.001 P=0.009 

Cochran-Armitage test P=0.254 

Fisher exact test P<0.001 P<0.001 P=0.006 

Liver: Hepatocellular Carcinoma or Hepatoblastoma 

Overall rate 12/50 (24%) 39/50 (78%) 40/50 (80%) 39/50 (78%) 

Adjusted rate 29.1% 81.3% 83.2% 100.0% 

Terminal rate 7/35 (20%) 25/34 (74%) 23/31 (74%) 19/19 (100%) 

First incidence (days) 526 614 422 470 

Life table test P<0.001 P<0.001 P<0.001 P<0.001 

Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001 

Cochran-Armitage test P<0.001 

Fisher exact test P<0.001 P<0.001 P<0.001 

Liver: Hepatocellular Adenoma, Hepatocellular Carcinoma, or Hepatoblastoma 

Overall rate 31/50 (62%) 49/50 (98%) 49/50 (98%) 46/50 (92%) 

Adjusted rate 73.5% 100.0% 100.0% 100.0% 

Terminal rate 24/35 (69%) 34/34 (100%) 31/31 (100%) 19/19 (100%) 

First incidence (days) 526 603 422 368 

Life table test P<0.001 P=0.001 P<0.001 P<0.001 

Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001 

Cochran-Armitage test P<0.001 

Fisher exact test P<0.001 P<0.001 P<0.001 

Lung: Alveolar/bronchiolar Adenoma 

Overall rate 7/50 (14%) 7/50 (14%) 5/50 (10%) 6/50 (12%) 

Adjusted rate 18.6% 19.4% 16.1% 20.7% 

Terminal rate 5/35 (14%) 6/34 (18%) 5/31 (16%) 1/19 (5%) 

First incidence (days) 583 617 726 (T) 368 

Life table test P=0.345 P=0.610 P=0.455N P=0.426 

Logistic regression test P=0.467N P=0.585N P=0.394N P=0.504N 

Cochran-Armitage test P=0.412N 

Fisher exact test P=0.613N P=0.380N P=0.500N 

Lung: Alveolar/bronchiolar Carcinoma 

Overall rate 4/50 (8%) 2/50 (4%) 1/50 (2%) 3/50 (6%) 

Adjusted rate 10.7% 5.9% 2.7% 10.3% 

Terminal rate 3/35 (9%) 2/34 (6%) 0/31 (0%) 1/19 (5%) 

First incidence (days) 578 726 (T) 658 625 

Life table test P=0.515 P=0.337N P=0.202N P=0.609 

Logistic regression test P=0.497N P=0.324N P=0.180N P=0.517N 

Cochran-Armitage test P=0.487N 

Fisher exact test P=0.339N P=0.181N P=0.500N 
170 Primidone, NTP TR 476 

TABLE C3 

Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Feed Study of Primidone 

0 ppm 300 ppm 600 ppm 1,300 ppm 

Lung: Alveolar/bronchiolar Adenoma or Carcinoma 

Overall rate 11/50 (22%) 9/50 (18%) 6/50 (12%) 9/50 (18%) 

Adjusted rate 28.4% 25.2% 18.4% 29.1% 

Terminal rate 8/35 (23%) 8/34 (24%) 5/31 (16%) 2/19 (11%) 

First incidence (days) 578 617 658 368 

Life table test P=0.322 P=0.404N P=0.200N P=0.411 

Logistic regression test P=0.415N P=0.367N P=0.144N P=0.413N 

Cochran-Armitage test P=0.364N 

Fisher exact test P=0.402N P=0.143N P=0.402N 

Thyroid Gland (Follicular Cell): Adenoma 

Overall rate 0/49 (0%) 3/48 (6%) 3/50 (6%) 6/50 (12%) 

Adjusted rate 0.0% 9.1% 9.7% 25.3% 

Terminal rate 0/35 (0%) 3/33 (9%) 3/31 (10%) 3/19 (16%) 

First incidence (days) — 726 (T) 726 (T) 674 

Life table test P<0.001 P=0.110 P=0.100 P=0.003 

Logistic regression test P=0.003 P=0.110 P=0.100 P=0.008 

Cochran-Armitage test P=0.016 

Fisher exact test P=0.117 P=0.125 P=0.014 

All Organs: Hemangiosarcoma 

Overall rate 3/50 (6%) 3/50 (6%) 0/50 (0%) 0/50 (0%) 

Adjusted rate 7.1% 8.8% 0.0% 0.0% 

Terminal rate 1/35 (3%) 3/34 (9%) 0/31 (0%) 0/19 (0%) 

First incidence (days) 552 726 (T) — — 

Life table test P=0.067N P=0.654N P=0.123N P=0.167N 

Logistic regression test P=0.033N P=0.616 P=0.131N P=0.085N 

Cochran-Armitage test P=0.039N 

Fisher exact test P=0.661N P=0.121N P=0.121N 

All Organs: Hemangioma or Hemangiosarcoma 

Overall rate 4/50 (8%) 3/50 (6%) 0/50 (0%) 0/50 (0%) 

Adjusted rate 9.9% 8.8% 0.0% 0.0% 

Terminal rate 2/35 (6%) 3/34 (9%) 0/31 (0%) 0/19 (0%) 

First incidence (days) 552 726 (T) — — 

Life table test P=0.037N P=0.495N P=0.070N P=0.114N 

Logistic regression test P=0.017N P=0.536N P=0.067N P=0.049N 

Cochran-Armitage test P=0.019N 

Fisher exact test P=0.500N P=0.059N P=0.059N 

All Organs: Histiocytic Sarcoma 

Overall rate 1/50 (2%) 0/50 (0%) 0/50 (0%) 5/50 (10%) 

Adjusted rate 2.9% 0.0% 0.0% 18.1% 

Terminal rate 1/35 (3%) 0/34 (0%) 0/31 (0%) 1/19 (5%) 

First incidence (days) 726 (T) — — 551 

Life table test P=0.001 P=0.506N P=0.524N P=0.040 

Logistic regression test P=0.008 P=0.506N P=0.524N P=0.101 

Cochran-Armitage test P=0.007 

Fisher exact test P=0.500N P=0.500N P=0.102 
Primidone, NTP TR 476 171 

TABLE C3 

Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Feed Study of Primidone 

0 ppm 300 ppm 600 ppm 1,300 ppm 

All Organs: Malignant Lymphoma 

Overall rate 7/50 (14%) 4/50 (8%) 2/50 (4%) 0/50 (0%) 

Adjusted rate 18.3% 10.6% 5.7% 0.0% 

Terminal rate 5/35 (14%) 3/34 (9%) 1/31 (3%) 0/19 (0%) 

First incidence (days) 432 603 646 — 

Life table test P=0.015N P=0.268N P=0.112N P=0.042N 

Logistic regression test P=0.004N P=0.278N P=0.082N P=0.010N 

Cochran-Armitage test P=0.004N 

Fisher exact test P=0.262N P=0.080N P=0.006N 

All Organs: Benign Neoplasms 

Overall rate 26/50 (52%) 42/50 (84%) 39/50 (78%) 36/50 (72%) 

Adjusted rate 68.1% 93.2% 92.6% 91.5% 

Terminal rate 23/35 (66%) 31/34 (91%) 28/31 (90%) 16/19 (84%) 

First incidence (days) 551 603 566 368 

Life table test P<0.001 P=0.001 P=0.002 P<0.001 

Logistic regression test P=0.023 P=0.001 P=0.005 P=0.010 

Cochran-Armitage test P=0.109 

Fisher exact test P<0.001 P=0.006 P=0.032 

All Organs: Malignant Neoplasms 

Overall rate 22/50 (44%) 42/50 (84%) 42/50 (84%) 42/50 (84%) 

Adjusted rate 49.3% 85.7% 85.7% 100.0% 

Terminal rate 13/35 (37%) 27/34 (79%) 24/31 (77%) 19/19 (100%) 

First incidence (days) 432 603 422 470 

Life table test P<0.001 P=0.002 P<0.001 P<0.001 

Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001 

Cochran-Armitage test P<0.001 

Fisher exact test P<0.001 P<0.001 P<0.001 

All Organs: Benign or Malignant Neoplasms 

Overall rate 40/50 (80%) 50/50 (100%) 50/50 (100%) 47/50 (94%) 

Adjusted rate 86.8% 100.0% 100.0% 100.0% 

Terminal rate 29/35 (83%) 34/34 (100%) 31/31 (100%) 19/19 (100%) 

First incidence (days) 432 603 422 368 

Life table test P<0.001 P=0.058 P=0.017 P<0.001 

Logistic regression test P=0.007 P=0.002 P=0.001 P=0.010 

Cochran-Armitage test P=0.042 

Fisher exact test P<0.001 P<0.001 P=0.036 

(T)Terminal sacrifice 

a Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for liver, lung, and 

thyroid gland; for other tissues, denominator is number of animals necropsied. b	 Kaplan-Meier estimated neoplasm incidence after adjustment for intercurrent mortality 

Observed incidence at terminal kill 

d	 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 

c 
172 Primidone, NTP TR 476 

TABLE C4a 

Historical Incidence of Liver Neoplasms in Untreated Male B6C3F1 Micea 

Incidence in Controls 

Study Hepatocellular Hepatocellular Hepatoblastoma Hepatocellular Adenoma, 

Adenoma Carcinoma Hepatocellular Carcinoma,

 or Hepatoblastoma 

Historical Incidence at Battelle Columbus Laboratories 

4,4N-Thiobis(6-t-butyl-m-cresol) 17/50 11/50 0/50 25/50 

5,5-Diphenylhydantoin 19/50 13/50 0/50 29/50 

Pentachlorophenol (Dowicide EC-7) 5/35 1/35 0/35 6/35 

Ethylene Thiourea 11/49 13/49 0/49 20/49 

Polybrominated Biphenyls (Firemaster FF-1®) 9/50 8/50 0/50 16/50 

Manganese (II) Sulfate Monohydrate 30/50 9/50 0/50 34/50 

Oxazepam 17/49 9/49 0/49 23/49 

Pentachlorophenol (Technical Grade) 5/32 2/32 0/32 7/32 

Triamterene 17/50 5/50 0/50 20/50 

Triamterene 21/50 9/50 0/50 25/50 

Tricresyl Phosphate 18/52 15/52 0/52 28/52 

Overall Historical Incidence 

Total 413/1,465 (28.2%) 252/1,465 (17.2%) 0/1,465 (0%) 596/1,465 (40.7%) 

Standard deviation 14.2% 7.1% 14.5% 

Range 4%-60% 3%-29% 10%-68% 

a Data as of 12 May 1995 
Primidone NTP TR 476 173 

TABLE C4b 

Historical Incidence of Thyroid Gland Follicular Cell Adenoma in Untreated Male B6C3F1 Micea 

Study Incidence in Controls 

Historical Incidence at Battelle Columbus Laboratories 

4,4N-Thiobis(6-t-butyl-m-cresol) 0/50 

5,5-Diphenylhydantoin 0/49 

Pentachlorophenol (Dowicide EC-7) 0/35 

Ethylene Thiourea 0/50 

Polybrominated Biphenyls (Firemaster FF-1®) 1/50 

Manganese (II) Sulfate Monohydrate 0/50 

Oxazepam 0/49 

Pentachlorophenol (Technical Grade) 1/31 

Triamterene 0/50 

Triamterene 1/50 

Tricresyl Phosphate 0/52 

Overall Historical Incidence 

Total 22/1,455 (1.5%) 

Standard deviation 1.5% 

Range 0%-4% 

a Data as of 12 May 1995 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

174 Primidone, NTP TR 476 

TABLE C5 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Feed Study of Primidonea 

0 ppm 300 ppm 600 ppm 1,300 ppm 

Disposition Summary 

Animals initially in study 50 50 50 50 

Early deaths 

Moribund 6 7 6 17 

Natural deaths 9 9 13 14 

Survivors 

Died last week of study 1 

Terminal sacrifice 35 33 31 19 

Animals examined microscopically 50 50 50 50 

Alimentary System 

Intestine small, duodenum (44) (48) (48) (45) 

Necrosis, focal 1 (2%) 

Ulcer 1 (2%) 1 (2%) 

Intestine small, jejunum (46) (49) (50) (47) 

Peyer’s patch, hyperplasia, lymphoid 1 (2%) 

Intestine small, ileum (42) (47) (49) (48) 

Congestion 1 (2%) 

Liver (50) (50) (50) (50) 

Angiectasis 2 (4%) 1 (2%) 

Basophilic focus 2 (4%) 1 (2%) 

Clear cell focus 11 (22%) 7 (14%) 8 (16%) 1 (2%) 

Clear cell focus, multiple 6 (12%) 4 (8%) 2 (4%) 

Eosinophilic focus 7 (14%) 4 (8%) 8 (16%) 5 (10%) 

Hematopoietic cell proliferation 1 (2%) 1 (2%) 2 (4%) 

Inflammation, chronic 2 (4%) 1 (2%) 

Mixed cell focus 1 (2%) 1 (2%) 1 (2%) 

Necrosis 6 (12%) 7 (14%) 6 (12%) 6 (12%) 

Thrombosis, acute 1 (2%) 

Centrilobular, hypertrophy 3 (6%) 30 (60%) 21 (42%) 18 (36%) 

Hepatocyte, vacuolization cytoplasmic 1 (2%) 8 (16%) 3 (6%) 2 (4%) 

Mesentery (1) (2) (1) (1) 

Fat, inflammation, chronic 1 (100%) 1 (50%) 1 (100%) 1 (100%) 

Pancreas (46) (48) (50) (47) 

Duct, cyst 1 (2%) 1 (2%) 1 (2%) 1 (2%) 

Stomach, forestomach (50) (49) (50) (49) 

Hyperplasia, diffuse, squamous 2 (4%) 

Hyperplasia, focal, squamous 6 (12%) 9 (18%) 9 (18%) 3 (6%) 

Stomach, glandular (50) (49) (50) (49) 

Epithelium, hyperplasia, focal 1 (2%) 

Cardiovascular System 

Heart (50) (50) (50) (50) 

Infiltration cellular, focal, lymphocyte 1 (2%) 

Inflammation, chronic active 1 (2%) 

Mineralization, diffuse 1 (2%) 

a Number of animals examined microscopically at the site and the number of animals with lesion 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 175 

TABLE C5 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Feed Study of Primidone 

0 ppm 300 ppm 600 ppm 1,300 ppm 

Endocrine System 

Adrenal cortex (50) (49) (50) (50) 

Hyperplasia, focal 2 (4%) 

Hypertrophy, focal 2 (4%) 2 (4%) 2 (4%) 1 (2%) 

Necrosis 1 (2%) 

Capsule, accessory adrenal cortical nodule 1 (2%) 

Capsule, hyperplasia 3 (6%) 4 (8%) 2 (4%) 

Islets, pancreatic (45) (48) (49) (47) 

Hyperplasia 32 (71%) 34 (71%) 18 (37%) 4 (9%) 

Pituitary gland (45) (46) (48) (47) 

Pars distalis, cyst 2 (4%) 1 (2%) 

Thyroid gland (49) (48) (50) (50) 

Follicle, cyst 1 (2%) 1 (2%) 

Follicular cell, hyperplasia 8 (16%) 20 (42%) 31 (62%) 42 (84%) 

General Body System 

None 

Genital System 

Epididymis (50) (50) (50) (50) 

Granuloma sperm, focal 1 (2%) 

Spermatocele, focal 1 (2%) 

Preputial gland (50) (50) (48) (50) 

Inflammation, chronic active 1 (2%) 

Duct, cyst 23 (46%) 32 (64%) 26 (54%) 30 (60%) 

Prostate (49) (50) (50) (50) 

Inflammation, chronic active 1 (2%) 

Testes (50) (50) (50) (50) 

Germinal epithelium, atrophy 1 (2%) 1 (2%) 

Hematopoietic System 

Bone marrow (50) (50) (50) (50) 

Myeloid cell, hyperplasia 2 (4%) 1 (2%) 1 (2%) 

Lymph node (1) (4) (1) (3) 

Hyperplasia, lymphoid 1 (33%) 

Inflammation, chronic 1 (33%) 

Mediastinal, hyperplasia, lymphoid 1 (25%) 

Renal, hematopoietic cell proliferation 1 (33%) 

Renal, hyperplasia, lymphoid 1 (100%) 

Lymph node, mandibular (39) (43) (46) (46) 

Hyperplasia, lymphoid 1 (2%) 

Infiltration cellular, plasma cell 1 (2%) 

Lymph node, mesenteric (48) (48) (49) (42) 

Angiectasis 1 (2%) 

Congestion 1 (2%) 

Hematopoietic cell proliferation 1 (2%) 1 (2%) 2 (5%) 

Hyperplasia, lymphoid 1 (2%) 

Spleen (48) (49) (50) (49) 

Atrophy 2 (4%) 3 (6%) 4 (8%) 

Hematopoietic cell proliferation 14 (29%) 15 (31%) 26 (52%) 37 (76%) 

Infiltration cellular, plasma cell 1 (2%) 

Capsule, fibrosis 1 (2%) 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

176 Primidone, NTP TR 476 

TABLE C5 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Feed Study of Primidone 

0 ppm 300 ppm 600 ppm 1,300 ppm 

Hematopoietic System (continued) 

Thymus 

Atrophy

Hyperplasia, lymphoid

(42) 

 39 (93%)

(40) 

 36 (90%)

 1 (3%)

(42) 

 35 (83%)

 1 (2%) 

(34) 

 29 (85%) 

Integumentary System 

Skin 

Hyperplasia, squamous

Inflammation, chronic active

Ulcer

Subcutaneous tissue, edema

(50) (50) 

 1 (2%)

(50) 

 1 (2%)

 1 (2%)

(50) 

 1 (2%) 

 2 (4%) 

 2 (4%) 

 1 (2%) 

Musculoskeletal System 

Bone 

Hyperostosis

(50) (50) 

 1 (2%) 

(50) (50) 

Nervous System 

None 

Respiratory System 

Lung (50) (50) (50) (50) 

Infiltration cellular, focal, mast cell 1 (2%) 

Alveolar epithelium, hyperplasia, focal 4 (8%) 6 (12%) 3 (6%) 3 (6%) 

Perivascular, inflammation, chronic 1 (2%) 

Special Senses System 

None 

Urinary System 

Kidney (50) (49) (50) (50) 

Hydronephrosis 1 (2%) 1 (2%) 

Infiltration cellular, focal, mast cell 1 (2%) 

Inflammation, acute 1 (2%) 

Nephropathy 34 (68%) 30 (61%) 25 (50%) 20 (40%) 

Cortex, cyst 1 (2%) 1 (2%) 

Cortex, infarct, focal 1 (2%) 

Renal tubule, pigmentation, hemosiderin 7 (14%) 8 (16%) 2 (4%) 
177 

APPENDIX D 

SUMMARY OF LESIONS IN FEMALE MICE 

IN THE 2-YEAR FEED STUDY 

OF PRIMIDONE 

TABLE D1 Summary of the Incidence of Neoplasms in Female Mice 

in the 2-Year Feed Study of Primidone ................................. 179 

TABLE D2 Individual Animal Tumor Pathology of Female Mice 

in the 2-Year Feed Study of Primidone ................................. 182 

TABLE D3 Statistical Analysis of Primary Neoplasms in Female Mice 

in the 2-Year Feed Study of Primidone ................................. 198 

TABLE D4 Historical Incidence of Liver Neoplasms in Untreated Female B6C3F1 Mice . . . . . . . . . 203 

TABLE D5 Summary of the Incidence of Nonneoplastic Lesions in Female Mice 

in the 2-Year Feed Study of Primidone ................................. 204 
178 Primidone, NTP TR 476
 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 179 

TABLE D1 

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Feed Study of Primidonea 

0 ppm 300 ppm 600 ppm 1,300 ppm 

Disposition Summary 

Animals initially in study 50 50 50 50 

Early deaths 

Moribund 7 1 3 8 

Natural deaths 2 7 2 3 

Survivors 

Terminal sacrifice 41 42 44 39 

Missing 1 

Animals examined microscopically 50 50 49 50 

Alimentary System 

Esophagus (50) (50) (49) (50) 

Squamous cell carcinoma 1 (2%) 

Intestine small, jejunum (50) (49) (49) (50) 

Intestine small, ileum (45) (49) (49) (49) 

Liver (50) (50) (49) (50) 

Fibrosarcoma, metastatic, skin 1 (2%) 

Hemangiosarcoma 1 (2%) 

Hepatoblastoma 1 (2%) 3 (6%) 3 (6%) 4 (8%) 

Hepatoblastoma, multiple 1 (2%) 1 (2%) 

Hepatocellular carcinoma 3 (6%) 7 (14%) 14 (29%) 13 (26%) 

Hepatocellular carcinoma, multiple 4 (8%) 5 (10%) 25 (50%) 

Hepatocellular adenoma 8 (16%) 5 (10%) 6 (12%) 5 (10%) 

Hepatocellular adenoma, multiple 7 (14%) 37 (74%) 39 (80%) 42 (84%) 

Histiocytic sarcoma 2 (4%) 1 (2%) 

Mesentery (5) (5) (6) (2) 

Histiocytic sarcoma 1 (17%) 

Pancreas (50) (48) (49) (50) 

Salivary glands (50) (48) (49) (50) 

Cardiovascular System 

None 

Endocrine System 

Adrenal cortex (50) (49) (49) (50) 

Capsule, adenoma 1 (2%) 

Islets, pancreatic (50) (47) (49) (50) 

Adenoma 1 (2%) 1 (2%) 

Pituitary gland (49) (48) (49) (48) 

Histiocytic sarcoma 1 (2%) 

Pars distalis, adenoma 3 (6%) 5 (10%) 6 (12%) 6 (13%) 

Pars intermedia, adenoma 1 (2%) 

Thyroid gland (50) (48) (48) (50) 

Follicular cell, adenoma 1 (2%) 1 (2%) 2 (4%) 

General Body System 

None 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

180 Primidone, NTP TR 476 

TABLE D1 

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Feed Study of Primidone 

0 ppm 300 ppm 600 ppm 1,300 ppm 

Genital System 

Ovary (49) (49) (48) (50) 

Cystadenoma 3 (6%) 2 (4%) 1 (2%) 

Hemangiosarcoma 1 (2%) 

Histiocytic sarcoma 1 (2%) 1 (2%) 

Uterus (50) (49) (49) (50) 

Histiocytic sarcoma 1 (2%) 1 (2%) 

Polyp stromal 1 (2%) 3 (6%) 1 (2%) 1 (2%) 

Sarcoma stromal 1 (2%) 

Cervix, histiocytic sarcoma 1 (2%) 

Hematopoietic System 

Bone marrow (50) (49) (49) (50) 

Hemangioma 1 (2%) 

Histiocytic sarcoma 1 (2%) 

Lymph node (6) (8) (7) (4) 

Deep cervical, alveolar/bronchiolar

 carcinoma, metastatic, lung 1 (13%) 

Lumbar, histiocytic sarcoma 1 (13%) 

Renal, histiocytic sarcoma 1 (13%) 

Lymph node, mandibular (48) (44) (45) (46) 

Histiocytic sarcoma 1 (2%) 1 (2%) 

Lymph node, mesenteric (48) (48) (47) (47) 

Histiocytic sarcoma 1 (2%) 1 (2%) 

Spleen (50) (48) (49) (50) 

Hemangiosarcoma 1 (2%) 

Histiocytic sarcoma 1 (2%) 1 (2%) 

Thymus (45) (47) (44) (44) 

Fibrosarcoma, metastatic, skin 1 (2%) 

Histiocytic sarcoma 1 (2%) 

Integumentary System 

Mammary gland (50) (50) (49) (47) 

Carcinoma 1 (2%) 

Skin (50) (50) (49) (50) 

Subcutaneous tissue, fibrosarcoma 3 (6%) 2 (4%) 1 (2%) 2 (4%) 

Subcutaneous tissue, hemangiosarcoma 1 (2%) 1 (2%) 

Musculoskeletal System 

Bone (50) (50) (49) (50) 

Maxilla, carcinoma, metastatic, harderian

 gland 1 (2%) 

Nervous System 

None 
 

 

 

 

 

 

 

 

 

 

181 

c 

Primidone, NTP TR 476 

TABLE D1 

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Feed Study of Primidone 

0 ppm 300 ppm 600 ppm 1,300 ppm 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma

Alveolar/bronchiolar adenoma, multiple

Alveolar/bronchiolar carcinoma

Fibrosarcoma, metastatic, skin

Hepatoblastoma, metastatic, liver

Hepatocellular carcinoma, metastatic, liver

Histiocytic sarcoma

(50) 

 4 (8%)

 4 (8%)

 1 (2%)

 1 (2%) 

(50) 

 2 (4%)

 3 (6%)

 1 (2%)

 1 (2%) 

(49) 

 3 (6%)

 1 (2%) 

 1 (2%)

(50) 

 2 (4%) 

 1 (2%) 

 1 (2%) 

 1 (2%) 

Special Senses System 

Harderian gland

Adenoma

Carcinoma

 (5)

 3 (60%)

 2 (40%) 

 (5)

 5 (100%)

 (3)

 3 (100%)

 (1) 

 1 (100%) 

Urinary System 

Kidney 

Urinary bladder 

Histiocytic sarcoma

(50) 

(50) 

(49) 

(49) 

(49) 

(48) 

 1 (2%) 

(50) 

(49) 

Systemic Lesions 

Multiple organsb 

Histiocytic sarcoma

Lymphoma malignant

(50) 

 8 (16%)

(50) 

 2 (4%)

 6 (12%)

(49) 

 1 (2%) 

 8 (16%)

(50) 

 5 (10%) 

Neoplasm Summary 

Total animals with primary neoplasmsc 

Total primary neoplasms 

Total animals with benign neoplasms 

Total benign neoplasms 

Total animals with malignant neoplasms 

Total malignant neoplasms 

Total animals with metastatic neoplasms 

Total metastatic neoplasms 

34 

57 

26 

32 

20 

25 

3 

3 

47 

89 

45 

59 

23 

30 

2 

2 

47 

97 

46 

62 

26 

35 

1 

3 

50 

111 

47 

61 

40 

50 

2 

2 

a Number of animals examined microscopically at the site and the number of animals with neoplasm b Number of animals with any tissue examined microscopically 

Primary neoplasms: all neoplasms except metastatic neoplasms 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

182 Primidone, NTP TR 476 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Primidone: 0 ppm 

4555666667777777777777777 

Number of Days on Study 2199224792222222222222222 

0956115347777888888888888 

2222222222222222222222222 

Carcass ID Number 0242011010223000111122333 

1310528768270269157918246 

Alimentary System 

Esophagus +++++++++++++++++++++++++ 

Gallbladder +++++++++++++++++++++++++ 

Intestine large, colon +++++++++++++++++++++++++ 

Intestine large, rectum +++++++++++++++++++++++++ 

Intestine large, cecum +++++++++++++++++++++++++ 

Intestine small, duodenum +++++++++++++++++++++++++ 

Intestine small, jejunum +++++++++++++++++++++++++ 

Intestine small, ileum ++++M++++++++++++++++++++ 

Liver +++++++++++++++++++++++++ 

Hepatoblastoma 

Hepatocellular carcinoma X X 

Hepatocellular adenoma X X 

Hepatocellular adenoma, multiple X X X X 

Mesentery + ++ + + 

Pancreas +++++++++++++++++++++++++ 

Salivary glands +++++++++++++++++++++++++ 

Stomach, forestomach +++++++++++++++++++++++++ 

Stomach, glandular +++++++++++++++++++++++++ 

Cardiovascular System 

Blood vessel 

Heart 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

Endocrine System 

Adrenal cortex +++++++++++++++++++++++++ 

Adrenal medulla +++++++++++++++++++++++++ 

Islets, pancreatic +++++++++++++++++++++++++ 

Adenoma 

Parathyroid gland +++++++++++++M++++++++++M 

Pituitary gland ++++++++++++M++++++++++++ 

Pars distalis, adenoma X 

Thyroid gland +++++++++++++++++++++++++ 

Follicular cell, adenoma 

General Body System 

None 

Genital System 

Clitoral gland MM+++++++++++++++++++++++ 

Ovary +++++++++++++++++++++++++ 

Cystadenoma X 

Uterus +++++++++++++++++++++++++ 

Polyp stromal 

Sarcoma stromal X 

+: Tissue examined microscopically M: Missing tissue X: Lesion present 

A: Autolysis precludes examination I: Insufficient tissue Blank: Not examined 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 183 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Primidone: 0 ppm 

Number of Days on Study 

7777777777777777777777777 

2222222222222222222222222 

8888889999999999999999999 

Carcass ID Number 

2222222222222222222222222 

3444440011122223333344445 

7034563403445691358927890 

Total 

Tissues/ 

Tumors 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatoblastoma

Hepatocellular carcinoma

Hepatocellular adenoma

Hepatocellular adenoma, multiple 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

+++++++++++++++++++++++++ 

+++++++++++M+++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++MM+MM+++ 

+++++++++++++++++++++++++ 

X 

X 

X X X X X X 

X X X 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

49 

50 

50 

50 

50 

50 

45 

50 

1 

3 

8 

7 

5 

50 

50 

50 

50 

Cardiovascular System 

Blood vessel + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Adenoma X 1 

Parathyroid gland + + + + M + + + + + + + + + + + + M + M + + + + + 45 

Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Pars distalis, adenoma X X 3 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Follicular cell, adenoma X 1 

General Body System 

None 

Genital System 

Clitoral gland + + + + + + + + + + + + + + + + + + + + + + + + M 47 

Ovary + M + + + + + + + + + + + + + + + + + + + + + + + 49 

Cystadenoma X X 3 

Uterus + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Polyp stromal X 1 

Sarcoma stromal 1 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

184 Primidone, NTP TR 476 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Primidone: 0 ppm 

4555666667777777777777777 

Number of Days on Study 2199224792222222222222222 

0956115347777888888888888 

2222222222222222222222222 

Carcass ID Number 0242011010223000111122333 

1310528768270269157918246 

Hematopoietic System 

Bone marrow 

Hemangioma 

Lymph node

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Hemangiosarcoma 

Thymus 

+++++++++++++++++++++++++ 

+ 

++++++++M++++++++++++++++ 

+ + M M +++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++M+ 

Integumentary System 

Mammary gland 

Carcinoma 

Skin 

Subcutaneous tissue, fibrosarcoma

Subcutaneous tissue, hemangiosarcoma

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

 X X 

X 

Musculoskeletal System 

Bone 

Maxilla, carcinoma, metastatic, 

harderian gland

+++++++++++++++++++++++++ 

X 

Nervous System 

Brain 

Peripheral nerve

Spinal cord

+++++++++++++++++++++++++ 

+ 

+ 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma

Alveolar/bronchiolar carcinoma

Fibrosarcoma, metastatic, skin

Hepatocellular carcinoma, metastatic, liver 

Nose 

Trachea 

+++++++++++++++++++++++++ 

X X 

X X 

X 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

Special Senses System 

Eye

Harderian gland

Adenoma

Carcinoma

 + 

+

 X

 +

 X 

+

 X

 + 

X 

Urinary System 

Kidney 

Urinary bladder 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

Systemic Lesions 

Multiple organs 

Lymphoma malignant

+++++++++++++++++++++++++ 

X X X 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 185 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Primidone: 0 ppm 

7777777777777777777777777 

Number of Days on Study 2222222222222222222222222 

8888889999999999999999999 

2222222222222222222222222 Total 

Carcass ID Number 3444440011122223333344445 

7034563403445691358927890 

Tissues/ 

Tumors 

Hematopoietic System 

Bone marrow 

Hemangioma

Lymph node

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Hemangiosarcoma

Thymus 

+++++++++++++++++++++++++ 

X 

+ + + + + 

++++++M++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

X 

+++++MM+++++++++M++M+++++ 

50 

1 

6 

48 

48 

50 

1 

45 

Integumentary System 

Mammary gland 

Carcinoma 

Skin 

Subcutaneous tissue, fibrosarcoma

Subcutaneous tissue, hemangiosarcoma 

+++++++++++++++++++++++++ 

X 

+++++++++++++++++++++++++ 

X 

50 

1 

50 

3 

1 

Musculoskeletal System 

Bone 

Maxilla, carcinoma, metastatic, 

harderian gland 

+++++++++++++++++++++++++ 50 

1 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

+++++++++++++++++++++++++ 50 

1 

1 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma

Alveolar/bronchiolar carcinoma

Fibrosarcoma, metastatic, skin 

Hepatocellular carcinoma, metastatic, liver

Nose 

Trachea 

+++++++++++++++++++++++++ 

X X 

 X X 

X 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

4 

4 

1 

1 

50 

50 

Special Senses System 

Eye 

Harderian gland

Adenoma

Carcinoma 

+ 

X 

1 

5 

3 

2 

Urinary System 

Kidney 

Urinary bladder 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

50 

Systemic Lesions 

Multiple organs 

Lymphoma malignant

+++++++++++++++++++++++++ 

X X X X X 

50 

8 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

186 Primidone, NTP TR 476 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Primidone: 300 ppm 

6667777777777777777777777 

Number of Days on Study 1790122222222222222222222 

3301804677777777777777788 

2222222222222222222222222 

Carcass ID Number 7865698655566777889999955 

1447737823903457260257815 

Alimentary System 

Esophagus +++++++++++++++++++++++++ 

Gallbladder + + + + A + + + + + + + + + + + + + + + + + + + + 

Intestine large, colon ++++A ++++++++++++++++++++ 

Intestine large, rectum ++++A ++++++++++++++++++++ 

Intestine large, cecum + + + + A + + + + + + + + + + + + + + + + + + + + 

Intestine small, duodenum + + + + A + + + + + + + + + + + + + + + + + + + + 

Intestine small, jejunum + + + + A + + + + + + + + + + + + + + + + + + + + 

Intestine small, ileum ++++A ++++++++++++++++++++ 

Liver +++++++++++++++++++++++++ 

Hepatoblastoma X 

Hepatoblastoma, multiple X 

Hepatocellular carcinoma X X X X 

Hepatocellular carcinoma, multiple X 

Hepatocellular adenoma X X X 

Hepatocellular adenoma, multiple X XXXXXXXXXX XXX XXX 

Histiocytic sarcoma X X 

Mesentery + + + 

Pancreas + + + + A + M + + + + + + + + + + + + + + + + + + 

Salivary glands +M++A ++++++++++++++++++++ 

Stomach, forestomach ++++A ++++++++++++++++++++ 

Stomach, glandular + + + + A + + + + + + + + + + + + + + + + + + + + 

Cardiovascular System 

Blood vessel 

Heart 

+ + + + A + + + + + + + + + + + + + + + + + + + + 

+++++++++++++++++++++++++ 

Endocrine System 

Adrenal cortex ++++A ++++++++++++++++++++ 

Capsule, adenoma 

Adrenal medulla ++++A ++++++++++++++++++++ 

Islets, pancreatic +A ++A +M++++++++++++++++++ 

Parathyroid gland +M++M+++M++++++++++M+++++ 

Pituitary gland ++++A +++++M++++++++++++++ 

Histiocytic sarcoma X 

Pars distalis, adenoma X X X 

Thyroid gland +A ++A ++++++++++++++++++++ 

Follicular cell, adenoma 

General Body System 

None 

Genital System 

Clitoral gland M+MMA M+++++++++++++++++++ 

Ovary + + + + A + + + + + + + + + + + + + + + + + + + + 

Hemangiosarcoma 

Histiocytic sarcoma X 

Uterus + + + + A + + + + + + + + + + + + + + + + + + + + 

Histiocytic sarcoma X 

Polyp stromal X X 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 187 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Primidone: 300 ppm 

7777777777777777777777777 

Number of Days on Study 2222222222222222222222222 

8888888888888899999999999 

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 Total 

Carcass ID Number 5 6 6 6 6 7 7 8 8 8 8 8 9 9 5 5 6 7 7 7 7 8 9 9 0 Tissues/ 

6 2 5 6 9 0 9 0 1 3 5 8 1 9 4 8 1 2 3 6 8 9 4 6 0 Tumors 

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Gallbladder + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Liver + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Hepatoblastoma X X 3 

Hepatoblastoma, multiple 1 

Hepatocellular carcinoma X X X 7 

Hepatocellular carcinoma, multiple X X X 4 

Hepatocellular adenoma X X 5 

Hepatocellular adenoma, multiple X X X X X X X X X X X X X X X X X X X X 37 

Histiocytic sarcoma 2 

Mesentery + + 5 

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 48 

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 48 

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Cardiovascular System 

Blood vessel + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Capsule, adenoma X 1 

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + 47 

Parathyroid gland + + + + + + + M + + + + + + + + + + + M + + + + + 44 

Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + 48 

Histiocytic sarcoma 1 

Pars distalis, adenoma X X 5 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 48 

Follicular cell, adenoma X 1 

General Body System 

None 

Genital System 

Clitoral gland + + + + + + + + + + + + + + + + + + + + + + + + + 45 

Ovary + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Hemangiosarcoma X 1 

Histiocytic sarcoma 1 

Uterus + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Histiocytic sarcoma 1 

Polyp stromal X 3 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

188 Primidone, NTP TR 476 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Primidone: 300 ppm 

6667777777777777777777777 

Number of Days on Study 1790122222222222222222222 

3301804677777777777777788 

2222222222222222222222222 

Carcass ID Number 7865698655566777889999955 

1447737823903457260257815 

Hematopoietic System 

Bone marrow 

Histiocytic sarcoma

Lymph node 

Deep cervical, alveolar/bronchiolar 

carcinoma, metastatic, lung

Lumbar, histiocytic sarcoma

Renal, histiocytic sarcoma

Lymph node, mandibular 

Histiocytic sarcoma

Lymph node, mesenteric 

Histiocytic sarcoma

Spleen 

Histiocytic sarcoma

Thymus 

Histiocytic sarcoma

++++A ++++++++++++++++++++ 

X 

+++ + + 

X 

X 

X 

+ M + + A +++MM++M+++++++++M++ 

X 

+ M + + A ++++++++++++++++++++ 

X 

+ A + + A + + + + + + + + + + + + + + + + + + + + 

X 

+ + + + A + + + + + + + + + + + M + + + + + + + + 

X 

Integumentary System 

Mammary gland 

Skin 

Subcutaneous tissue, fibrosarcoma 

Subcutaneous tissue, hemangiosarcoma

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

X 

Musculoskeletal System 

Bone +++++++++++++++++++++++++ 

Nervous System 

Brain + + + + A + + + + + + + + + + + + + + + + + + + + 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma

Alveolar/bronchiolar carcinoma

Hepatoblastoma, metastatic, liver

Histiocytic sarcoma

Nose 

Trachea 

+++++++++++++++++++++++++ 

X 

X X 

X 

X 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

Special Senses System 

Harderian gland

Adenoma

 ++ 

 X X 

Urinary System 

Kidney 

Urinary bladder 

+ + + + A + + + + + + + + + + + + + + + + + + + + 

++++A ++++++++++++++++++++ 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma

Lymphoma malignant

+++++++++++++++++++++++++ 

X X 

 X X X 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 189 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Primidone: 300 ppm 

7777777777777777777777777 

Number of Days on Study 2222222222222222222222222 

8888888888888899999999999 

2222222222222222222222223 Total 

Carcass ID Number 5666677888889955677778990 

6256909013581948123689460 

Tissues/ 

Tumors 

Hematopoietic System 

Bone marrow +++++++++++++++++++++++++ 49 

Histiocytic sarcoma 

Lymph node

Deep cervical, alveolar/bronchiolar 

carcinoma, metastatic, lung 

Lumbar, histiocytic sarcoma 

Renal, histiocytic sarcoma 

Lymph node, mandibular 

Histiocytic sarcoma 

Lymph node, mesenteric 

Histiocytic sarcoma 

Spleen 

Histiocytic sarcoma 

Thymus 

Histiocytic sarcoma 

+ + + 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++M+++++++++++++ 

1 

8 

1 

1 

1 

44 

1 

48 

1 

48 

1 

47 

1 

Integumentary System 

Mammary gland 

Skin 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

50 

Subcutaneous tissue, fibrosarcoma

Subcutaneous tissue, hemangiosarcoma 

X X 2 

1 

Musculoskeletal System 

Bone +++++++++++++++++++++++++ 50 

Nervous System 

Brain +++++++++++++++++++++++++ 49 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma

Alveolar/bronchiolar carcinoma

Hepatoblastoma, metastatic, liver 

Histiocytic sarcoma 

Nose 

+++++++++++++++++++++++++ 

X 

X 

+++++++++++++++++++++++++ 

50 

2 

3 

1 

1 

50 

Trachea +++++++++++++++++++++++++ 50 

Special Senses System 

Harderian gland

Adenoma

 +

 X

 +

 X

 + 

X 

5 

5 

Urinary System 

Kidney 

Urinary bladder 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

49 

49 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant

+++++++++++++++++++++++++ 

X X X 

50 

2 

6 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

190 Primidone, NTP TR 476 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Primidone: 600 ppm 

Number of Days on Study 

466667777777777777777777 

978992222222222222222222 

870497777777777777777777 

Carcass ID Number 

333333333333333333333333 

420110000111222333344445 

693672569345345248934570 

Alimentary System 

Esophagus 

Squamous cell carcinoma

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Fibrosarcoma, metastatic, skin

Hemangiosarcoma

Hepatoblastoma

Hepatoblastoma, multiple 

Hepatocellular carcinoma

Hepatocellular carcinoma, multiple

Hepatocellular adenoma

Hepatocellular adenoma, multiple

Histiocytic sarcoma

Mesentery 

Histiocytic sarcoma

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

++++++++++++++++++++++++ 

X 

++++++++++++++++++++++++ 

++++++++++++++++++++++++ 

++++++M+++++++++++++++++ 

++++++++++++++++++++++++ 

++++++++++++++++++++++++ 

++++++++++++++++++++++++ 

++++++++++++++++++++++++ 

++++++++++++++++++++++++ 

X 

X 

X X 

XX XXXX X X 

X 

X X X 

X X X XXXXXXXXXXXXXX 

X 

+ + + + 

X 

++++++++++++++++++++++++ 

++++++++++++++++++++++++ 

++++++++++++++++++++++++ 

++++++++++++++++++++++++ 

Cardiovascular System 

Blood vessel ++++++++++++++++++++++++ 

Heart ++++++++++++++++++++++++ 

Endocrine System 

Adrenal cortex ++++++++++++++++++++++++ 

Adrenal medulla ++++++++++++++++++++++++ 

Islets, pancreatic ++++++++++++++++++++++++ 

Adenoma 

Parathyroid gland ++MMM++MM+++++++++++++++ 

Pituitary gland ++++++++++++++++++++++++ 

Pars distalis, adenoma X X X 

Thyroid gland ++++++++++++++++++++++++ 

General Body System 

None 

Genital System 

Clitoral gland +M++++++++++++++++++++++ 

Ovary M+++++++++++++++++++++++ 

Cystadenoma X 

Histiocytic sarcoma X 

Uterus ++++++++++++++++++++++++ 

Histiocytic sarcoma X 

Polyp stromal 

Cervix, histiocytic sarcoma X 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 191 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Primidone: 600 ppm 

7777777777777777777777777 

Number of Days on Study 2222222222222222222222222 

8888888888889999999999999 

3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Total 

Carcass ID Number 1 1 1 2 2 2 2 2 3 3 4 4 0 0 0 0 1 1 2 3 3 3 4 4 4 Tissues/ 

1 2 8 0 1 6 7 8 0 5 2 8 1 4 7 8 0 9 2 1 3 6 0 1 9 Tumors 

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Squamous cell carcinoma 1 

Gallbladder + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Intestine large, rectum + + + + + + + + + + + + M + + + + + + + + + + + + 47 

Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Liver + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Fibrosarcoma, metastatic, skin 1 

Hemangiosarcoma 1 

Hepatoblastoma X 3 

Hepatoblastoma, multiple X 1 

Hepatocellular carcinoma XX XXX X 14 

Hepatocellular carcinoma, multiple X X X X 5 

Hepatocellular adenoma X X X 6 

Hepatocellular adenoma, multiple X X X X X X X X X X X X X X X X X X X X X X 39 

Histiocytic sarcoma 1 

Mesentery + + 6 

Histiocytic sarcoma 1 

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Cardiovascular System 

Blood vessel + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Adenoma X 1 

Parathyroid gland + + + + M + + + + + + + + + + + + + M + + + + + + 42 

Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Pars distalis, adenoma X X X 6 

Thyroid gland + + + + M + + + + + + + + + + + + + + + + + + + + 48 

General Body System 

None 

Genital System 

Clitoral gland + + + + + + + + + + + + + + + + + + + + + + + + + 48 

Ovary + + + + + + + + + + + + + + + + + + + + + + + + + 48 

Cystadenoma X 2 

Histiocytic sarcoma 1 

Uterus + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Histiocytic sarcoma 1 

Polyp stromal X 1 

Cervix, histiocytic sarcoma 1 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

192 Primidone, NTP TR 476 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Primidone: 600 ppm 

Number of Days on Study 

466667777777777777777777 

978992222222222222222222 

870497777777777777777777 

Carcass ID Number 

333333333333333333333333 

420110000111222333344445 

693672569345345248934570 

Hematopoietic System 

Bone marrow 

Lymph node

Lymph node, mandibular 

Histiocytic sarcoma

Lymph node, mesenteric 

Histiocytic sarcoma

Spleen 

Histiocytic sarcoma

Thymus 

Fibrosarcoma, metastatic, skin

++++++++++++++++++++++++ 

+ + + 

M +++++M++++++M++M+++++++ 

X 

+ M ++++++++++++++++++M+++ 

X 

++++++++++++++++++++++++ 

X 

++++M+++++++++M+++++++++ 

X 

Integumentary System 

Mammary gland 

Skin 

Subcutaneous tissue, fibrosarcoma

++++++++++++++++++++++++ 

++++++++++++++++++++++++ 

X 

Musculoskeletal System 

Bone ++++++++++++++++++++++++ 

Nervous System 

Brain ++++++++++++++++++++++++ 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma

Alveolar/bronchiolar adenoma, multiple

Fibrosarcoma, metastatic, skin

Nose 

Pleura 

Trachea 

++++++++++++++++++++++++ 

X X 

X 

X 

++++++++++++++++++++++++ 

++++++++++++++++++++++++ 

Special Senses System 

Harderian gland 

Adenoma 

Urinary System 

Kidney 

Urinary bladder 

Histiocytic sarcoma

++++++++++++++++++++++++ 

++++M+++++++++++++++++++ 

X 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma

Lymphoma malignant

++++++++++++++++++++++++ 

X 

X X 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 193 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Primidone: 600 ppm 

Number of Days on Study 

7777777777777777777777777 

2222222222222222222222222 

8888888888889999999999999 

Carcass ID Number 

3333333333333333333333333 

1112222233440000112333444 

1280167805281478092136019 

Total 

Tissues/ 

Tumors 

Hematopoietic System 

Bone marrow 

Lymph node

Lymph node, mandibular 

Histiocytic sarcoma 

Lymph node, mesenteric 

Histiocytic sarcoma 

Spleen 

Histiocytic sarcoma 

Thymus 

Fibrosarcoma, metastatic, skin 

+++++++++++++++++++++++++ 

 + + + + 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

++++++M+++M++++++++++M+++ 

49 

7 

45 

1 

47 

1 

49 

1 

44 

1 

Integumentary System 

Mammary gland 

Skin 

Subcutaneous tissue, fibrosarcoma 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

49 

49 

1 

Musculoskeletal System 

Bone +++++++++++++++++++++++++ 49 

Nervous System 

Brain +++++++++++++++++++++++++ 49 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma

Alveolar/bronchiolar adenoma, multiple 

Fibrosarcoma, metastatic, skin 

Nose 

Pleura

Trachea 

+++++++++++++++++++++++++ 

X 

+++++++++++++++++++++++++ 

+ 

+++++++++++++++++++++++++ 

49 

3 

1 

1 

49 

1 

49 

Special Senses System 

Harderian gland

Adenoma

 +

 X

 +

 X

 + 

X 

3 

3 

Urinary System 

Kidney 

Urinary bladder 

Histiocytic sarcoma 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

49 

48 

1 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant

+++++++++++++++++++++++++ 

 X X X X X X 

49 

1 

8 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

194 Primidone, NTP TR 476 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Primidone: 1,300 ppm 

5566666666777777777777777 

Number of Days on Study 8933777779222222222222222 

3718333373177777777777777 

3333333333333333333333333 

Carcass ID Number 7887888969655555666667789 

2627178663512568234894841 

Alimentary System 

Esophagus +++++++++++++++++++++++++ 

Gallbladder ++++M++++++++++++M+++++++ 

Intestine large, colon +++++++++++++++++++++++++ 

Intestine large, rectum +++++++++++++++++++++++++ 

Intestine large, cecum +++++++++++++++++++++++++ 

Intestine small, duodenum +++++++++++++++++++++++++ 

Intestine small, jejunum +++++++++++++++++++++++++ 

Intestine small, ileum +++++++++++M+++++++++++++ 

Liver +++++++++++++++++++++++++ 

Hepatoblastoma X 

Hepatocellular carcinoma X XX X X XX X 

Hepatocellular carcinoma, multiple X X XXX XXXX 

Hepatocellular adenoma X X X X 

Hepatocellular adenoma, multiple XX XXX X XXXXXXXXX XXXX 

Mesentery + +
 

Pancreas +++++++++++++++++++++++++ 
 

Salivary glands +++++++++++++++++++++++++ 
 

Stomach, forestomach +++++++++++++++++++++++++ 
 

Stomach, glandular +++++++++++++++++++++++++ 
 

Cardiovascular System 

Blood vessel 

Heart 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

Endocrine System 

Adrenal cortex +++++++++++++++++++++++++ 

Adrenal medulla +++++++++++++++++++++++++ 

Islets, pancreatic +++++++++++++++++++++++++ 

Parathyroid gland +++++++++M++++M+M+++++++M 

Pituitary gland ++++++++M++++++++++I +++++ 

Pars distalis, adenoma X X 

Pars intermedia, adenoma X 

Thyroid gland +++++++++++++++++++++++++ 

Follicular cell, adenoma X 

General Body System 

None 

Genital System 

Clitoral gland +++++++++++++++++++++++++ 

Ovary +++++++++++++++++++++++++ 

Cystadenoma X 

Uterus +++++++++++++++++++++++++ 

Polyp stromal 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 195 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Primidone: 1,300 ppm 

7777777777777777777777777 

Number of Days on Study 2222222222222222222222222 

7778888888888999999999999 

3333333333334333333333333 Total 

Carcass ID Number 9995667778890555677788999 Tissues/ 

2599071563540347103909078 Tumors 

Alimentary System 

Esophagus +++++++++++++++++++++++++ 50 

Gallbladder +++++++++++++++++++++++++ 48 

Intestine large, colon +++++++++++++++++++++++++ 50 

Intestine large, rectum ++++++++++++M++++++++++++ 49 

Intestine large, cecum +++++++++++++++++++++++++ 50 

Intestine small, duodenum +++++++++++++++++++++++++ 50 

Intestine small, jejunum +++++++++++++++++++++++++ 50 

Intestine small, ileum +++++++++++++++++++++++++ 49 

Liver +++++++++++++++++++++++++ 50 

Hepatoblastoma X X X 4 

Hepatocellular carcinoma X X X X X 13 

Hepatocellular carcinoma, multiple XX X XX XXXX X XXXXXX 25 

Hepatocellular adenoma X 5 

Hepatocellular adenoma, multiple XXXXXX XXXXXXX XXXXXXXXXX 42 

Mesentery 2 

Pancreas +++++++++++++++++++++++++ 50 

Salivary glands +++++++++++++++++++++++++ 50 

Stomach, forestomach +++++++++++++++++++++++++ 50 

Stomach, glandular +++++++++++++++++++++++++ 50 

Cardiovascular System 

Blood vessel 

Heart 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

50 

Endocrine System 

Adrenal cortex +++++++++++++++++++++++++ 50 

Adrenal medulla +++++++++++++++++++++++++ 50 

Islets, pancreatic +++++++++++++++++++++++++ 50 

Parathyroid gland M+++M+++++++++++++++M++++ 43 

Pituitary gland +++++++++++++++++++++++++ 48 

Pars distalis, adenoma X X X X 6 

Pars intermedia, adenoma 1 

Thyroid gland +++++++++++++++++++++++++ 50 

Follicular cell, adenoma X 2 

General Body System 

None 

Genital System 

Clitoral gland M+++++M++++++++++++++++++ 48 

Ovary +++++++++++++++++++++++++ 50 

Cystadenoma 1 

Uterus +++++++++++++++++++++++++ 50 

Polyp stromal X 1 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

196 Primidone, NTP TR 476 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Primidone: 1,300 ppm 

Number of Days on Study 

5566666666777777777777777 

8933777779222222222222222 

3718333373177777777777777 

Carcass ID Number 

3333333333333333333333333 

7887888969655555666667789 

2627178663512568234894841 

Hematopoietic System 

Bone marrow 

Lymph node

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

+++++++++++++++++++++++++ 

+ 

+ M M ++++++++++++++++++++++ 

+++++++++++M++++++M++++++ 

+++++++++++++++++++++++++ 

+M+++++++++++++++++++++M+ 

Integumentary System 

Mammary gland 

Skin 

Subcutaneous tissue, fibrosarcoma 

M+++M+++++M++++++++++++++ 

+++++++++++++++++++++++++ 

X X 

Musculoskeletal System 

Bone +++++++++++++++++++++++++ 

Nervous System 

Brain +++++++++++++++++++++++++ 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Alveolar/bronchiolar carcinoma 

Fibrosarcoma, metastatic, skin 

Hepatoblastoma, metastatic, liver 

Nose 

Trachea 

+++++++++++++++++++++++++ 

X 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

Special Senses System 

Harderian gland 

Adenoma 

Urinary System 

Kidney 

Urinary bladder 

+++++++++++++++++++++++++ 

+++++M+++++++++++++++++++ 

Systemic Lesions 

Multiple organs 

Lymphoma malignant

+++++++++++++++++++++++++ 

X X X 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 197 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Primidone: 1,300 ppm 

Number of Days on Study 

7777777777777777777777777 

2222222222222222222222222 

7778888888888999999999999 

Carcass ID Number 

3333333333334333333333333 

9995667778890555677788999 

2599071563540347103909078 

Total 

Tissues/ 

Tumors 

Hematopoietic System 

Bone marrow 

Lymph node

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

+++++++++++++++++++++++++ 

+ + + 

++++++M++++++M+++++++++++ 

+++++++++++++++++++++++M+ 

+++++++++++++++++++++++++ 

++++++M+++MM+M+++++++++++ 

50 

4 

46 

47 

50 

44 

Integumentary System 

Mammary gland 

Skin 

Subcutaneous tissue, fibrosarcoma 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

47 

50 

2 

Musculoskeletal System 

Bone +++++++++++++++++++++++++ 50 

Nervous System 

Brain +++++++++++++++++++++++++ 50 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma

Alveolar/bronchiolar carcinoma

Fibrosarcoma, metastatic, skin 

Hepatoblastoma, metastatic, liver

Nose 

Trachea 

+++++++++++++++++++++++++ 

X X 

X 

X 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

2 

1 

1 

1 

50 

50 

Special Senses System 

Harderian gland

Adenoma

 + 

X 

1 

1 

Urinary System 

Kidney 

Urinary bladder 

+++++++++++++++++++++++++ 

+++++++++++++++++++++++++ 

50 

49 

Systemic Lesions 

Multiple organs 

Lymphoma malignant

+++++++++++++++++++++++++ 

X X 

50 

5 
198 Primidone, NTP TR 476 

TABLE D3 

Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Feed Study of Primidone 

0 ppm 300 ppm 600 ppm 1,300 ppm 

Harderian Gland: Adenoma 

Overall ratea 

Adjusted rateb 3/50 (6%) 

7.3% 

5/50 (10%) 

11.6% 

3/49 (6%) 

6.8% 

1/50 (2%) 

2.6% 

Terminal ratec 3/41 (7%) 4/42 (10%) 3/44 (7%) 1/39 (3%) 

First incidence (days) 

Life table testd 727 (T) 

P=0.168N 

726 

P=0.372 

727 (T) 

P=0.630N 

727 (T) 

P=0.323N 

Logistic regression testd 

Cochran-Armitage testd 

Fisher exact testd 

P=0.167N 

P=0.155N 

P=0.399 

P=0.357 

P=0.630N 

P=0.651 

P=0.323N 

P=0.309N 

Harderian Gland: Adenoma or Carcinoma 

Overall rate 5/50 (10%) 5/50 (10%) 3/49 (6%) 1/50 (2%) 

Adjusted rate 11.2% 11.6% 6.8% 2.6% 

Terminal rate 3/41 (7%) 4/42 (10%) 3/44 (7%) 1/39 (3%) 

First incidence (days) 519 726 727 (T) 727 (T) 

Life table test P=0.064N P=0.609N P=0.329N P=0.113N 

Logistic regression test P=0.064N P=0.534 P=0.444N P=0.134N 

Cochran-Armitage test P=0.058N 

Fisher exact test P=0.630N P=0.369N P=0.102N 

Liver: Hepatocellular Adenoma 

Overall rate 15/50 (30%) 42/50 (84%) 45/49 (92%) 47/50 (94%) 

Adjusted rate 34.9% 91.3% 97.8% 97.9% 

Terminal rate 13/41 (32%) 38/42 (90%) 43/44 (98%) 38/39 (97%) 

First incidence (days) 673 701 680 583 

Life table test P<0.001 P<0.001 P<0.001 P<0.001 

Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001 

Cochran-Armitage test P<0.001 

Fisher exact test P<0.001 P<0.001 P<0.001 

Liver: Hepatocellular Carcinoma 

Overall rate 3/50 (6%) 11/50 (22%) 19/49 (39%) 38/50 (76%) 

Adjusted rate 7.1% 24.9% 42.2% 84.4% 

Terminal rate 2/41 (5%) 9/42 (21%) 18/44 (41%) 32/39 (82%) 

First incidence (days) 694 701 699 638 

Life table test P<0.001 P=0.029 P<0.001 P<0.001 

Logistic regression test P<0.001 P=0.032 P<0.001 P<0.001 

Cochran-Armitage test P<0.001 

Fisher exact test P=0.020 P<0.001 P<0.001 

Liver: Hepatocellular Adenoma or Carcinoma 

Overall rate 16/50 (32%) 42/50 (84%) 45/49 (92%) 50/50 (100%) 

Adjusted rate 37.2% 91.3% 97.8% 100.0% 

Terminal rate 14/41 (34%) 38/42 (90%) 43/44 (98%) 39/39 (100%) 

First incidence (days) 673 701 680 583 

Life table test P<0.001 P<0.001 P<0.001 P<0.001 

Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001 

Cochran-Armitage test P<0.001 

Fisher exact test P<0.001 P<0.001 P<0.001 
Primidone NTP TR 476 199 

TABLE D3 

Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Feed Study of Primidone 

0 ppm 300 ppm 600 ppm 1,300 ppm 

Liver: Hepatoblastoma 

Overall rate 1/50 (2%) 4/50 (8%) 4/49 (8%) 4/50 (8%) 

Adjusted rate 2.4% 9.2% 8.8% 9.7% 

Terminal rate 1/41 (2%) 3/42 (7%) 3/44 (7%) 3/39 (8%) 

First incidence (days) 727 (T) 718 677 673 

Life table test P=0.199 P=0.195 P=0.208 P=0.174 

Logistic regression test P=0.220 P=0.212 P=0.182 P=0.185 

Cochran-Armitage test P=0.220 

Fisher exact test P=0.181 P=0.175 P=0.181 

Liver: Hepatocellular Carcinoma or Hepatoblastoma 

Overall rate 4/50 (8%) 12/50 (24%) 20/49 (41%) 39/50 (78%) 

Adjusted rate 9.5% 26.5% 43.4% 84.7% 

Terminal rate 3/41 (7%) 9/42 (21%) 18/44 (41%) 32/39 (82%) 

First incidence (days) 694 701 677 638 

Life table test P<0.001 P=0.040 P<0.001 P<0.001 

Logistic regression test P<0.001 P=0.042 P<0.001 P<0.001 

Cochran-Armitage test P<0.001 

Fisher exact test P=0.027 P<0.001 P<0.001 

Liver: Hepatocellular Adenoma, Hepatocellular Carcinoma, or Hepatoblastoma 

Overall rate 16/50 (32%) 42/50 (84%) 46/49 (94%) 50/50 (100%) 

Adjusted rate 37.2% 91.3% 97.9% 100.0% 

Terminal rate 14/41 (34%) 38/42 (90%) 43/44 (98%) 39/39 (100%) 

First incidence (days) 673 701 677 583 

Life table test P<0.001 P<0.001 P<0.001 P<0.001 

Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001 

Cochran-Armitage test P<0.001 

Fisher exact test P<0.001 P<0.001 P<0.001 

Lung: Alveolar/bronchiolar Adenoma 

Overall rate 4/50 (8%) 2/50 (4%) 4/49 (8%) 2/50 (4%)
 

Adjusted rate 9.8% 4.8% 8.9% 5.1%
 

Terminal rate 4/41 (10%) 2/42 (5%) 3/44 (7%) 2/39 (5%)
 

First incidence (days) 727 (T) 727 (T) 699 727 (T)
 

Life table test P=0.369N P=0.326N P=0.602N P=0.360N 

Logistic regression test P=0.355N P=0.326N P=0.608N P=0.360N 

Cochran-Armitage test P=0.345N
 

Fisher exact test P=0.339N P=0.631 P=0.339N 

Lung: Alveolar/bronchiolar Carcinoma 

Overall rate 4/50 (8%) 3/50 (6%) 0/49 (0%) 1/50 (2%) 

Adjusted rate 9.1% 7.1% 0.0% 2.6% 

Terminal rate 2/41 (5%) 3/42 (7%) 0/44 (0%) 1/39 (3%) e First incidence (days) 621 727 (T) — 727 (T) 

Life table test P=0.087N P=0.476N P=0.057N P=0.186N 

Logistic regression test P=0.088N P=0.543N P=0.079N P=0.192N 

Cochran-Armitage test P=0.084N 

Fisher exact test P=0.500N P=0.061N P=0.181N 
200 Primidone, NTP TR 476 

TABLE D3 

Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Feed Study of Primidone 

0 ppm 300 ppm 600 ppm 1,300 ppm 

Lung: Alveolar/bronchiolar Adenoma or Carcinoma 

Overall rate 7/50 (14%) 5/50 (10%) 4/49 (8%) 3/50 (6%) 

Adjusted rate 16.1% 11.9% 8.9% 7.7% 

Terminal rate 5/41 (12%) 5/42 (12%) 3/44 (7%) 3/39 (8%) 

First incidence (days) 621 727 (T) 699 727 (T) 

Life table test P=0.140N P=0.356N P=0.227N P=0.174N 

Logistic regression test P=0.124N P=0.372N P=0.276N P=0.154N 

Cochran-Armitage test P=0.126N 

Fisher exact test P=0.380N P=0.274N P=0.159N 

Ovary: Cystadenoma 

Overall rate 3/49 (6%) 0/49 (0%) 2/48 (4%) 1/50 (2%) 

Adjusted rate 7.5% 0.0% 4.5% 2.6% 

Terminal rate 3/40 (8%) 0/42 (0%) 2/44 (5%) 1/39 (3%) 

First incidence (days) 727 (T) — 727 (T) 727 (T) 

Life table test P=0.359N P=0.113N P=0.456N P=0.314N 

Logistic regression test P=0.359N P=0.113N P=0.456N P=0.314N 

Cochran-Armitage test P=0.343N 

Fisher exact test P=0.121N P=0.510N P=0.301N 

Pituitary Gland (Pars Distalis): Adenoma 

Overall rate 3/49 (6%) 5/48 (10%) 6/49 (12%) 6/48 (13%) 

Adjusted rate 7.5% 12.2% 13.3% 15.8% 

Terminal rate 3/40 (8%) 5/41 (12%) 5/44 (11%) 6/38 (16%) 

First incidence (days) 727 (T) 727 (T) 694 727 (T) 

Life table test P=0.190 P=0.369 P=0.293 P=0.216 

Logistic regression test P=0.199 P=0.369 P=0.286 P=0.216 

Cochran-Armitage test P=0.216 

Fisher exact test P=0.346 P=0.243 P=0.233 

Skin (Subcutaneous Tissue): Fibrosarcoma 

Overall rate 3/50 (6%) 2/50 (4%) 1/49 (2%) 2/50 (4%) 

Adjusted rate 6.5% 4.8% 2.2% 4.1% 

Terminal rate 1/41 (2%) 2/42 (5%) 0/44 (0%) 0/39 (0%) 

First incidence (days) 595 727 (T) 694 583 

Life table test P=0.434N P=0.484N P=0.293N P=0.489N 

Logistic regression test P=0.358N P=0.660N P=0.428N P=0.683 

Cochran-Armitage test P=0.427N 

Fisher exact test P=0.500N P=0.316N P=0.500N 

Uterus: Stromal Polyp 

Overall rate 1/50 (2%) 3/50 (6%) 1/49 (2%) 1/50 (2%) 

Adjusted rate 2.4% 7.1% 2.3% 2.6% 

Terminal rate 1/41 (2%) 3/42 (7%) 1/44 (2%) 1/39 (3%) 

First incidence (days) 727 (T) 727 (T) 727 (T) 727 (T) 

Life table test P=0.468N P=0.314 P=0.746N P=0.751 

Logistic regression test P=0.468N P=0.314 P=0.746N P=0.751 

Cochran-Armitage test P=0.449N 

Fisher exact test P=0.309 P=0.747 P=0.753N 
Primidone NTP TR 476 201 

TABLE D3 

Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Feed Study of Primidone 

0 ppm 300 ppm 600 ppm 1,300 ppm 

Uterus: Stromal Polyp or Stromal Sarcoma 

Overall rate 2/50 (4%) 3/50 (6%) 1/49 (2%) 1/50 (2%) 

Adjusted rate 4.9% 7.1% 2.3% 2.6% 

Terminal rate 2/41 (5%) 3/42 (7%) 1/44 (2%) 1/39 (3%) 

First incidence (days) 727 (T) 727 (T) 727 (T) 727 (T) 

Life table test P=0.303N P=0.511 P=0.475N P=0.518N 

Logistic regression test P=0.303N P=0.511 P=0.475N P=0.517N 

Cochran-Armitage test P=0.289N 

Fisher exact test P=0.500 P=0.508N P=0.500N 

All Organs: Hemangioma or Hemangiosarcoma 

Overall rate 3/50 (6%) 2/50 (4%) 1/49 (2%) 0/50 (0%) 

Adjusted rate 7.1% 4.7% 2.3% 0.0% 

Terminal rate 2/41 (5%) 1/42 (2%) 1/44 (2%) 0/39 (0%) 

First incidence (days) 673 726 727 (T) — 

Life table test P=0.069N P=0.478N P=0.279N P=0.125N 

Logistic regression test P=0.065N P=0.487N P=0.305N P=0.119N 

Cochran-Armitage test P=0.067N 

Fisher exact test P=0.500N P=0.316N P=0.121N 

All Organs: Malignant Lymphoma 

Overall rate 8/50 (16%) 6/50 (12%) 8/49 (16%) 5/50 (10%) 

Adjusted rate 18.4% 13.6% 17.4% 12.4% 

Terminal rate 6/41 (15%) 4/42 (10%) 6/44 (14%) 4/39 (10%) 

First incidence (days) 621 724 680 693 

Life table test P=0.311N P=0.361N P=0.542N P=0.301N 

Logistic regression test P=0.272N P=0.368N P=0.597 P=0.268N 

Cochran-Armitage test P=0.275N 

Fisher exact test P=0.387N P=0.590 P=0.277N 

All Organs: Benign Neoplasms 

Overall rate 26/50 (52%) 45/50 (90%) 46/49 (94%) 47/50 (94%) 

Adjusted rate 60.5% 97.8% 97.9% 97.9% 

Terminal rate 24/41 (59%) 41/42 (98%) 43/44 (98%) 38/39 (97%) 

First incidence (days) 673 701 680 583 

Life table test P<0.001 P<0.001 P<0.001 P<0.001 

Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001 

Cochran-Armitage test P<0.001 

Fisher exact test P<0.001 P<0.001 P<0.001 

All Organs: Malignant Neoplasms 

Overall rate 20/50 (40%) 23/50 (46%) 26/49 (53%) 40/50 (80%) 

Adjusted rate 42.3% 47.9% 54.2% 85.0% 

Terminal rate 14/41 (34%) 17/42 (40%) 22/44 (50%) 32/39 (82%) 

First incidence (days) 519 673 677 583 

Life table test P<0.001 P=0.421 P=0.271 P<0.001 

Logistic regression test P<0.001 P=0.288 P=0.080 P<0.001 

Cochran-Armitage test P<0.001 

Fisher exact test P=0.343 P=0.135 P<0.001 
202 

c 

Primidone, NTP TR 476 

TABLE D3 

Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Feed Study of Primidone 

0 ppm 300 ppm 600 ppm 1,300 ppm 

All Organs: Benign or Malignant Neoplasms 

Overall rate 34/50 (68%) 47/50 (94%) 47/49 (96%) 50/50 (100%) 

Adjusted rate 72.2% 97.9% 97.9% 100.0% 

Terminal rate 28/41 (68%) 41/42 (98%) 43/44 (98%) 39/39 (100%) 

First incidence (days) 519 673 677 583 

Life table test P<0.001 P=0.014 P=0.025 P<0.001 

Logistic regression test P<0.001 P=0.003 P<0.001 P<0.001 

Cochran-Armitage test P<0.001 

Fisher exact test P<0.001 P<0.001 P<0.001 

(T)Terminal sacrifice 

a Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for liver, lung, ovary, 

and pituitary gland; for other tissues, denominator is number of animals necropsied. b	 Kaplan-Meier estimated neoplasm incidence after adjustment for intercurrent mortality 

Observed incidence at terminal kill 

d	 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 
Primidone, NTP TR 476 203 

TABLE D4 

Historical Incidence of Liver Neoplasms in Untreated Female B6C3F1 Micea 

Incidence in Controls 

Study Hepatocellular Hepatocellular Hepatoblastoma Hepatocellular Adenoma, 

Adenoma Carcinoma Hepatocellular Carcinoma,

 or Hepatoblastoma 

Historical Incidence at Battelle Columbus Laboratories 

4,4N-Thiobis(6-t-butyl-m-cresol) 17/51 4/51 0/51 20/51 

5,5-Diphenylhydantoin 5/48 0/48 0/48 5/48 

Pentachlorophenol (Dowicide EC-7) 1/34 0/34 0/34 1/34 

Ethylene Thiourea 2/50 2/50 0/50 4/50 

Polybrominated Biphenyls (Firemaster FF-1®) 4/50 1/50 0/50 5/50 

Manganese (II) Sulfate Monohydrate 12/51 3/51 0/51 13/51 

Oxazepam 25/50 9/50 0/50 28/50 

Pentachlorophenol (Technical Grade) 3/33 0/33 0/33 3/33 

Triamterene 10/50 4/50 0/50 13/50 

Triamterene 7/50 5/50 0/50 10/50 

Tricresyl Phosphate 12/50 10/50 0/50 21/50 

Overall Historical Incidence 

Total 213/1,464 (15.8%) 108/1,464 (7.4%) 1/1,464 (0.1%) 313/1,464 (21.4%) 

Standard deviation 10.6% 6.1% 0.4% 13.0% 

Range 2%-50% 0%-20% 0%-2% 3%-56% 

a Data as of 12 May 1995 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

204 Primidone, NTP TR 476 

TABLE D5 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Feed Study of Primidonea 

0 ppm 300 ppm 600 ppm 1,300 ppm 

Disposition Summary 

Animals initially in study 50 50 50 50 

Early deaths 

Moribund 7 1 3 8 

Natural deaths 2 7 2 3 

Survivors 

Terminal sacrifice 41 42 44 39 

Missing 1 

Animals examined microscopically 50 50 49 50 

Alimentary System 

Intestine small, duodenum (50) (49) (49) (50) 

Ulcer, chronic, focal 1 (2%) 

Intestine small, jejunum (50) (49) (49) (50) 

Peyer’s patch, hyperplasia, lymphoid 1 (2%) 

Serosa, inflammation, chronic, granulomatous 1 (2%) 

Liver (50) (50) (49) (50) 

Angiectasis 1 (2%) 

Basophilic focus 1 (2%) 1 (2%) 1 (2%) 

Clear cell focus 1 (2%) 4 (8%) 1 (2%) 4 (8%) 

Congestion 1 (2%) 

Eosinophilic focus 8 (16%) 23 (46%) 24 (49%) 17 (34%) 

Hematopoietic cell proliferation 3 (6%) 2 (4%) 2 (4%) 

Inflammation, chronic 1 (2%) 

Mixed cell focus 1 (2%) 1 (2%) 

Necrosis 4 (8%) 2 (4%) 3 (6%) 

Thrombosis, diffuse 1 (2%) 

Centrilobular, hypertrophy 1 (2%) 11 (22%) 11 (22%) 21 (42%) 

Centrilobular, necrosis 1 (2%) 

Hepatocyte, vacuolization cytoplasmic 3 (6%) 35 (70%) 39 (80%) 28 (56%) 

Mesentery (5) (5) (6) (2) 

Fat, inflammation, chronic 4 (80%) 5 (100%) 5 (83%) 2 (100%) 

Pancreas (50) (48) (49) (50) 

Inflammation, chronic 1 (2%) 

Acinus, atrophy 2 (4%) 2 (4%) 1 (2%) 1 (2%) 

Duct, cyst 1 (2%) 

Salivary glands (50) (48) (49) (50) 

Duct, cyst 1 (2%) 

Stomach, forestomach (50) (49) (49) (50) 

Hyperplasia, focal, squamous 4 (8%) 11 (22%) 3 (6%) 7 (14%) 

Stomach, glandular (50) (49) (49) (50) 

Necrosis, focal 1 (2%) 

Polyp, inflammatory 1 (2%) 

Cardiovascular System 

Blood vessel (50) (49) (49) (50) 

Inflammation, chronic 1 (2%) 

a Number of animals examined microscopically at the site and the number of animals with lesion 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primidone, NTP TR 476 205 

TABLE D5 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Feed Study of Primidone 

0 ppm 300 ppm 600 ppm 1,300 ppm 

Endocrine System 

Adrenal cortex (50) (49) (49) (50) 

Hematopoietic cell proliferation 1 (2%) 

Hyperplasia, focal 1 (2%) 

Hypertrophy, focal 2 (4%) 2 (4%) 1 (2%) 

Capsule, hyperplasia 2 (4%) 1 (2%) 

Zona reticularis, angiectasis, focal 1 (2%) 

Zona reticularis, vacuolization cytoplasmic 1 (2%) 

Adrenal medulla (50) (49) (49) (50) 

Hyperplasia 1 (2%) 

Islets, pancreatic (50) (47) (49) (50) 

Hyperplasia 15 (30%) 17 (36%) 6 (12%) 5 (10%) 

Pituitary gland (49) (48) (49) (48) 

Pars distalis, angiectasis 1 (2%) 1 (2%) 

Pars distalis, hemorrhage 1 (2%) 

Pars distalis, hyperplasia 13 (27%) 12 (25%) 19 (39%) 10 (21%) 

Pars intermedia, hyperplasia 1 (2%) 1 (2%) 

Rathke’s cleft, cyst 1 (2%) 

Thyroid gland (50) (48) (48) (50) 

Inflammation, chronic 1 (2%) 

Follicle, cyst 1 (2%) 

Follicular cell, hyperplasia 13 (26%) 12 (25%) 28 (58%) 49 (98%) 

General Body System 

None 

Genital System 

Clitoral gland (47) (45) (48) (48) 

Inflammation, chronic 1 (2%) 

Ovary (49) (49) (48) (50) 

Angiectasis 1 (2%) 

Atrophy 37 (76%) 47 (96%) 39 (81%) 42 (84%) 

Hematocyst 5 (10%) 1 (2%) 1 (2%) 

Hemorrhage 1 (2%) 

Hyperplasia, cystic 1 (2%) 

Inflammation, chronic, granulomatous 1 (2%) 

Follicle, cyst 12 (24%) 7 (14%) 9 (19%) 9 (18%) 

Periovarian tissue, cyst 2 (4%) 

Uterus (50) (49) (49) (50) 

Angiectasis 1 (2%) 

Hydrometra 28 (56%) 23 (47%) 17 (35%) 20 (40%) 

Inflammation, suppurative 2 (4%) 2 (4%) 

Endometrium, hyperplasia, cystic 36 (72%) 39 (80%) 42 (86%) 42 (84%) 

Endometrium, inflammation, chronic active 1 (2%) 

Hematopoietic System 

Bone marrow (50) (49) (49) (50) 

Depletion cellular 1 (2%) 

Myelofibrosis, focal 1 (2%) 

Myeloid cell, hyperplasia 1 (2%) 1 (2%) 3 (6%) 2 (4%) 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

206 Primidone, NTP TR 476 

TABLE D5 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Feed Study of Primidone 

0 ppm 300 ppm 600 ppm 1,300 ppm 

Hematopoietic System (continued) 

Lymph node (6) (8) (7) (4) 

Lumbar, infiltration cellular, histiocyte 1 (17%) 1 (14%) 

Lumbar, pigmentation 1 (17%) 

Mediastinal, hyperplasia, lymphoid 1 (14%) 3 (75%) 

Renal, infiltration cellular, histiocyte 1 (14%) 

Lymph node, mandibular (48) (44) (45) (46) 

Cyst 1 (2%) 

Hyperplasia, lymphoid 1 (2%) 1 (2%) 2 (4%) 

Infiltration cellular, plasma cell 1 (2%) 

Infiltration cellular, histiocyte 1 (2%) 

Lymph node, mesenteric (48) (48) (47) (47) 

Hematopoietic cell proliferation 1 (2%) 

Hyperplasia, lymphoid 5 (10%) 2 (4%) 4 (9%) 

Spleen (50) (48) (49) (50) 

Atrophy 1 (2%) 

Hematopoietic cell proliferation 14 (28%) 13 (27%) 13 (27%) 23 (46%) 

Hyperplasia, lymphoid 5 (10%) 5 (10%) 7 (14%) 4 (8%) 

Thrombosis 1 (2%) 

Thymus (45) (47) (44) (44) 

Angiectasis 1 (2%) 

Atrophy 30 (67%) 35 (74%) 37 (84%) 35 (80%) 

Hyperplasia, lymphoid 6 (13%) 5 (11%) 3 (7%) 2 (5%) 

Integumentary System 

Skin (50) (50) (49) (50) 

Inflammation, chronic active 1 (2%) 

Ulcer, chronic active 1 (2%) 

Subcutaneous tissue, cyst, chronic active 1 (2%) 

Subcutaneous tissue, edema 1 (2%) 

Musculoskeletal System 

None 

Nervous System 

None 

Respiratory System 

Lung 

Alveolar epithelium, hyperplasia, focal

(50) 

 2 (4%)

(50) 

 3 (6%)

(49) (50) 

 1 (2%) 

Special Senses System 

Eye

Retina, atrophy

 (1) 

 1 (100%) 
 

 

 

 

 

Primidone, NTP TR 476 207 

TABLE D5 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Feed Study of Primidone 

0 ppm 300 ppm 600 ppm 1,300 ppm 

Urinary System 

Kidney 

Cyst

Hydronephrosis

Infiltration cellular, lymphocyte

Nephropathy

Renal tubule, pigmentation, hemosiderin

(50) 

 1 (2%)

 7 (14%)

 1 (2%)

(49) 

 3 (6%)

(49) 

 1 (2%) 

 1 (2%) 

 6 (12%)

 1 (2%)

(50) 

 2 (4%) 

 3 (6%) 

 1 (2%) 
208 Primidone, NTP TR 476
 
209 

APPENDIX E
 

GENETIC TOXICOLOGY
 

SALMONELLA MUTAGENICITY TEST PROTOCOL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 
 

CHINESE HAMSTER OVARY CELL CYTOGENETICS PROTOCOLS . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 
 

MOUSE BONE MARROW MICRONUCLEUS TEST PROTOCOL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 
 

RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212 
 

TABLE E1 Mutagenicity of Primidone in Salmonella typhimurium . . . . . . . . . . . . . . . . . . . . . . . . 213 
 

TABLE E2 Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells
 

by Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 
 

TABLE E3 Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells
 

by Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216 
 

TABLE E4 Frequency of Micronuclei in Bone Marrow Polychromatic Erythrocytes of Mice
 

Treated with Primidone by Intraperitoneal Injection . . . . . . . . . . . . . . . . . . . . . . . . . 217 
 
210 Primidone, NTP TR 476 

GENETIC TOXICOLOGY 

SALMONELLA TYPHIMURIUM MUTAGENICITY TEST PROTOCOL 

Testing was performed as reported by Mortelmans et al. (1986). Primidone was sent to the laboratory 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 37E C. Top agar supplemented with l-histidine and 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 37E C. 

Each trial consisted of triplicate plates of concurrent positive and negative controls and of at least three 

doses of primidone. The high dose was limited by experimental design to 10,000 μg/plate. All trials were 

repeated. 

In this assay, a positive response is defined as a reproducible, dose-related increase in histidine-independent 

(revertant) colonies in any one strain/activation 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 was no minimum percentage or fold increase required for a 

chemical to be judged positive or weakly positive. 

CHINESE HAMSTER OVARY CELL CYTOGENETICS PROTOCOLS 

Testing was performed as reported by Galloway et al. (1987). Primidone 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 three doses of primidone; the high dose was limited by 

toxicity. A single flask per dose was used. 

Sister Chromatid Exchange Test: In the SCE test without S9, CHO cells were incubated for 26 hours with 

primidone in McCoy’s 5A medium. Bromodeoxyuridine (BrdU) was added 2 hours after culture initiation. 

After 26 hours, the medium containing primidone 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 

primidone, serum-free medium, and S9 for 2 hours. The medium was then removed and replaced with 

medium containing serum and BrdU and no primidone 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 SCE/cell from each dose level. Because 

significant chemical-induced cell cycle delay was seen, incubation time was lengthened to ensure a sufficient 

number of scorable (second-division metaphase) cells. 

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 
Primidone, NTP TR 476 211 

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 statistically 

significant trend (P<0.005) in the absence of any responses reaching 20% above background led to a call 

of equivocal. 

Chromosomal Aberrations Test: In the test without S9, cells were incubated in McCoy’s 5A medium with 

primidone for 12 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 primidone and S9 for 2 hours, after which the treatment medium was removed and the cells were 

incubated for 10.3 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: if cell cycle delay was anticipated, the incubation 

period was extended. 

Cells were selected for scoring on the basis of good morphology and completeness of karyotype 

(21 ± 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 (P#0.05) difference for one dose point and a significant trend 

(P#0.015) was 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 resulted in 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. 

MOUSE BONE MARROW MICRONUCLEUS TEST PROTOCOL 

Preliminary range-finding studies were performed. Factors affecting dose selection included chemical 

solubility and toxicity and the extent of cell cycle delay induced by primidone exposure. The standard 

three-exposure protocol is described in detail by Shelby et al. (1993). Male B6C3F1 mice were injected 

intraperitoneally three times at 24-hour intervals with primidone dissolved in corn oil. The total dosing 

volume was 0.4 mL. Solvent control animals were injected with 0.4 mL of corn oil only. The positive 

control mice received injections of dimethybenzanthracene (12.5 mg/kg body weight). The mice were 

killed 24 hours after the final injection, and blood smears were prepared from bone marrow cells obtained 

from the femurs. Air-dried smears were fixed and stained; 2,000 polychromatic erythrocytes (PCEs) were 

scored for the frequency of micronucleated cells in each of up to five animals per dose group. 

The results were tabulated as the mean of the pooled results from all animals within a treatment group plus 

or minus the standard error of the mean. The frequency of micronucleated cells among PCEs was analyzed 

by a statistical software package that tested for increasing trend over dose groups using a one-tailed 

Cochran-Armitage trend test, followed by pairwise comparisons between each dose group and the control 

group (Margolin and Risko, 1988). In the presence of excess binomial variation, as detected by a binomial 

dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to 

the excess variation. In the micronucleus test, an individual trial was considered positive if the trend test 

P value was less than or equal to 0.025 or the P value for any single dose group was less than or equal to 

0.025 divided by the number of dose groups. A final call of positive for micronucleus induction was 

preferably based on reproducibly positive trials (as noted above). Ultimately, the final call was determined 
212 Primidone, NTP TR 476 

by the scientific staff after considering the results of statistical analyses, the reproducibility of any effects 

observed, and the magnitudes of those effects. 

RESULTS 

Primidone (33 to 10,000 μg/plate) induced mutations in Salmonella typhimurium strain TA1535 in trials 

conducted in the absence of exogenous metabolic activation (S9); no mutagenic response was detected in 

TA1535 with S9 (Mortelmans et al., 1986; Table E1). Negative results were obtained in the 

S. typhimurium assay with strains TA98, TA100, and TA1537, with and without S9. No induction of 

SCEs (Table E2) or Abs (Table E3) was noted in cultured CHO cells treated with concentrations of 

primidone ranging from 125 to 1,250 Fg/mL, with or without S9. In vivo, no significant increase was 

observed in the frequency of micronucleated PCEs in bone marrow of male mice treated with 87.5 to 

350 mg primidone/kg body weight three times at 24-hour intervals in either of two trials (Table E4). 

In summary, primidone induced gene mutations in S. typhimurium in the absence of S9 activation but did 

not induce chromosomal damage in mammalian cells, in vitro or in vivo, even at doses associated with 

marked toxicity. 
Primidone, NTP TR 476 213 

TABLE E1 

Mutagenicity of Primidone in Salmonella typhimuriuma 

Strain Dose 

(μg/plate) Trial 1 

-S9 

Trial 2 

Revertants/plateb 

+10% hamster S9 

Trial 1 Trial 2 

+10% rat S9 

Trial 1 Trial 2 

TA100 0 

33 

100 

333 

1,000 

3,333 

6,666 

10,000 

131 ± 2.1 

153 ± 14.7 

140 ± 2.4 

148 ± 3.3 

152 ± 3.7 

177 ± 9.6 

123 ± 3.8 

136 ± 1.7 

131 ± 1.5 

143 ± 3.7 

135 ± 14.6 

151 ± 6.9 

137 ± 14.4 

160 ± 12.7 

120 ± 8.5 

153 ± 6.4 

134 ± 8.2 

164 ± 7.5 

121 ± 11.3 

132 ± 6.1 

124 ± 4.4 

117 ± 2.3 

131 ± 3.4 

121 ± 11.2 

156 ± 9.0 

157 ± 7.4 

148 ± 0.7 

155 ± 9.8 

158 ± 2.0 

147 ± 8.5 

127 ± 7.2 

128 ± 2.1 

143 ± 2.6 

136 ± 0.9 

131 ± 10.1 

134 ± 13.4 

Trial summary 

Positive controlc Equivocal 

325 ± 12.3 

Negative 

327 ± 2.2 

Negative 

2,147 ± 92.9 

Negative 

1,639 ± 24.4 

Negative 

839 ± 41.0 

Negative 

591 ± 30.6 

– S9 +10% hamster S9 

Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 

TA1535 0 

33 

100 

333 

1,000 

3,333 

6,666 

10,000 

23 ± 4.0 

28 ± 1.9 

22 ± 4.2 

31 ± 3.0 

43 ± 0.7 

47 ± 4.0 

18 ± 4.4 

28 ± 4.9 

30 ± 3.0 

39 ± 1.0 

53 ± 5.3 

56 ± 5.2 

19 ± 2.8 

17 ± 2.0 

17 ± 2.2 

34 ± 4.2 

44 ± 4.1 

54 ± 2.2 

7 ± 1.2 

12 ± 2.4 

12 ± 0.0 

5 ± 1.0 

9 ± 2.7 

8 ± 2.7 

9 ± 1.8 

6 ± 1.2 

10 ± 3.1 

7 ± 0.7 

5 ± 1.5 

10 ± 1.7 

Trial summary 

Positive control 

Weakly Positive 

288 ± 13.0 

Positive 

305 ± 14.2 

Positive 

385 ± 7.1 

Negative 

530 ± 28.3 

Negative 

342 ± 28.3 

+10% rat S9 

Trial 1 Trial 2 

TA1535 0 

(continued) 33 

100 

333 

1,000 

3,333 

6,666 

10,000 

6 ± 0.7 

9 ± 2.0 

9 ± 2.6 

9 ± 3.5 

8 ± 1.8 

7 ± 0.6 

6 ± 0.9 

8 ± 0.9 

10 ± 1.7 

15 ± 1.8 

10 ± 3.2 

10 ± 2.0 

Trial summary 

Positive control 

Negative 

183 ± 6.4 

Negative 

146 ± 7.5 
214 

c 

Primidone, NTP TR 476 

TABLE E1 

Mutagenicity of Primidone in Salmonella typhimurium 

Revertants/plate 

Strain Dose 

(μg/plate) Trial 1 

-S9 

Trial 2 

+10% hamster S9 

Trial 1 Trial 2 

+10% rat S9 

Trial 1 Trial 2 

TA1537 0 

33 

100 

333 

1,000 

3,333 

6,666 

10,000 

4 ± 0.3 

7 ± 3.2 

6 ± 0.6 

6 ± 3.2 

4 ± 1.2 

10 ± 2.3 

9 ± 1.8 

6 ± 0.9 

9 ± 2.7 

9 ± 2.0 

6 ± 1.8 

5 ± 1.0 

6 ± 1.5 

8 ± 1.9 

7 ± 2.2 

8 ± 3.2 

9 ± 1.8 

8 ± 3.2 

8 ± 0.9 

14 ± 0.6 

13 ± 0.3 

13 ± 2.6 

9 ± 1.7 

11 ± 2.7 

8 ± 1.7 

7 ± 0.0 

8 ± 2.3 

7 ± 0.7 

6 ± 1.8 

7 ± 1.3 

9 ± 0.9 

7 ± 1.2 

11 ± 2.5 

10 ± 1.5 

13 ± 0.0 

12 ± 2.0 

Trial summary 

Positive control 

Negative 

114 ± 13.6 

Negative 

150 ± 10.6 

Negative 

316 ± 36.2 

Negative 

546 ± 26.3 

Negative 

158 ± 27.3 

Negative 

133 ± 3.6 

TA98 0 

33 

100 

333 

1,000 

3,333 

6,666 

10,000 

22 ± 2.5 

18 ± 1.2 

19 ± 0.7 

13 ± 2.6 

20 ± 3.3 

18 ± 1.2 

23 ± 1.3 

15 ± 1.8 

17 ± 2.0 

20 ± 2.0 

19 ± 4.0 

16 ± 4.4 

31 ± 3.5 

30 ± 4.5 

34 ± 3.2 

31 ± 2.6 

30 ± 1.8 

36 ± 2.1 

30 ± 2.0 

32 ± 5.7 

30 ± 1.2 

37 ± 2.3 

36 ± 2.9 

31 ± 6.4 

30 ± 5.5 

31 ± 2.9 

30 ± 3.0 

26 ± 2.7 

27 ± 3.1 

29 ± 3.8 

26 ± 5.2 

31 ± 6.8 

35 ± 2.2 

37 ± 5.9 

26 ± 3.2 

26 ± 1.2 

Trial summary 

Positive control 

Negative 

809 ± 12.7 

Negative 

989 ± 15.5 

Negative 

1,919 ± 26.9 

Negative 

1,466 ± 63.4 

Negative 

585 ± 43.1 

Negative 

437 ± 19.6 

a Study was performed at SRI International. The detailed protocol and these data are presented by Mortelmans et al. (1986). b	 Revertants are presented as mean ± standard error from three plates. 

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-aminoanthracene. 
215 

c 

Primidone, NTP TR 476 

TABLE E2 

Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells by Primidonea 

Total No. of SCEs/ Relative 

Dose Cells Chromo- No. of Chromo- SCEs/ Hrs Change of SCEs/ 

Compound (μg/mL) Scored somes SCEs some Cell in BrdU Chromosomeb 

(%) 

–S9 

Summary: Negative 

Dimethylsulfoxide 50 1,048 365 0.34 7.3 26.0 

50 1,048 417 0.39 8.3 31.0c 

Mitomycin-C 0.001 50 1,052 672 0.63 13.4 26.0 60.54 

0.005 10 210 269 1.28 26.9 26.0 221.93 

Primidone 125 50 1,048 411 0.39 8.2 26.0 –1.44 

417 50 1,048 416 0.39 8.3 31.0c –0.24 

1,250 50 1,037 454 0.43 9.1 31.0c 10.03 

P=0.082d 

+S9 

Summary: Negative 

Dimethylsulfoxide 50 1,048 427 0.40 8.5 26.0 

Cyclophosphamide 0.125 50 1,053 567 0.53 11.3 26.0 32.16 

0.500 10 210 184 0.87 18.4 26.0 115.05 

Primidone 125 50 1,046 457 0.43 9.1 26.0 7.23

 417 50 1,045 418 0.40 8.4 26.0 –1.83 

1,250 50 1,045 368 0.35 7.4 26.0 –13.57 

P=0.989 

a Study was performed at SITEK Research Laboratories. A detailed description of the protocol is presented by Galloway et al. (1987). 

SCE=sister chromatid exchange; BrdU=bromodeoxyuridine b	 SCEs/chromosome in treated cells versus SCEs/chromosome in solvent control cells 

Because a delay in the cell division cycle was induced, harvest time was extended to maximize the proportion of second-division 

metaphase cells available for analysis. d	 Significance of relative SCEs/chromosome tested by the linear regression trend versus log of the dose 
216 Primidone, NTP TR 476 

TABLE E3 

Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells by Primidonea 

–S9 +S9 

Dose Total No. of Abs/ Cells with Dose Total No. of Abs/ Cells with 

(μg/mL) Cells Abs Cell Abs (%) (μg/mL) Cells Abs Cell Abs (%) 

Scored 

Harvest time: 14.0 hours Harvest time: 12.3 hours 

Summary: Negative Summary: Negative 

Dimethylsulfoxide Dimethylsulfoxide 

200 0 0.00 0.0 200 2 0.01 1.0 

Mitomycin-C Cyclophosphamide 

0.4 50 28 0.56 46.0 15 50 61 1.22 46.0 

Primidone Primidone 

270 200 0 0.00 0.0 270 200 2 0.01 1.0 

581 200 3 0.02 1.5 581 200 7 0.04 2.0 

1,250 200 0 0.00 0.0 1,250 200 3 0.02 1.5 

P=0.219b P=0.249 

a Study was performed at SITEK Research Laboratories. The detailed protocol is presented by Galloway et al. 1987. Abs=aberrations. 

b Significance of percent cells with aberrations tested by the linear regression trend test versus log of the dose 
Primidone, NTP TR 476 217 

TABLE E4 

Frequency of Micronuclei in Bone Marrow Polychromatic Erythrocytes of Mice 

Treated with Primidone by Intraperitoneal Injectiona 

Dose Number of Mice Micronucleated PCEs/1,000 Cellsb 

Trial 1 

Corn oil 

0.4 mL 5 0.6 ± 0.2 

Dimethylbenzanthracenec 

12.5 mg/kg 5 2.4 ± 0.3 

Primidone 

87.5 mg/kg 

175 mg/kg 

300 mg/kg 

5 

5 

2d 

0.4 ± 0.2 

0.9 ± 0.2 

1.2 ± 0.2 

Trial 2 

P=0.200e 

Corn oil 

0.4 mL 5 0.6 ± 0.2 

Dimethylbenzanthracene 

12.5 mg/kg 4 4.2 ± 1.5 

Primidone 

87.5 mg/kg 

175 mg/kg 

300 mg/kg 

350 mg/kg 

5 

5 

3 

3 

1.1 ± 0.3 

1.1 ± 0.4 

1.3 ± 0.3 

1.5 ± 0.6 

P=0.041 

a Study was performed at Environmental Health Research and Testing, Inc. The detailed protocol is presented by Shelby et al. (1993). b Mean ± standard error c Positive control 

d The 300 mg/kg dose group was omitted from the trend analysis because fewer than three animals survived in this group. e Significance of micronucleated cells/1,000 cells tested by the one-tailed trend test; significant at P#0.025 (Margolin et al., 1986) 
218 Primidone, NTP TR 476
 
219 

APPENDIX F 

ORGAN WEIGHTS AND 

ORGAN-WEIGHT-TO-BODY-WEIGHT RATIOS 

TABLE F1 

TABLE F2 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats 

in the 14-Week Feed Study of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice 

in the 14-Week Feed Study of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

220 

221 
220 Primidone, NTP TR 476 

TABLE F1 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in the 14-Week Feed Study of Primidonea 

0 ppm 300 ppm 600 ppm 1,300 ppm 2,500 ppm 5,000 ppm 

Male 

n 10 10 10 10 5b 5b 

Necropsy body wt 363 ± 6 376 ± 8 373 ± 3 359 ± 6 334 ± 8** 332 ± 3** 

Heart 

Absolute 1.090 ± 0.029 1.101 ± 0.023 1.095 ± 0.008 1.096 ± 0.037 1.058 ± 0.042 1.023 ± 0.021 

Relative 3.00 ± 0.06 2.94 ± 0.06 2.94 ± 0.02 3.06 ± 0.10 3.17 ± 0.09 3.09 ± 0.05 

R. Kidney 

Absolute 1.259 ± 0.029 1.326 ± 0.026 1.367 ± 0.021* 1.337 ± 0.037 1.314 ± 0.024 1.336 ± 0.034 

Relative 3.46 ± 0.05 3.53 ± 0.06 3.67 ± 0.06* 3.72 ± 0.05** 3.94 ± 0.05** 4.03 ± 0.10** 

Liver 

Absolute 15.069 ± 0.416 16.742 ± 0.685 18.184 ± 0.580** 18.729 ± 1.146** 22.977 ± 0.419** 25.264 ± 0.399** 

Relative 41.45 ± 0.77 44.48 ± 1.29 48.79 ± 1.44** 51.92 ± 2.58** 68.85 ± 1.30** 76.26 ± 1.54** 

Lung 

Absolute 1.739 ± 0.033 1.768 ± 0.043 1.818 ± 0.061 1.760 ± 0.063 1.516 ± 0.091 1.542 ± 0.052 

Relative 4.79 ± 0.09 4.74 ± 0.19 4.88 ± 0.16 4.91 ± 0.19 4.54 ± 0.26 4.66 ± 0.19 

R. Testis 

Absolute 1.417 ± 0.051 1.477 ± 0.027 1.467 ± 0.021 1.463 ± 0.022 1.440 ± 0.027 1.357 ± 0.118 

Relative 3.90 ± 0.13 3.94 ± 0.06 3.94 ± 0.07 4.08 ± 0.06 4.32 ± 0.11 4.09 ± 0.34 

Thymus 

Absolute 0.410 ± 0.010 0.416 ± 0.016 0.448 ± 0.016 0.413 ± 0.020 0.395 ± 0.021 0.423 ± 0.019 

Relative 1.13 ± 0.03 1.11 ± 0.04 1.21 ± 0.05 1.15 ± 0.05 1.18 ± 0.06 1.28 ± 0.06 

Thyroid Gland 

Absolute 0.028 ± 0.001 0.028 ± 0.001 0.029 ± 0.002 0.028 ± 0.002 0.030 ± 0.002 0.028 ± 0.003 

Relative 0.08 ± 0.00 0.08 ± 0.00 0.08 ± 0.01 0.08 ± 0.01 0.09 ± 0.01 0.09 ± 0.01 

Female 

n 10 10 10 10 10 10 

Necropsy body wt 201 ± 3 201 ± 3 199 ± 3 195 ± 3 190 ± 3** 187 ± 2** 

Heart 

Absolute 0.720 ± 0.022 0.683 ± 0.012 0.695 ± 0.017 0.684 ± 0.010 0.699 ± 0.018 0.704 ± 0.016 

Relative 3.58 ± 0.10 3.40 ± 0.07 3.50 ± 0.06 3.51 ± 0.04 3.69 ± 0.09 3.76 ± 0.10 

R. Kidney 

Absolute 0.725 ± 0.016 0.739 ± 0.012 0.744 ± 0.015 0.752 ± 0.014 0.755 ± 0.015 0.739 ± 0.013 

Relative 3.61 ± 0.06 3.68 ± 0.05 3.75 ± 0.04 3.86 ± 0.04** 3.98 ± 0.06** 3.95 ± 0.06** 

Liver 

Absolute 6.628 ± 0.146 7.280 ± 0.138* 7.219 ± 0.229* 8.173 ± 0.161** 9.578 ± 0.225** 10.913 ± 0.306** 

Relative 32.94 ± 0.54 36.24 ± 0.46* 36.33 ± 0.85* 41.99 ± 0.58** 50.54 ± 1.13** 58.22 ± 1.45** 

Lung 

Absolute 1.256 ± 0.050 1.137 ± 0.032 1.119 ± 0.030 1.194 ± 0.023 1.168 ± 0.059 1.174 ± 0.036 

Relative 6.23 ± 0.20 5.66 ± 0.12 5.63 ± 0.12 6.14 ± 0.10 6.15 ± 0.28 6.26 ± 0.18 

Thymus 

Absolute 0.305 ± 0.011 0.292 ± 0.012 0.307 ± 0.014 0.313 ± 0.008 0.312 ± 0.014 0.333 ± 0.009 

Relative 1.52 ± 0.05 1.46 ± 0.06 1.54 ± 0.06 1.61 ± 0.05 1.64 ± 0.06 1.78 ± 0.05** 

Thyroid Gland 

Absolute 0.027 ± 0.002 0.020 ± 0.002 0.023 ± 0.002 0.028 ± 0.003 0.025 ± 0.002 0.027 ± 0.002 

Relative 0.13 ± 0.01 0.10 ± 0.01 0.12 ± 0.01 0.14 ± 0.02 0.13 ± 0.01 0.15 ± 0.01 

* Significantly different (P#0.05) from the control group by Williams’ or Dunnett’s test 

** P#0.01 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 ± standard error). b Five animals omitted from analysis due to dehydration 
Primidone, NTP TR 476 221 

TABLE F2 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice in the 14-Week Feed Study of Primidonea 

0 ppm 300 ppm 600 ppm 1,300 ppm 2,500 ppm 5,000 ppm 

Male 

n 10 10 10 10 10 7 

Necropsy body wt 32.5 ± 0.7 32.5 ± 0.8 33.2 ± 1.1 32.1 ± 0.7 32.6 ± 0.8 29.9 ± 0.6 

Heart 

Absolute 0.167 ± 0.005 0.156 ± 0.005 0.178 ± 0.010 0.175 ± 0.009 0.166 ± 0.007 0.188 ± 0.009 

Relative 5.16 ± 0.18 4.79 ± 0.09 5.39 ± 0.31 5.45 ± 0.26 5.11 ± 0.18 6.32 ± 0.38** 

R. Kidney 

Absolute 0.294 ± 0.006 0.281 ± 0.005 0.295 ± 0.009 0.292 ± 0.006 0.302 ± 0.010 0.280 ± 0.008 

Relative 9.08 ± 0.22 8.69 ± 0.18 8.93 ± 0.16 9.11 ± 0.15 9.29 ± 0.20 9.36 ± 0.17 

Liver 

Absolute 1.587 ± 0.036 1.714 ± 0.053 1.989 ± 0.087** 2.225 ± 0.076** 2.709 ± 0.117** 2.956 ± 0.111** 

Relative 48.97 ± 1.12 52.74 ± 1.06 59.91 ± 1.11** 69.26 ± 1.64** 82.97 ± 1.97** 98.60 ± 2.11** 

Lung 

Absolute 0.243 ± 0.011 0.230 ± 0.007 0.243 ± 0.011 0.236 ± 0.007 0.236 ± 0.016 0.239 ± 0.017 

Relative 7.53 ± 0.45 7.09 ± 0.21 7.34 ± 0.23 7.36 ± 0.22 7.22 ± 0.39 8.01 ± 0.61 

R. Testis 

Absolute 0.123 ± 0.004 0.122 ± 0.002 0.124 ± 0.003 0.120 ± 0.003 0.126 ± 0.003 0.123 ± 0.003 

Relative 3.80 ± 0.13 3.76 ± 0.08 3.75 ± 0.10 3.75 ± 0.05 3.86 ± 0.06 4.11 ± 0.11 

Thymus 

Absolute 0.046 ± 0.003 0.050 ± 0.002 0.047 ± 0.003 0.044 ± 0.002 0.040 ± 0.004 0.042 ± 0.003 

Relative 1.43 ± 0.07 1.53 ± 0.07 1.42 ± 0.07 1.37 ± 0.07 1.22 ± 0.10 1.40 ± 0.11 

Thyroid Gland 

Absolute 0.011 ± 0.001 0.013 ± 0.001 0.011 ± 0.001 0.013 ± 0.001 0.013 ± 0.001 0.010 ± 0.001 

Relative 0.32 ± 0.04 0.41 ± 0.04 0.34 ± 0.04 0.42 ± 0.04 0.41 ± 0.03 0.33 ± 0.05 

Female 

n 10 10 10 10 10 8 

Necropsy body wt 28.4 ± 0.6 29.9 ± 0.6 31.0 ± 0.8 29.9 ± 1.0 29.3 ± 0.6 27.6 ± 0.6 

Heart 

Absolute 0.142 ± 0.006 0.141 ± 0.004 0.149 ± 0.006 0.143 ± 0.006 0.145 ± 0.006 0.143 ± 0.006 

Relative 5.05 ± 0.24 4.72 ± 0.10 4.84 ± 0.17 4.80 ± 0.16 4.95 ± 0.14 5.17 ± 0.21 

R. Kidney 

Absolute 0.202 ± 0.007 0.216 ± 0.009 0.223 ± 0.005 0.223 ± 0.005 0.217 ± 0.005 0.215 ± 0.007 

Relative 7.14 ± 0.31 7.23 ± 0.31 7.22 ± 0.19 7.48 ± 0.19 7.39 ± 0.08 7.79 ± 0.17 

Liver 

Absolute 1.382 ± 0.027 1.519 ± 0.032 1.739 ± 0.032** 1.981 ± 0.073** 2.299 ± 0.053** 2.746 ± 0.112** 

Relative 48.82 ± 1.03 50.95 ± 0.94 56.31 ± 0.87** 66.17 ± 1.26** 78.42 ± 1.04** 99.20 ± 2.23** 

Lung 

Absolute 0.226 ± 0.008 0.234 ± 0.010 0.230 ± 0.011 0.233 ± 0.014 0.226 ± 0.009 0.251 ± 0.016 

Relative 8.02 ± 0.38 7.85 ± 0.26 7.44 ± 0.34 7.77 ± 0.29 7.72 ± 0.28 9.17 ± 0.79 

Thymus 

Absolute 0.061 ± 0.005 0.066 ± 0.003 0.064 ± 0.004 0.063 ± 0.003 0.059 ± 0.003 0.058 ± 0.004 

Relative 2.16 ± 0.14 2.21 ± 0.12 2.06 ± 0.10 2.12 ± 0.08 2.03 ± 0.11 2.09 ± 0.13 

Thyroid Gland 

Absolute 0.006 ± 0.001 0.007 ± 0.001 0.007 ± 0.002 0.007 ± 0.001 0.006 ± 0.001 0.009 ± 0.002 

Relative 0.22 ± 0.04 0.25 ± 0.05 0.22 ± 0.05 0.23 ± 0.04 0.22 ± 0.04 0.31 ± 0.08 

** Significantly different (P#0.01) from the control group by Williams’ or Dunnett’s test 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 ± standard error). 
222 Primidone, NTP TR 476
 
223 

APPENDIX G
 

HEMATOLOGY 

AND CLINICAL CHEMISTRY RESULTS
 

TABLE G1	 Hematology and Clinical Chemistry Data for Rats 

in the 14-Week Feed Study of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224 
224 Primidone, NTP TR 476 

TABLE G1 

Hematology and Clinical Chemistry Data for Rats in the 14-Week Feed Study of Primidonea 

0 ppm 300 ppm 600 ppm 1,300 ppm 2,500 ppm 5,000 ppm 

Male 

n 10 10 10 10 10 10 

Hematology 

Hematocrit (%) 

Day 4 40.0 ± 0.4 38.7 ± 0.2 39.0 ± 0.4b 39.0 ± 0.3 39.0 ± 0.4 40.2 ± 0.5 

Day 22 

Week 14 

44.6 ± 0.5 

45.6 ± 0.4 

46.0 ± 0.4 

44.9 ± 0.4b 

45.4 ± 0.5 

46.3 ± 0.5 

44.8 ± 0.3 

47.6 ± 0.7 

45.0 ± 0.5 

44.6 ± 0.3 

44.4 ± 0.5 

44.6 ± 0.3 

Hemoglobin (g/dL) 

Day 4 13.3 ± 0.1 13.0 ± 0.1 13.2 ± 0.1 13.1 ± 0.1 13.1 ± 0.1 13.4 ± 0.2 

Day 22 14.7 ± 0.2 15.0 ± 0.1 14.9 ± 0.1 14.6 ± 0.1 14.4 ± 0.1 14.3 ± 0.2 

Week 14 15.0 ± 0.1 14.9 ± 0.1 15.1 ± 0.2 15.3 ± 0.1 14.5 ± 0.1** 14.4 ± 0.1** 

Erythrocytes (106

/μL) 

Day 4 6.28 ± 0.07 6.05 ± 0.04 6.14 ± 0.07 6.12 ± 0.07 6.15 ± 0.07 6.41 ± 0.09 

Day 22 7.29 ± 0.10 7.39 ± 0.11 7.26 ± 0.08 7.13 ± 0.05 7.10 ± 0.09 7.01 ± 0.11 

Week 14 8.23 ± 0.06 8.12 ± 0.07 8.24 ± 0.09 8.43 ± 0.11 8.09 ± 0.06 8.13 ± 0.07 

Reticulocytes (106

/μL) 

Day 4 0.42 ± 0.03 0.40 ± 0.02 0.38 ± 0.02 0.38 ± 0.02 0.40 ± 0.02 0.35 ± 0.02 

Day 22 0.22 ± 0.02 0.21 ± 0.01 0.22 ± 0.02 0.23 ± 0.01 0.22 ± 0.01 0.22 ± 0.01 

Week 14 0.12 ± 0.01 0.13 ± 0.01 0.13 ± 0.01 0.14 ± 0.01 0.14 ± 0.01 0.14 ± 0.01 

Nucleated erythrocytes (103

/μL) 

Day 4 0.12 ± 0.03 0.04 ± 0.02 0.14 ± 0.03 0.08 ± 0.02 0.12 ± 0.03 0.07 ± 0.03 

Day 22 0.02 ± 0.01 0.00 ± 0.00 0.05 ± 0.02 0.03 ± 0.01 0.04 ± 0.03 0.04 ± 0.02 

Week 14 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 

Mean cell volume (fL) 

Day 4 63.7 ± 0.4 63.9 ± 0.3 63.6 ± 0.2 63.7 ± 0.2 63.3 ± 0.3 62.7 ± 0.3* 

Day 22 61.2 ± 0.3 62.2 ± 0.4 62.6 ± 0.4* 62.8 ± 0.3** 63.4 ± 0.4** 63.3 ± 0.4** 

Week 14 55.5 ± 0.3 55.3 ± 0.3 56.3 ± 0.3 56.5 ± 0.3 55.2 ± 0.1 54.8 ± 0.2 

Mean cell hemoglobin (pg) 

Day 4 21.2 ± 0.2 21.5 ± 0.2 21.4 ± 0.1 21.4 ± 0.1 21.3 ± 0.1 21.0 ± 0.2 

Day 22 20.2 ± 0.1 20.3 ± 0.1 20.5 ± 0.1 20.4 ± 0.1 20.3 ± 0.2 20.4 ± 0.1 

Week 14 18.2 ± 0.1 18.4 ± 0.1 18.3 ± 0.1 18.1 ± 0.1 17.9 ± 0.1* 17.8 ± 0.1** 

Mean cell hemoglobin concentration (g/dL) 

Day 4 33.4 ± 0.2 33.6 ± 0.1 33.7 ± 0.1 33.6 ± 0.2 33.6 ± 0.1 33.5 ± 0.2 

Day 22 32.9 ± 0.1 32.7 ± 0.1 32.7 ± 0.2 32.5 ± 0.1* 32.1 ± 0.3* 32.3 ± 0.1** 

Week 14 32.9 ± 0.3 33.2 ± 0.2 32.6 ± 0.2 32.1 ± 0.3* 32.4 ± 0.1 32.4 ± 0.1 

Platelets (103

/μL) 

Day 4 910.5 ± 23.3 956.1 ± 9.6 968.4 ± 15.7 922.8 ± 10.4 911.3 ± 13.1 936.9 ± 10.0 

Day 22 822.7 ± 13.1 885.5 ± 15.1** 874.9 ± 13.1* 915.6 ± 10.8** 926.1 ± 10.3** 900.1 ± 8.1** 

Week 14 684.3 ± 13.4 697.4 ± 8.3 755.2 ± 9.3** 785.1 ± 15.0** 804.2 ± 9.7** 818.5 ± 12.9** 

Leukocytes (103

/μL) 

Day 4 7.59 ± 0.48 7.38 ± 0.28 7.79 ± 0.37 7.48 ± 0.49 7.54 ± 0.51 7.22 ± 0.37 

Day 22 7.13 ± 0.31 7.10 ± 0.35 8.77 ± 0.46* 8.60 ± 0.62* 9.16 ± 0.33** 9.50 ± 0.46** 

Week 14 10.75 ± 0.32 8.88 ± 0.72 10.16 ± 0.50 9.80 ± 0.65 8.19 ± 0.32** 8.83 ± 0.28 

Segmented neutrophils (103

/μL) 

Day 4 0.92 ± 0.09 0.76 ± 0.09 1.03 ± 0.09 1.00 ± 0.11 0.91 ± 0.11 0.97 ± 0.06 

Day 22 0.80 ± 0.06 1.00 ± 0.14 0.81 ± 0.06 0.81 ± 0.07 0.95 ± 0.07 0.91 ± 0.11 

Week 14 1.70 ± 0.18 1.08 ± 0.12* 1.25 ± 0.13 1.28 ± 0.09 1.23 ± 0.12 1.38 ± 0.07 

Lymphocytes (103

/μL) 

Day 4 6.51 ± 0.42 6.46 ± 0.26 6.57 ± 0.36 6.35 ± 0.46 6.50 ± 0.41 6.13 ± 0.36 

Day 22 6.14 ± 0.30 6.00 ± 0.31 7.74 ± 0.47* 7.61 ± 0.62* 8.11 ± 0.34** 8.36 ± 0.43** 

Week 14 8.79 ± 0.18 7.51 ± 0.61 8.63 ± 0.50 8.24 ± 0.58 6.75 ± 0.27** 7.17 ± 0.27 
Primidone, NTP TR 476 225 

TABLE G1 

Hematology and Clinical Chemistry Data for Rats in the 14-Week Feed Study of Primidone 

0 ppm 300 ppm 600 ppm 1,300 ppm 2,500 ppm 5,000 ppm 

Male (continued) 

n 10 10 10 10 10 10 

Hematology (continued) 

Monocytes (103

/μL) 

Day 4 0.05 ± 0.02 0.10 ± 0.02 0.13 ± 0.02 0.07 ± 0.02 0.10 ± 0.03 0.09 ± 0.02 

Day 22 0.15 ± 0.03 0.07 ± 0.02 0.13 ± 0.03 0.16 ± 0.03 0.07 ± 0.02 0.16 ± 0.04 

Week 14 0.19 ± 0.04 0.18 ± 0.05 0.14 ± 0.02 0.17 ± 0.05 0.11 ± 0.03 0.17 ± 0.04 

Eosinophils (103

/μL) 

Day 4 0.06 ± 0.02 0.06 ± 0.02 0.05 ± 0.01 0.07 ± 0.03 0.03 ± 0.01 0.03 ± 0.01 

Day 22 0.04 ± 0.02 0.04 ± 0.02 0.09 ± 0.03 0.02 ± 0.01 0.03 ± 0.01 0.07 ± 0.03 

Week 14 0.08 ± 0.03 0.11 ± 0.03 0.10 ± 0.03 0.12 ± 0.03 0.10 ± 0.03 0.11 ± 0.04 

Clinical Chemistry 

Urea nitrogen (mg/dL) 

Day 4 22.4 ± 0.8 21.3 ± 0.7 21.5 ± 0.3 21.1 ± 0.4 20.9 ± 0.3 22.8 ± 0.4 

Day 22 23.6 ± 0.7 20.9 ± 0.9 23.9 ± 0.7 23.3 ± 0.4 22.9 ± 0.6 23.3 ± 0.6 

Week 14 22.2 ± 0.6 22.4 ± 0.4 21.5 ± 0.3 22.5 ± 0.5 23.1 ± 0.8 23.2 ± 0.6 

Creatinine (mg/dL) 

Day 4 0.63 ± 0.03 0.61 ± 0.01 0.63 ± 0.02 0.61 ± 0.01 0.62 ± 0.01 0.61 ± 0.02 

Day 22 0.65 ± 0.02 0.61 ± 0.01 0.66 ± 0.02 0.67 ± 0.02 0.60 ± 0.00 0.63 ± 0.02 

Week 14 0.72 ± 0.02 0.78 ± 0.07 0.74 ± 0.02 0.71 ± 0.02 0.68 ± 0.01 0.71 ± 0.01 

Total protein (g/dL) 

Day 4 5.8 ± 0.1 5.8 ± 0.0 5.8 ± 0.1 5.7 ± 0.0 5.5 ± 0.1* 5.7 ± 0.1 

Day 22 6.6 ± 0.0 6.9 ± 0.1** 7.0 ± 0.1** 7.1 ± 0.1** 7.1 ± 0.1** 7.1 ± 0.1** 

Week 14 7.2 ± 0.1 7.1 ± 0.1 7.5 ± 0.1* 7.8 ± 0.1** 8.0 ± 0.1** 8.1 ± 0.1** 

Albumin (g/dL) 

Day 4 4.3 ± 0.1 4.2 ± 0.0 4.2 ± 0.1 4.1 ± 0.0* 4.0 ± 0.0** 4.1 ± 0.1** 

Day 22 4.7 ± 0.0 4.8 ± 0.1 4.9 ± 0.1 4.9 ± 0.1 4.9 ± 0.1 4.9 ± 0.1 

Week 14 5.0 ± 0.0 5.0 ± 0.0 5.2 ± 0.1* 5.4 ± 0.0** 5.5 ± 0.0** 5.4 ± 0.1** 

Alanine aminotransferase (IU/L) 

Day 4 44 ± 2 41 ± 1 42 ± 2 46 ± 1 47 ± 1 50 ± 1** 

Day 22 45 ± 1 40 ± 2* 42 ± 2 35 ± 1** 32 ± 1** 33 ± 1** 

Week 14 45 ± 1 47 ± 2 41 ± 2 43 ± 1 49 ± 3 50 ± 5 

Alkaline phosphatase (IU/L) 

Day 4 1,645 ± 40 1,684 ± 30 1,652 ± 30 1,732 ± 48 1,682 ± 26 1,698 ± 52 

Day 22 1,225 ± 27 1,131 ± 26* 1,232 ± 28 1,141 ± 26 1,054 ± 15** 981 ± 19** 

Week 14 553 ± 13 527 ± 12 552 ± 13 423 ± 30** 476 ± 16** 427 ± 10** 

Creatine kinase (IU/L) 

Day 4 273 ± 30 250 ± 14 245 ± 21 314 ± 42 245 ± 12 273 ± 23 

Day 22 404 ± 85 358 ± 65 410 ± 57 401 ± 61 366 ± 56 419 ± 90 

Week 14 252 ± 32 322 ± 28 296 ± 33 361 ± 36* 500 ± 42** 507 ± 30** 

Sorbitol dehydrogenase (IU/L) 

Day 4 18 ± 1 17 ± 1 18 ± 1 19 ± 1 20 ± 1 23 ± 1** 

Day 22 21 ± 1 18 ± 1 21 ± 1 22 ± 2 23 ± 3 23 ± 1 

Week 14 22 ± 1 25 ± 1 24 ± 3 21 ± 2 18 ± 1 20 ± 1 

Bile salts (μmol/L) 

Day 4 25.1 ± 3.6 22.8 ± 1.8 26.3 ± 3.1 37.0 ± 2.8* 38.8 ± 7.2 45.3 ± 6.2* 

Day 22 22.1 ± 3.2 17.2 ± 1.8 17.7 ± 1.3 27.8 ± 4.7 21.0 ± 2.3 21.9 ± 1.4 

Week 14 11.0 ± 2.1 12.2 ± 1.2 10.9 ± 1.9 8.4 ± 0.8 11.0 ± 1.4 9.4 ± 1.0 
226 Primidone, NTP TR 476 

TABLE G1 

Hematology and Clinical Chemistry Data for Rats in the 14-Week Feed Study of Primidone 

0 ppm 300 ppm 600 ppm 1,300 ppm 2,500 ppm 5,000 ppm 

Female 

n 10 10 10 10 10 10 

Hematology 

Hematocrit (%) 

Day 4 40.5 ± 0.3 40.4 ± 0.4 41.2 ± 0.4 40.9 ± 0.4 40.6 ± 0.5 42.8 ± 0.7 

Day 22 47.8 ± 0.4 47.7 ± 0.5 48.6 ± 0.3 47.7 ± 0.4 47.2 ± 0.3 46.5 ± 0.4 

Week 14 45.6 ± 0.5 45.7 ± 0.3 45.5 ± 0.3 45.8 ± 0.3 45.3 ± 0.3 44.5 ± 0.4 

Hemoglobin (g/dL) 

Day 4 13.6 ± 0.1 13.5 ± 0.1 13.6 ± 0.1 13.7 ± 0.1 13.6 ± 0.2 14.1 ± 0.3 

Day 22 15.7 ± 0.1 15.5 ± 0.1 15.6 ± 0.1 15.5 ± 0.1 15.2 ± 0.1* 15.1 ± 0.1** 

Week 14 15.0 ± 0.1 15.2 ± 0.1 15.0 ± 0.1 15.0 ± 0.1 14.8 ± 0.1 14.5 ± 0.1** 

Erythrocytes (106

/μL) 

Day 4 6.35 ± 0.08 6.34 ± 0.07 6.49 ± 0.08 6.50 ± 0.09 6.41 ± 0.09 6.90 ± 0.10** 

Day 22 7.62 ± 0.09 7.51 ± 0.09 7.66 ± 0.06 7.55 ± 0.08 7.40 ± 0.07 7.45 ± 0.07 

Week 14 7.66 ± 0.07 7.71 ± 0.06 7.65 ± 0.06 7.76 ± 0.04 7.75 ± 0.05 7.66 ± 0.08 

Reticulocytes (106

/μL) 

Day 4 0.29 ± 0.02 0.31 ± 0.02 0.30 ± 0.02 0.30 ± 0.02 0.31 ± 0.02 0.28 ± 0.02 

Day 22 0.12 ± 0.01 0.11 ± 0.01 0.12 ± 0.01 0.11 ± 0.01 0.13 ± 0.01 0.14 ± 0.01 

Week 14 0.10 ± 0.01 0.12 ± 0.01 0.10 ± 0.01 0.09 ± 0.01 0.11 ± 0.01 0.12 ± 0.01 

Nucleated erythrocytes (103

/μL) 

Day 4 0.07 ± 0.02 0.09 ± 0.02 0.08 ± 0.03 0.04 ± 0.02 0.07 ± 0.02 0.03 ± 0.02 

Day 22 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 

Week 14 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 

Mean cell volume (fL) 

Day 4 63.7 ± 0.5 63.8 ± 0.3 63.5 ± 0.3 63.0 ± 0.4 63.3 ± 0.5 62.0 ± 0.2** 

Day 22 62.8 ± 0.3 63.6 ± 0.3 63.4 ± 0.3 63.2 ± 0.3 63.7 ± 0.3 62.5 ± 0.3 

Week 14 59.5 ± 0.2 59.3 ± 0.3 59.5 ± 0.1 59.0 ± 0.2 58.4 ± 0.2** 58.1 ± 0.1** 

Mean cell hemoglobin (pg) 

Day 4 21.4 ± 0.2 21.3 ± 0.1 21.0 ± 0.2 21.1 ± 0.2 21.3 ± 0.1 20.5 ± 0.1** 

Day 22 20.6 ± 0.1 20.6 ± 0.2 20.4 ± 0.1 20.5 ± 0.1 20.6 ± 0.1 20.3 ± 0.1 

Week 14 19.6 ± 0.1 19.7 ± 0.1 19.6 ± 0.1 19.3 ± 0.1** 19.1 ± 0.1** 18.9 ± 0.1** 

Mean cell hemoglobin concentration (g/dL) 

Day 4 33.5 ± 0.1 33.4 ± 0.1 33.2 ± 0.2 33.4 ± 0.2 33.6 ± 0.1 33.0 ± 0.2 

Day 22 32.8 ± 0.1 32.5 ± 0.2 32.2 ± 0.2** 32.5 ± 0.1 32.3 ± 0.1** 32.5 ± 0.1 

Week 14 33.0 ± 0.3 33.1 ± 0.3 32.9 ± 0.1 32.8 ± 0.2 32.7 ± 0.1 32.6 ± 0.2 

Platelets (103

/μL) 

Day 4 938.9 ± 15.2 967.1 ± 17.9 961.9 ± 14.1 960.6 ± 20.8 920.3 ± 17.9 959.9 ± 19.5 

Day 22 697.9 ± 28.0 721.7 ± 14.6 754.7 ± 14.9 804.6 ± 18.4** 813.3 ± 13.5** 840.3 ± 17.8** 

Week 14 698.0 ± 8.4 684.8 ± 9.0 710.7 ± 14.0 754.9 ± 6.6** 752.2 ± 15.2** 768.3 ± 17.4** 

Leukocytes (103

/μL) 

Day 4 7.39 ± 0.41 8.11 ± 0.21 8.10 ± 0.35 8.49 ± 0.40 7.68 ± 0.26 9.31 ± 0.42* 

Day 22 6.28 ± 0.38 7.19 ± 0.31* 7.36 ± 0.37* 7.09 ± 0.22 6.88 ± 0.24 8.13 ± 0.23** 

Week 14 5.89 ± 0.47 6.42 ± 0.31 6.63 ± 0.46 6.51 ± 0.31 6.02 ± 0.30 6.76 ± 0.27 

Segmented neutrophils (103

/μL) 

Day 4 0.84 ± 0.12 0.78 ± 0.08 0.94 ± 0.09 0.77 ± 0.09 0.80 ± 0.07 1.10 ± 0.09 

Day 22 0.69 ± 0.11 0.83 ± 0.06 0.86 ± 0.10 0.70 ± 0.07 0.70 ± 0.06 1.04 ± 0.12 

Week 14 1.02 ± 0.13 1.07 ± 0.12 1.06 ± 0.12 0.99 ± 0.10 0.88 ± 0.09 0.76 ± 0.10 

Lymphocytes (103

/μL) 

Day 4 6.42 ± 0.33 7.19 ± 0.24 7.03 ± 0.35 7.55 ± 0.39 6.77 ± 0.29 7.96 ± 0.35* 

Day 22 5.45 ± 0.29 6.22 ± 0.34 6.35 ± 0.28 6.20 ± 0.22 6.08 ± 0.25 6.95 ± 0.23** 

Week 14 4.68 ± 0.35 5.23 ± 0.31 5.32 ± 0.34 5.35 ± 0.29 5.04 ± 0.30 5.84 ± 0.22* 
Primidone, NTP TR 476 227 

TABLE G1 

Hematology and Clinical Chemistry Data for Rats in the 14-Week Feed Study of Primidone 

0 ppm 300 ppm 600 ppm 1,300 ppm 2,500 ppm 5,000 ppm 

Female (continued) 

n 10 10 10 10 10 10 

Hematology (continued) 

Monocytes (103

/μL) 

Day 4 0.08 ± 0.01 0.10 ± 0.02 0.11 ± 0.03 0.12 ± 0.03 0.08 ± 0.02 0.19 ± 0.04 

Day 22 0.09 ± 0.02 0.11 ± 0.03 0.05 ± 0.02 0.15 ± 0.04 0.07 ± 0.02 0.10 ± 0.03 

Week 14 0.11 ± 0.03 0.07 ± 0.03 0.18 ± 0.03 0.06 ± 0.02 0.06 ± 0.02 0.09 ± 0.03 

Eosinophils (103

/μL) 

Day 4 0.06 ± 0.02 0.04 ± 0.02 0.02 ± 0.01 0.05 ± 0.02 0.03 ± 0.02 0.06 ± 0.03 

Day 22 0.04 ± 0.02 0.03 ± 0.02 0.09 ± 0.03 0.04 ± 0.02 0.03 ± 0.01 0.05 ± 0.02 

Week 14 0.09 ± 0.02 0.05 ± 0.02 0.07 ± 0.03 0.11 ± 0.02 0.04 ± 0.02 0.07 ± 0.02 

Clinical Chemistry 

Urea nitrogen (mg/dL) 

Day 4 22.2 ± 0.6 22.7 ± 0.7 24.4 ± 1.0 23.6 ± 0.6 23.4 ± 0.9 23.4 ± 0.9 

Day 22 23.9 ± 0.7 23.7 ± 0.5 23.6 ± 0.8 23.1 ± 0.7 23.1 ± 0.7 23.4 ± 0.6 

Week 14 20.4 ± 0.6 20.2 ± 0.7 18.7 ± 0.5 17.8 ± 0.2* 17.5 ± 0.6** 19.5 ± 0.7 

Creatinine (mg/dL) 

Day 4 0.56 ± 0.02 0.58 ± 0.01 0.55 ± 0.02 0.56 ± 0.02 0.58 ± 0.01 0.57 ± 0.02 

Day 22 0.66 ± 0.02 0.62 ± 0.01 0.63 ± 0.02 0.64 ± 0.02 0.63 ± 0.02 0.64 ± 0.02 

Week 14 0.64 ± 0.02 0.66 ± 0.02 0.66 ± 0.02 0.66 ± 0.02 0.68 ± 0.01 0.68 ± 0.01 

Total protein (g/dL) 

Day 4 5.7 ± 0.0 5.6 ± 0.1 5.7 ± 0.0 5.7 ± 0.1 5.6 ± 0.1 5.8 ± 0.1 

Day 22 6.5 ± 0.1 6.6 ± 0.1 6.6 ± 0.1 6.8 ± 0.1 6.7 ± 0.0 6.8 ± 0.1** 

Week 14 7.1 ± 0.1 7.2 ± 0.1 7.2 ± 0.1 7.3 ± 0.1 7.5 ± 0.1** 7.6 ± 0.1** 

Albumin (g/dL) 

Day 4 4.2 ± 0.0 4.2 ± 0.0 4.3 ± 0.0 4.2 ± 0.1 4.2 ± 0.1 4.2 ± 0.1 

Day 22 4.8 ± 0.1 4.8 ± 0.1 4.7 ± 0.1 4.8 ± 0.1 4.7 ± 0.0 4.7 ± 0.0 

Week 14 5.2 ± 0.1 5.2 ± 0.1 5.2 ± 0.1 5.2 ± 0.0 5.2 ± 0.1 5.4 ± 0.0 

Alanine aminotransferase (IU/L) 

Day 4 33 ± 1 36 ± 1** 38 ± 1** 39 ± 1** 39 ± 1** 39 ± 1** 

Day 22 34 ± 1 32 ± 1 32 ± 2 30 ± 2 30 ± 1 32 ± 1 

Week 14 32 ± 2 32 ± 1 32 ± 1 29 ± 1 27 ± 1** 30 ± 1 

Alkaline phosphatase (IU/L) 

Day 4 1,316 ± 22 1,354 ± 31 1,385 ± 34 1,345 ± 31 1,369 ± 27 1,220 ± 32 

Day 22 869 ± 16 853 ± 29 831 ± 15 738 ± 14** 719 ± 18** 714 ± 15** 

Week 14 434 ± 13 379 ± 7** 373 ± 10** 308 ± 9** 303 ± 8** 282 ± 7** 

Creatine kinase (IU/L) 

Day 4 216 ± 20 186 ± 18 231 ± 32 267 ± 25 237 ± 34 214 ± 13 

Day 22 

Week 14 

342 ± 37 

201 ± 23b 

434 ± 56 

290 ± 35 

374 ± 51 

280 ± 68 

306 ± 30 

224 ± 34 

338 ± 46 

216 ± 17 

368 ± 57 

312 ± 40 

Sorbitol dehydrogenase (IU/L) 

Day 4 15 ± 1 15 ± 1 17 ± 1 20 ± 1** 20 ± 1** 24 ± 1** 

Day 22 22 ± 1 21 ± 1 20 ± 1 27 ± 3 24 ± 1 26 ± 1 

Week 14 19 ± 2 19 ± 1 21 ± 1 22 ± 1** 20 ± 1 22 ± 1* 

Bile salts (μmol/L) 

Day 4 21.9 ± 3.0 23.9 ± 1.0 17.9 ± 2.6 18.1 ± 2.7 30.7 ± 3.1 22.1 ± 3.3 

Day 22 25.2 ± 4.7 20.6 ± 2.3 11.8 ± 1.5* 14.3 ± 1.8 16.5 ± 1.6 15.5 ± 1.3 

Week 14 21.5 ± 4.4 20.5 ± 2.6 21.2 ± 2.5 13.8 ± 2.0 17.9 ± 1.8 21.5 ± 4.9 

* Significantly different (P#0.05) from the control group by Dunn’s or Shirley’s test 

** P#0.01 a Mean ± standard error. Statistical tests were performed on unrounded data. b n=9 for the male 300 ppm group at week 14 and for the 600 ppm group on day 4 for hematology data. 
228 Primidone, NTP TR 476
 
229 

APPENDIX H 

DETERMINATIONS 

OF PRIMIDONE AND PHENOBARBITAL 

IN PLASMA 

TABLE H1 

TABLE H2 

FIGURE H1 

FIGURE H2 

TABLE H3 

TABLE H4 

FIGURE H3 

FIGURE H4 

Plasma Concentrations of Primidone in Female Rats 

in the 2-Year Feed Study of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

Plasma Concentrations of Phenobarbital in Female Rats 

in the 2-Year Feed Study of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

Plasma Concentrations of Primidone in Female Rats after Exposure 

to 600, 1,300, or 2,500 ppm Primidone in Feed for 2 Years .................... 

Plasma Concentrations of Phenobarbital in Female Rats after Exposure 

to 600, 1,300, or 2,500 ppm Primidone in Feed for 2 Years .................... 

Plasma Concentrations of Phenobarbital in Male Mice 

in the 2-Year Feed Study of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

Plasma Concentrations of Phenobarbital in Female Mice 

in the 2-Year Feed Study of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

Plasma Concentrations of Phenobarbital in Male Mice after Exposure 

to 300, 600, or 1,300 ppm Primidone in Feed for 2 Years ..................... 

Plasma Concentrations of Phenobarbital in Female Mice after Exposure 

to 300, 600, or 1,300 ppm Primidone in Feed for 2 Years ..................... 

230 

230 

231 

231 

232 

232 

233 

233 
230 Primidone, NTP TR 476 

TABLE H1 

Plasma Concentrations of Primidone in Female Rats in the 2-Year Feed Study of Primidonea 

Time of Dayb 600 ppm 1,300 ppm 2,500 ppm 

n 3 3 3 

Day 721 

8:30 a.m. 

2:30 p.m. 

8:00 p.m. 

10:00 p.m. 

0.73 ± 0.52 

0.95 ± 0.39 

1.37 ± 0.71 

1.62 ± 0.56 

1.46 ± 0.95 

1.52 ± 1.64 

2.54 ± 0.45 

3.28 ± 0.93 

2.55 ± 1.10 

1.98 ± 1.28 

8.27 ± 5.56 

6.32 ± 2.24 

Day 722 

8:30 a.m. 1.22 ± 0.34 2.16 ± 0.63 2.95 ± 1.74 

a Data are given in μg/mL as mean ± standard deviation. b Samples were collected on the last 2 days of the study. 

TABLE H2 

Plasma Concentrations of Phenobarbital in Female Rats in the 2-Year Feed Study of Primidonea 

Time of Dayb 600 ppm 1,300 ppm 2,500 ppm 

n 3 3 3 

Day 721 

8:30 a.m. 

2:30 p.m. 

8:00 p.m. 

10:00 p.m. 

5.84 ± 0.88 

4.52 ± 0.47 

4.47 ± 1.33 

5.84 ± 0.30 

10.3 ± 1.7 

9.1 ± 1.4 

12.6 ± 3.5 

12.2 ± 0.7 

17.9 ± 0.1 

18.0 ± 1.3 

19.7 ± 2.5 

19.2 ± 2.1 

Day 722 

8:30 a.m. 4.68 ± 2.13 10.5 ± 0.6 16.6 ± 1.4 

a Data are given in μg/mL as mean ± standard deviation. b Samples were collected on the last 2 days of the study. 
Primidone, NTP TR 476 231 

Figure H1 

Plasma Concentrations of Primidone in Female Rats after Exposure 

to 600, 1,300, or 2,500 ppm Primidone in feed for 2 Years 

Figure H2 

Plasma Concentrations of Phenobarbital in Female Rats after Exposure 

to 600, 1,300, or 2,500 ppm Primidone in feed for 2 Years 
232 

c 

Primidone, NTP TR 476 

TABLE H3 

Plasma Concentrations of Phenobarbital in Male Mice in the 2-Year Feed Study of Primidonea 

Time of Dayb 300 ppm 600 ppm 1,300 ppm 

n 3 3 3 

Day 726 

8:30 a.m. 

2:30 p.m. 

8:00 p.m. 

10:00 p.m. 

2.73 ± 1.80 

1.83 ± 1.23 

3.31 ± 1.31 

3.82 ± 0.23 

5.50 ± 1.89 

1.61 ± 0.51 

3.33 ± 1.47 

6.07 ± 1.07 

7.88 ± 4.27 

1.86 ± 1.50 

5.88 ± 1.63 

11.8 ± 2.5 

Day 727 

8:30 a.m. 3.43 ± 0.25 6.17 ± 2.69 12.0 ± 3.2 

a Data are given in μg/mL as mean ± standard deviation. b Samples were collected on the last 2 days of the study. 

TABLE H4 

Plasma Concentrations of Phenobarbital in Female Mice in the 2-Year Feed Study of Primidonea 

Time of Dayb 300 ppm 600 ppm 1,300 ppm 

n 3 3 3 

Day 727 

8:30 a.m. 

2:30 p.m. 

8:00 p.m. 

10:00 p.m. 

3.41 ± 1.07 

2.16 ± 0.62 

0.95 ± 0.61c 

1.03 ± 0.26 

5.68 ± 1.99 

4.14 ± 1.07 

2.28 ± 0.85 

2.13 ± 0.79 

13.0 ± 3.5 

3.05 ± 2.35 

1.12 ± 0.76 

3.36 ± 2.12 

Day 728 

8:30 a.m. 2.17 ± 0.72 7.57 ± 1.86 16.6 ± 11.6 

a Data are given in μg/mL as mean ± standard deviation. b Samples were collected on the last 2 days of the study. 

n=2 
Figure H3 

Plasma Concentrations of Phenobarbital in Male Mice after Exposure 

to 300, 600, or 1,300 ppm Primidone in Feed for 2 Years 

Primidone, NTP TR 476 233 

Figure H4 

Plasma Concentrations of Phenobarbital in Female Mice after Exposure 

to 300, 600, or 1,300 ppm Primidone in Feed for 2 Years 
234 Primidone, NTP TR 476
 
235 

APPENDIX I 

REPRODUCTIVE TISSUE EVALUATIONS 

AND ESTROUS CYCLE CHARACTERIZATION 

TABLE I1 

TABLE I2 

Summary of Reproductive Tissue Evaluations and Estrous Cycle Characterization 

for Rats in the 14-Week Feed Study of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . 

Summary of Reproductive Tissue Evaluations and Estrous Cycle Characterization 

for Mice in the 14-Week Feed Study of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . 

236 

237 
236 Primidone, NTP TR 476 

TABLE I1 

Summary of Reproductive Tissue Evaluations and Estrous Cycle Characterization for Rats 

in the 14-Week Feed Study of Primidonea 

0 ppm 1,300 ppm 2,500 ppm 5,000 ppm 

Male 

n 10 10 10 10 

Weights (g) 

Necropsy body wt 363 ± 6 359 ± 6 334 ± 8** 332 ± 3** 

L. cauda 0.1532 ± 0.0044 0.1529 ± 0.0043 0.1536 ± 0.0039 0.1414 ± 0.0061 

L. epididymis 0.4272 ± 0.0068 0.4355 ± 0.0077 0.4265 ± 0.0069 0.4146 ± 0.0143 

L. testis 1.4584 ± 0.0307 1.5167 ± 0.0274 1.5011 ± 0.0168 1.4495 ± 0.0530 

Spermatid measurements 

Spermatid heads (107

/g testis) 9.36 ± 0.40 8.62 ± 0.24 8.20 ± 0.31 9.37 ± 0.30 

Spermatid heads (107

/testis) 13.64 ± 0.61 13.06 ± 0.34 12.30 ± 0.45 13.61 ± 0.72 

Spermatid count 

(mean/10-4 mL suspension) 68.20 ± 3.03 65.28 ± 1.71 61.48 ± 2.25 68.03 ± 3.59 

Epididymal spermatozoal measurements 

Motility (%) 70.69 ± 2.55 73.18 ± 1.68 73.38 ± 1.57 70.34 ± 2.17 

Concentration 

(106

/g cauda epididymal tissue) 719 ± 64 611 ± 60 824 ± 71 699 ± 63 

Female 

n 10 10 10 10 

Necropsy body wt (g) 

Estrous cycle length (days) 

201 ± 3 

4.95 ± 0.16 

195 ± 3 

5.00 ± 0.00 

190 ± 3** 

5.10 ± 0.10 

187 ± 2** 

5.17 ± 0.22b 

Estrous stages (% of cycle) 

Diestrus 41.7 38.3 38.3 40.8 

Proestrus 15.0 16.7 17.5 15.8 

Estrus 24.2 23.3 25.8 23.3 

Metestrus 19.2 21.7 18.3 20.0 

** Significantly different (P#0.01) from the control group by Williams’ or Dunnett’s test a Weights, spermatid and epididymal spermatozoal parameters, and estrous cycle lengths are presented as mean ± standard error. 

Differences from the control group for organ weights are not significant by Dunnett’s test; differences from the control group for 

spermatid and epididymal spermatozoal parameters and estrous cycle length are not significant by Dunn’s test. By multivariate analysis 

of variance, exposed females do not differ significantly from the control females in relative length of time spent in the estrous stages. b Estrous cycle was longer than 12 days or unclear in 1 of 10 animals. 
 

 

237 

c 

Primidone, NTP TR 476 

TABLE I2 

Summary of Reproductive Tissue Evaluations and Estrous Cycle Characterization for Mice 

in the 14-Week Feed Study of Primidonea 

0 ppm 1,300 ppm 2,500 ppm 5,000 ppm 

Male 

n 10 10 10 7 

Weights (g) 

Necropsy body wt 32.5 ± 0.7 32.1 ± 0.7 32.6 ± 0.8 29.9 ± 0.6 

L. cauda 0.0133 ± 0.0006 0.0135 ± 0.0004 0.0132 ± 0.0004 0.0131 ± 0.0006 

L. epididymis 0.0387 ± 0.0009 0.0388 ± 0.0007 0.0389 ± 0.0006 0.0379 ± 0.0008 

L. testis 0.1125 ± 0.0022 0.1121 ± 0.0028 0.1151 ± 0.0022 0.1116 ± 0.0037 

Spermatid measurements 

Spermatid heads (107

/g testis) 18.55 ± 0.50 19.84 ± 0.73 18.73 ± 0.73 19.00 ± 0.99 

Spermatid heads (107

/testis) 2.08 ± 0.04 2.22 ± 0.08 2.15 ± 0.08 2.10 ± 0.07 

Spermatid count 

(mean/10-4 mL suspension) 65.00 ± 1.31 69.25 ± 2.43 67.20 ± 2.50 65.71 ± 2.21 

Epididymal spermatozoal measurements 

Motility (%) 72.92 ± 2.35 72.58 ± 2.51 69.58 ± 3.50 69.63 ± 2.62 

Concentration 

(106

/g cauda epididymal tissue) 1,248 ± 161 1,261 ± 212 1,647 ± 170 1,385 ± 225 

Female 

n 10 10 10 8 

Necropsy body wt (g) 28.4 ± 0.6 29.9 ± 1.0 29.3 ± 0.6 27.6 ± 0.6 

Estrous cycle length (days) 

Estrous stagesb

 (% of cycle) 

4.2 ± 0.1 4.9 ± 0.1** 4.9 ± 0.1** 5.1 ± 1.1**c 

Diestrus 28.3 21.7 24.2 28.1 

Proestrus 24.2 16.7 20.0 16.7 

Estrus 26.7 42.5 37.5 38.5 

Metestrus 20.8 19.2 18.3 16.7 

** Significantly different from the control group (P#0.01) by Shirley’s test a Weights, spermatid and epididymal spermatozoal parameters, and estrous cycle length are presented as mean ± standard error. 

Differences from the control group for weights were not significant by Dunnett’s test; differences from the control group for spermatid 

and epididymal spermatozoal parameters were not significant by Dunn’s test. b	 Evidence shows that females exposed to 1,300, 2,500, or 5,000 ppm differ significantly (Wilk’s Criterion, P#0.01) from the control 

females in the relative length of time spent in the estrous stages. Exposed females spent more time in estrus and less time in proestrus 

and metestrus than control females. 

Estrous cycle was longer than 12 days or unclear in one of eight animals. 
 

238 Primidone, NTP TR 476
 
239 

APPENDIX J
 

CHEMICAL CHARACTERIZATION AND
 

DOSE FORMULATION STUDIES
 

PROCUREMENT AND CHARACTERIZATION OF PRIMIDONE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240 
 

PREPARATION AND ANALYSIS OF DOSE FORMULATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240 
 

FIGURE J1 Infrared Absorption Spectrum of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242 
 

FIGURE J2 Nuclear Magnetic Resonance Spectrum of Primidone . . . . . . . . . . . . . . . . . . . . . . . . 243 
 

TABLE J1 Preparation and Storage of Dose Formulations in the Feed Studies of Primidone . . . . . 244 
 

TABLE J2 Results of Analyses of Dose Formulations Administered to Rats and Mice
 

in the 14-Week Feed Studies of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245 
 

TABLE J3 Results of Analyses of Dose Formulations Administered to Rats and Mice
 

in the 2-Year Feed Studies of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 
 
240 Primidone, NTP TR 476 

CHEMICAL CHARACTERIZATION AND 

DOSE FORMULATION STUDIES 

PROCUREMENT AND CHARACTERIZATION OF PRIMIDONE 

Primidone was obtained from Siegfried, LTD (Zofingen, Switzerland) in one lot (G041889), which was 

used during the 14-day, 14-week, and 2-year studies. Identity, purity, and stability analyses were 

conducted by the analytical chemistry laboratory, Research Triangle Institute (Research Triangle Park, 

NC). Reports on analyses performed in support of the primidone studies are on file at the National Institute 

of Environmental Health Sciences. 

The chemical, a white crystalline powder, was identified as primidone by infrared, ultraviolet/visible, 

nuclear magnetic resonance spectroscopy, and low- and high-resolution mass spectroscopy. All spectra 

were consistent with those expected for the structure of primidone; the low-resolution mass spectrum was 

also consistent with a literature reference (NIST Database). The infrared and nuclear magnetic spectra are 

presented in Figures J1 and J2. The melting point range of 282E to 283E C was consistent with a literature 

reference range of 281E to 282E C (Merck Index, 1983). 

The purity of lot G041889 was determined by Karl Fischer water analysis, thin-layer chromatography 

(TLC), and high-performance liquid chromatography (HPLC). TLC was performed on silica gel 60 F-254 

plates with two solvent systems: A) acetone:chloroform:t-butanol (30:68:2) and B) hexane:ethyl 

acetate:ethanol (50:40:10). Visualization was accomplished with I2 vapors, ultraviolet light, and a 

PMA/ceric sulfate spray. HPLC analyses were performed with two column systems: 1) a DuPont Zorbax® 

C8 column using ultraviolet detection (210 nm) and a gradient solvent system of 0.01M aqueous ammonium 

acetate:methanol (90:10 to 10:90) and 2) a DuPont Zorbax CN column using ultraviolet detection (210 nm) 

and a gradient solvent system of hexane:ethanol (90:10 to 10:90). The flow rate was 1.0 mL/minute for 

each system. 

Karl Fischer water analysis indicated 0.27% ± 0.01% water. TLC by each system indicated a major 

product spot and no impurities. HPLC indicated one major peak and one impurity with an area of 0.06% 

(first HPLC system) or 0.32% (second HPLC system) relative to the major peak area. The overall purity 

was determined to be greater than 99%. 

Accelerated stability studies of the bulk chemical were performed by the analytical chemistry laboratory. 

HPLC was performed with system 1 but with ultraviolet detection at 215 nm and a solvent ratio of 70:30. 

These studies indicated that primidone was stable as a bulk chemical for 2 weeks when stored protected 

from light at room temperature (22E to 27E C). To ensure stability, the bulk chemical was stored at room 

temperature protected from light in plastic bags in metal containers. Stability was monitored during the 

14-day, 14-week, and 2-year studies with HPLC, using the same system as the bulk chemical stability 

studies. No degradation of the bulk chemical was detected. 

PREPARATION AND ANALYSIS OF DOSE FORMULATIONS 

The dose formulations were prepared once during the 14-day studies, five times during the 14-week studies, 

and approximately every 6 weeks during the 2-year studies by mixing primidone with feed (Table J1). 

Mixtures were made by preparing by hand a primidone/feed premix, which was then blended with 

additional feed in a Patterson-Kelly twin-shell blender (East Stroudsberg, PA) for 15 minutes, using an 
Primidone, NTP TR 476 241 

intensifier bar for the initial 5 minutes. The dose formulations were stored in plastic containers in the dark 

at 5E C for up to 4 weeks (14-week studies) or up to 35 days (2-year studies). 

Homogeneity studies of the 300 and 5,000 ppm dose formulations for the 14-week studies and of 300 and 

2,500 ppm dose formulations for the 2-year studies were performed by the study laboratory. Primidone 

was extracted from the dose formulations with methanol with phthalamide added as an internal standard. 

Extracted feed samples were analyzed by HPLC with the system described for the bulk purity analyses. 

The analytical chemistry laboratory tested the homogeneity and stability of a 0.2 mg/g formulation with 

HPLC by the methods described for the bulk stability analyses. Homogeneity was confirmed, and stability 

was confirmed for at least 28 days for dose formulations stored frozen (-14E to -19E C) or refrigerated (3E

to 7E C) in sealed glass bottles. The study laboratory confirmed stability for dose formulations stored in 

plastic containers at 5E C for at least 35 days. 

Periodic analyses of the dose formulations of primidone were conducted at the study laboratory with the 

HPLC system used for the homogeneity studies. Dose formulations were analyzed once during the 14-day 

studies, three times during the 14-week studies (Table J2), and approximately every 8 weeks during the 

2-year studies (Table J3). All dose formulations for rats and mice were within 10% of the target 

concentration during the 14-day studies. All dose formulations for rats and mice were within 10% of the 

target concentration during the 14-week studies with no concentration less than 92% or greater than 107% 

of the target concentration; all animal room samples for rats and 14 of 15 animal room samples for mice 

were also within 10% of the target concentration (Table J2). During the 2-year studies, 56 of 57 dose 

formulations for rats and 36 of 38 dose formulations for mice were within 10% of the target concentration; 

all animal room samples for rats and 9 of 12 animal room samples for mice were also within 10% of the 

target concentration. One dose formulation that was 111% of the target concentration was fed to mice; no 

dose formulation given to rats was greater than 110% of the target concentration. 
242 Primidone, NTP TR 476
 

Figure J1 

Infrared Absorption Spectrum of Primidone 
Primidone, NTP TR 476
 243 

Figure J2 

Nuclear Magnetic Resonance Spectrum of Primidone 
 

244 Primidone, NTP TR 476 

TABLE J1 

Preparation and Storage of Dose Formulations in the Feed Studies of Primidone 

14-Day Studies 14-Week Studies 2-Year Studies 

Preparation 

A premix of feed and primidone was Same as the 14-day studies except dose Same as the 14-day studies except dose 

prepared by hand, then layered into the formulations were prepared monthly. formulations were blended for 

remaining feed and blended in a 15 minutes, with the intensifier bar on 

Patterson-Kelly twin-shell blender with for the first 5 minutes for formulations 

the intensifier bar on for 5 minutes and blended prior to 18 March 1993. Dose 

off for 10 minutes. Dose formulations formulations were prepared every 

were prepared once. 2 months. 

Chemical Lot Number 

G041889 G041889 G041889 

Maximum Storage Time 

4 weeks 4 weeks 35 days 

Storage Conditions 

Stored at 5° C (container not specified) Stored in plastic bags inside buckets at Stored in plastic containers protected 

5E C from light at 5E C 

Study Laboratory 

Battelle Columbus Laboratories Battelle Columbus Laboratories Battelle Columbus Laboratories 

(Columbus, OH) (Columbus, OH) (Columbus, OH) 
Primidone, NTP TR 476 245 

TABLE J2 

Results of Analyses of Dose Formulations Administered to Rats and Mice in the 14-Week Feed Studies 

of Primidone 

Date Prepared Date Analyzed 

Target 

Concentration 

(mg/g) 

Determined 

Concentrationa 

(mg/g) 

Difference 

from Target 

(%) 

Rats 

3 December 1990 6 December 1990 0.3b 

0.3c 

0.3d 

0.6 

1.3 

2.5 

5.0b 

5.0c 

5.0d 

0.308 

0.303 

0.314 

0.64 

1.36 

2.46 

5.04 

5.14 

5.06 

+3 

+1 

+5 

+7 

+5 

!2 

+1 

+3 

+1 

10 January 1991e 0.3 

0.6 

1.3 

2.5 

5.0 

0.283 

0.588 

1.28 

2.43 

5.09 

!6 

!2 

!2 

!3 

+2 

9 January 1991 10 January 1991 0.3 

0.6 

1.3 

2.5 

5.0 

0.296 

0.609 

1.34 

2.38 

5.12 

!1 

+2 

+3 

!5 

+2 

7 February 1991e 0.3 

0.6 

1.3 

2.5 

5.0 

0.289 

0.625 

1.31 

2.55 

5.17 

!4 

+4 

+1 

+2 

+3 

19 February 1991 21 February 1991 0.3 

0.6 

1.3 

2.5 

5.0 

0.293 

0.600 

1.35 

2.42 

5.00 

!2 

0 

+4 

!3 

0 

22 March 1991e 0.3 

0.6 

1.3 

2.5 

5.0 

0.311 

0.627 

1.35 

2.50 

4.99 

+4 

+5 

+4 

0 

0 
246 Primidone, NTP TR 476 

TABLE J2 

Results of Analyses of Dose Formulations Administered to Rats and Mice in the 14-Week Feed Studies 

of Primidone 

Target Determined Difference 

Date Prepared Date Analyzed Concentration Concentration from Target 

(mg/g) (mg/g) (%) 

Mice 

3 December 1990 6 December 1990	 0.3b 0.296 !1 

0.3c 0.295 !2 

0.3d 0.294 !2 

0.6	 0.64 +7 

1.3	 1.36 +5 

2.5 2.29 !8 

5.0b 4.99 0 

5.0c 5.03 +1 

5.0d 4.97 !1 

10 January 1991e 0.3 0.300	 0 

0.6	 0.595 !1 

1.3 1.29	 !1 

2.5 2.27	 !9 

5.0	 5.14 +3 

9 January 1991 10 January 1991	 0.3 0.302 +1 

0.6 0.599	 0 

1.3	 1.32 +2 

2.5 2.40	 !4 

5.0	 5.21 +4 

7 February 1991e 0.3	 0.297 !1 

0.6	 0.629 +5 

1.3	 1.33 +2 

2.5	 2.56 +2 

5.0 4.94	 !1 

19 February 1991 21 February 1991	 0.3 0.295 !2 

0.6	 0.612 +2 

1.3	 1.31 +1 

2.5 2.50	 0 

5.0 4.99	 0 

5 April 1991e 0.3	 0.268 !11 

0.6	 0.563 !6 

1.3	 1.31 +1 

2.5	 2.70 +8 

5.0	 5.10 +2 

a Results of duplicate analyses. 0.3 mg/g = 300 ppm; 0.6 mg/g = 600 ppm; 1.3 mg/g = 1,300 ppm; 2.5 mg/g = 2,500 ppm; and 

5.0 mg/g = 5,000 ppm b Sample selection from top left of twin-shell blender 

Sample selection from top right of twin-shell blender d Sample selection from bottom of twin-shell blender e Animal room samples 

c 
Primidone, NTP TR 476 247 

TABLE J3 

Results of Analyses of Dose Formulations Administered to Rats and Mice in the 2-Year Feed Studies 

of Primidone 

Target Determined Difference 

Date Prepared Date Analyzed Concentration Concentrationa from Target 

(mg/g) (mg/g) (%) 

Rats 

26 March 1992 26 March 1992 0.6 0.587 !2 

0.6 0.598 0 

1.3 1.33 +2 

1.3 1.29 !1 

2.5 2.52 +1 

2.5 2.46 !2 

27-29 April 1992b 0.6 0.605 +1 

0.6 0.610 +2 

1.3 1.35 +4 

1.3 1.32 +2 

2.5 2.47 !1 

2.5 2.51 0 

28 May 1992 28-30 May 1992 0.6 0.650 +8 

0.6 0.681 +14 

1.3 1.30 0 

1.3 1.37 +5 

2.5 2.54 +2 

2.5 2.53 +1 

2 June 1992c 2 June 1992 0.6 0.633 +6 

30 July 1992 6-11 August 1992 0.6 0.612 +2 

0.6 0.604 +1 

1.3 1.31 +1 

1.3 1.29 !1 

2.5 2.54 +2 

2.5 2.48 !1 

1 October 1992 5-6 October 1992 0.6 0.624 +4 

0.6 0.629 +5 

1.3 1.36 +5 

1.3 1.37 +5 

2.5 2.63 +5 

2.5 2.58 +3 

30 October 1992b 0.6 0.583 !3 

3 November 1992 0.6 0.598 0 

1.3 1.26 !3 

1.3 1.34 +3 

2.5 2.49 0 

2.5 2.43 !3 
248 Primidone, NTP TR 476 

TABLE J3 

Results of Analyses of Dose Formulations Administered to Rats and Mice in the 2-Year Feed Studies 

of Primidone 

Target Determined Difference 

Date Prepared Date Analyzed Concentration Concentration from Target 

(mg/g) (mg/g) (%) 

Rats (continued) 

3 December 1992 4-7 December 1992 0.6 0.585 !2 

0.6 0.589 !2 

1.3 1.28 !2 

1.3 1.28 !2 

2.5 2.49 0 

2.5 2.46 !2

 3 February 1993 4-5 February 1993 0.6 0.599 0 

0.6 0.601 0 

1.3 1.34 +3 

1.3 1.30 0 

2.5 2.54 +2 

2.5 2.51 0 

8 April 1993 9-12 April 1993 0.6 0.618 +3 

1.3 1.35 +4 

2.5 2.57 +3 

6-7 May 1993b 0.6 0.563 !6 

1.3 1.27 !2 

2.5 2.50 0 

10 June 1993 11 and 14 June 1993 0.6 0.563 !6 

1.3 1.33 +2 

2.5 2.53 +1 

12 August 1993 13-14 August 1993 0.6 0.604 +1 

1.3 1.29 !1 

2.5 2.43 !3 

14 October 1993 18-19 October 1993 0.6 0.610 +2 

1.3 1.31 +1 

2.5 2.54 +2 

18-19 November 1993b 0.6 0.586 !2 

1.3 1.28 !2 

2.5 2.50 0 

16 December 1993 24 December 1993 0.6 0.593 !1 

0.6 0.584 !3 

1.3 1.26 !3 

1.3 1.28 !2 

2.5 2.44 !2 

2.5 2.51 0 

17 February 1994 18-19 February 1994 0.6 0.614 +2 

1.3 1.43 +10 

2.5 2.47 !1 
Primidone, NTP TR 476 249 

TABLE J3 

Results of Analyses of Dose Formulations Administered to Rats and Mice in the 2-Year Feed Studies 

of Primidone 

Target Determined Difference 

Date Prepared Date Analyzed Concentration Concentration from Target 

(mg/g) (mg/g) (%) 

Mice 

26 March 1992 26 Mrch 1992 0.3 0.242 !19 

0.6 0.563 !6 

1.3 1.34 +3 

27-29 April 1992b 0.6 0.570 !5 

1.3 1.17 !10 

31 March 1992c 2 April 1992 0.3 0.305 +2 

27-29 April 1992b 0.3 0.269 -10 

28 May 1992 28-30 May 1992 0.3 0.333 +11 

0.6 0.650 +8 

1.3 1.37 +5 

30 July 1992 6-11 August 1992 0.3 0.306 +2 

0.6 0.598 0 

1.3 1.31 +1 

1 October 1992 5-6 October 1992 0.3 0.308 +3 

0.6 0.625 +4 

1.3 1.37 +5 

30 October 1992b 0.3 0.272 !9 

3 November 1992 0.6 0.503 !16 

1.3 1.28 !2 

3 December 1992 4-7 December 1992 0.3 0.314 +5 

0.6 0.597 0 

1.3 1.31 +1 

3 February 1993 4-5 February 1993 0.3 0.317 +6 

0.6 0.620 +3 

1.3 1.29 !1 

8 April 1993 9-12 April 1993 0.3 0.322 +7 

0.6 0.618 +3 

1.3 1.35 +4 

6-7 May 1993b 0.3 0.255 !15 

0.6 0.550 !8 

1.3 1.26 !3 

10 June 1993 11 and 14 June 1993 0.3 0.278 !7 

0.6 0.563 !6 

1.3 1.33 +2 
250 

c 

Primidone, NTP TR 476 

TABLE J3 

Results of Analyses of Dose Formulations Administered to Rats and Mice in the 2-Year Feed Studies 

of Primidone 

Target Determined Difference 

Date Prepared Date Analyzed Concentration Concentration from Target 

(mg/g) (mg/g) (%) 

Mice (continued) 

12 August 1993 13-16 August 1993 0.3 0.307 +2 

0.6 0.604 +1 

1.3 1.29 !1

 14 October 1993 18-19 October 1993 0.3 0.294 !2 

0.6 0.610 +2 

1.3 1.31 +1 

18-19 October 1993b 0.3 0.263 !12 

0.6 0.544 !9 

1.3 1.30 0 

16 December 1993 24 December 1993 0.3 0.282 !6 

0.6 0.593 !1 

0.6 0.584 !3 

1.3 1.26 !3 

1.3 1.28 !2 

17 February 1994 18-19 February 1994 0.3 0.288 !4 

0.6 0.614 +2 

1.3 1.43 +10 

a Results of duplicate analyses. 0.3 mg/g = 300 ppm; 0.6 mg/g = 600 ppm; 1.3 mg/g = 1,300 ppm; and 2.5 mg/g = 2,500 ppm b Animal room samples 

Results of remix 
251 

APPENDIX K 

FEED AND COMPOUND CONSUMPTION 

IN THE 2-YEAR FEED STUDIES 

OF PRIMIDONE 

TABLE K1 Feed and Compound Consumption by Male Rats in the 2-Year Feed Study 

of Primidone ................................................... 252 

TABLE K2 Feed and Compound Consumption by Female Rats in the 2-Year Feed Study 

of Primidone ................................................... 253 

TABLE K3 Feed and Compound Consumption by Male Mice in the 2-Year Feed Study 

of Primidone ................................................... 254 

TABLE K4 Feed and Compound Consumption by Female Mice in the 2-Year Feed Study 

of Primidone ................................................... 255 
252 Primidone, NTP TR 476 

TABLE K1 

Feed and Compound Consumption by Male Rats in the 2-Year Feed Study of Primidone 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

Feed 

(g/day)a Body 

Weight 

Feed 

(g/day) 

Body 

Weight 

Dose/ 

Dayb 

Feed 

(g/day) 

Body 

Weight 

Dose/ 

Day 

Feed 

(g/day) 

Body 

Weight 

Dose/ 

Day 

Week (g) (g) (mg/kg) (g) (mg/kg) (g) (mg/kg) 

1 16.8 151 17.7 151 70 17.8 150 154 17.4 150 291 

2 17.8 191 17.7 192 55 17.7 191 121 18.3 190 241 

4 16.7 256 17.8 258 41 17.9 256 91 17.8 258 173 

5 17.2 271 18.6 276 41 18.4 276 87 18.8 278 169 

8 17.3 327 17.4 330 32 17.7 329 70 18.0 324 139 

9 17.5 336 17.1 342 30 17.3 337 67 17.0 329 129 

12 18.1 363 17.2 369 28 17.8 364 63 17.4 358 121 

13 16.9 372 17.5 378 28 18.0 376 62 17.8 369 121 

17 16.4 395 16.9 401 25 17.0 398 55 17.1 386 111 

21 17.5 421 17.2 424 24 17.7 423 54 17.5 405 108 

25 16.4 440 16.9 442 23 17.6 438 52 17.2 418 103 

29 17.0 459 16.6 456 22 16.9 450 49 17.1 432 99 

33 16.0 471 16.1 467 21 16.7 458 47 17.1 440 97 

37 17.0 480 17.1 474 22 16.6 466 46 18.1 441 103 

41 15.9 490 16.3 485 20 17.5 476 48 16.8 454 92 

45 17.2 493 17.1 489 21 17.3 479 47 17.1 458 94 

49 16.4 506 16.8 498 20 17.5 488 47 15.7 455 86 

53 17.4 514 17.0 508 20 15.5 488 41 15.5 464 84 

57 16.5 517 16.4 510 19 16.2 496 42 17.1 471 91 

60 16.4 518 16.4 509 19 17.3 494 45 16.4 468 88 

65 15.6 521 16.6 515 19 16.6 500 43 17.2 468 92 

69 16.6 521 15.8 505 19 15.7 493 41 17.4 460 94 

73 15.9 522 16.0 510 19 16.9 492 45 17.0 459 92 

77 15.1 521 15.3 505 18 15.3 485 41 15.2 444 86 

81 16.0 513 16.2 503 19 15.4 481 42 17.0 443 96 

85 15.1 510 15.0 500 18 14.7 471 40 11.1 390 71 

89 13.6 498 13.4 479 17 14.8 469 41 

93 13.4 485 15.0 477 19 14.0 445 41 

97 15.0 483 14.1 462 18 15.6 432 47 

101 13.8 468 13.8 444 19 

Mean for weeks 

1-13 17.3 283 17.6 287 41 17.8 285 89 17.8 282 173 

14-52 16.6 462 16.8 460 22 17.2 453 50 17.1 432 99 

53-101 15.4 507 15.5 494 19 15.7 479 43 16.0 452 88 

a Grams of feed consumed per animal per day b Milligrams of primidone consumed per kilogram body weight per day 
Primidone, NTP TR 476 253 

TABLE K2 

Feed and Compound Consumption by Female Rats in the 2-Year Feed Study of Primidone 

0 ppm 600 ppm 1,300 ppm 2,500 ppm 

Feed 

(g/day)a Body 

Weight 

Feed 

(g/day) 

Body 

Weight 

Dose/ 

Dayb 

Feed 

(g/day) 

Body 

Weight 

Dose/ 

Day 

Feed 

(g/day) 

Body 

Weight 

Dose/ 

Day 

Week (g) (g) (mg/kg) (g) (mg/kg) (g) (mg/kg) 

1 12.4 118 13.9 118 71 13.7 117 152 13.6 118 288 

2 11.5 133 11.4 133 51 11.7 135 113 11.8 135 219 

4 11.2 160 11.6 158 44 12.0 159 98 11.2 157 178 

5 11.9 167 11.5 166 41 11.4 166 90 11.2 165 170 

8 10.8 186 11.2 184 37 10.9 182 78 10.6 182 146 

9 10.9 190 10.6 186 34 10.7 187 74 10.3 184 140 

12 10.9 204 10.2 199 31 10.5 197 70 10.1 195 130 

13 10.7 207 10.7 201 32 10.5 199 68 10.5 198 133 

17 10.9 219 11.0 212 31 10.9 210 67 10.3 206 125 

21 10.7 229 10.4 223 28 10.7 219 64 9.7 212 114 

25 10.7 236 10.8 230 28 10.6 225 61 10.0 218 115 

29 10.9 247 10.7 242 27 11.0 237 61 10.2 225 113 

33 10.4 255 10.6 252 25 10.5 243 56 10.4 233 112 

37 11.0 265 10.9 259 25 10.8 252 56 10.5 238 111 

41 11.0 273 11.2 268 25 10.6 258 54 10.5 244 108 

45 11.3 280 11.2 274 24 10.9 268 53 10.8 251 108 

49 10.8 294 10.9 284 23 11.0 278 51 10.9 260 105 

53 11.1 301 10.7 288 22 10.8 284 50 10.5 266 98 

57 11.6 308 11.3 296 23 11.0 291 49 11.2 276 102 

60 11.8 317 11.3 304 22 12.0 300 52 10.7 281 95 

65 11.0 322 10.8 309 21 11.1 304. 47 10.8 291 93 

69 12.1 328 12.1 317 23 11.7 310 49 11.2 295 95 

73 12.4 339 11.9 326 22 11.5 319 47 10.9 302 91 

77 11.2 341 10.9 328 20 10.8 322 44 10.6 304 87 

81 11.5 342 11.0 328 20 11.8 325 47 11.2 310 91 

85 12.0 350 10.9 330 20 10.7 327 43 10.2 308 82 

89 11.3 347 11.3 329 21 11.4 330 45 11.4 314 91 

93 12.3 358 11.8 340 21 11.8 338 45 11.3 320 88 

97 12.5 363 12.1 347 21 12.0 338 46 11.0 315 87 

101 11.9 361 11.9 348 21 11.7 337 45 10.9 317 86 

Mean for weeks 

1-13 11.3 171 11.4 168 43 11.4 168 93 11.2 167 175 

14-52 10.9 255 10.8 249 26 10.8 243 58 10.4 232 112 

53-101 11.7 337 11.4 322 21 11.4 317 47 10.9 300 91 

a Grams of feed consumed per animal per day b Milligrams of primidone consumed per kilogram body weight per day 
254 Primidone, NTP TR 476 

TABLE K3 

Feed and Compound Consumption by Male Mice in the 2-Year Feed Study of Primidone 

0 ppm 300 ppm 600 ppm 1,300 ppm 

Feed 

(g/day)a Body 

Weight 

Feed 

(g/day) 

Body 

Weight 

Dose/ 

Dayb 

Feed 

(g/day) 

Body 

Weight 

Dose/ 

Day 

Feed 

(g/day) 

Body 

Weight 

Dose/ 

Day 

Week (g) (g) (mg/kg) (g) (mg/kg) (g) (mg/kg) 

2 4.6 23.8 4.6 23.8 58 4.3 23.7 108 5.0 23.6 273 

5 5.7 28.0 5.8 28.7 61 5.1 28.6 107 5.8 28.6 265 

9 5.4 32.6 5.1 33.8 45 4.8 32.9 87 5.1 32.5 203 

13 5.4 37.8 5.3 38.0 42 5.0 36.9 82 5.0 36.3 180 

17 4.8 42.3 4.7 42.2 34 4.6 41.2 67 4.5 39.1 149 

21 4.6 44.5 4.6 45.3 31 4.6 43.7 63 4.6 41.8 142 

25 4.7 46.0 4.6 46.6 29 4.4 45.4 58 4.5 43.7 133 

29 4.4 47.2 4.4 47.2 28 4.4 46.1 57 4.3 44.3 127 

33 4.5 47.5 4.5 48.3 28 4.4 47.3 56 4.4 45.2 126 

36 4.8 47.8 4.6 48.4 29 4.7 47.6 59 4.6 45.4 131 

41 4.6 49.9 4.4 49.6 27 4.6 48.5 56 4.7 46.8 130 

45 4.7 49.2 4.7 49.1 29 4.5 48.2 56 4.7 47.5 127 

49 4.3 50.4 4.3 51.3 25 4.2 50.8 49 4.2 49.5 110 

53 5.0 50.3 4.8 51.4 28 4.8 51.0 56 4.7 48.8 126 

57 4.8 50.5 4.8 52.3 28 4.7 51.8 54 4.6 49.3 121 

61 4.7 49.7 4.7 51.3 27 4.5 50.7 53 4.5 48.3 120 

65 5.1 50.7 5.0 52.0 29 4.9 52.2 56 4.9 48.4 131 

69 5.0 49.9 4.8 51.7 28 4.9 51.5 57 4.8 46.4 135 

73 5.1 50.3 4.9 52.1 28 5.1 52.4 59 4.9 45.3 140 

77 5.4 49.7 5.1 51.5 30 5.1 50.7 60 5.2 43.4 156 

81 5.0 50.2 4.9 51.8 28 5.1 49.5 62 5.0 41.8 157 

84 5.0 50.8 5.0 52.2 29 5.1 48.9 62 5.1 41.9 158 

88 5.0 51.2 5.0 52.0 29 5.2 46.9 66 5.1 40.7 163 

93 4.7 49.6 4.9 50.7 29 5.2 44.1 71 5.1 39.5 169 

97 4.5 49.1 5.0 51.1 29 5.3 43.9 72 5.0 38.3 169 

101 4.7 48.9 5.0 48.3 31 5.3 43.0 74 5.2 37.9 178 

Mean for weeks 

1-13 5.3 30.5 5.2 31.1 51 4.8 30.5 96 5.2 30.3 230 

14-52 4.6 47.2 4.5 47.5 29 4.5 46.5 58 4.5 44.8 131 

53-101 4.9 50.1 4.9 51.4 29 5.0 49.0 62 4.9 43.8 148 

a Grams of feed consumed per animal per day b Milligrams of primidone consumed per kilogram body weight per day 
Primidone, NTP TR 476 255 

TABLE K4 

Feed and Compound Consumption by Female Mice in the 2-Year Feed Study of Primidone 

0 ppm 300 ppm 600 ppm 1,300 ppm 

Feed 

(g/day)a Body 

Weight 

Feed 

(g/day) 

Body 

Weight 

Dose/ 

Dayb 

Feed 

(g/day) 

Body 

Weight 

Dose/ 

Day 

Feed 

(g/day) 

Body 

Weight 

Dose/ 

Day 

Week (g) (g) (mg/kg) (g) (mg/kg) (g) (mg/kg) 

1 2.6 18.1 2.4 18.1 39 2.4 18.1 80 2.2 18.0 160 

2 3.1 19.0 2.9 19.0 46 3.1 18.8 100 3.5 19.0 236 

3 4.5 19.9 4.9 20.5 72 4.8 20.4 140 4.9 20.2 314 

4 4.9 21.4 5.0 22.0 68 5.3 21.5 147 5.2 21.6 314 

5 5.0 22.8 5.2 23.3 67 5.1 23.0 134 4.7 23.3 262 

8 4.9 25.9 4.4 27.8 47 4.2 27.0 93 4.5 26.5 222 

9 5.2 27.8 4.7 29.1 48 4.8 28.6 101 4.5 28.2 206 

12 4.6 31.5 4.1 33.5 37 4.4 31.8 83 4.3 31.0 180 

13 5.0 32.9 4.5 34.3 39 4.9 33.0 89 4.6 32.2 186 

17 4.4 37.7 3.8 39.9 29 4.0 37.9 63 3.7 37.1 131 

21 4.0 40.7 3.8 42.7 27 4.0 40.2 59 3.5 39.4 117 

25 4.0 43.3 3.8 45.5 25 3.8 42.5 54 3.6 41.8 113 

29 4.5 43.4 3.6 47.2 23 3.7 43.9 51 3.7 43.0 111 

33 3.3 45.2 3.0 48.3 19 2.9 44.8 38 2.9 43.1 87 

36 3.8 46.7 3.6 49.6 22 3.7 46.0 48 3.5 44.5 102 

41 3.9 49.1 3.8 51.5 22 3.6 48.2 45 3.6 46.3 101 

45 3.3 51.1 3.4 53.4 19 3.2 49.8 39 3.4 47.7 93 

49 3.8 53.4 3.5 55.3 19 3.7 52.4 42 3.7 49.7 98 

53 4.0 54.7 4.0 55.7 21 3.8 53.2 43 3.9 50.4 102 

57 4.0 55.6 3.9 57.2 21 3.8 54.5 42 3.8 51.7 95 

61 3.7 55.8 3.8 57.1 20 3.9 54.8 42 3.8 52.2 94 

65 4.2 56.2 4.2 57.5 22 4.0 54.8 44 4.1 51.8 103 

69 3.8 57.4 3.9 58.8 20 3.8 55.8 40 3.8 53.1 94 

73 4.2 59.0 4.3 60.0 21 4.2 57.0 44 4.0 54.5 95 

77 3.8 58.1 4.0 59.8 20 4.0 56.2 43 4.3 53.5 104 

81 4.2 59.3 4.4 61.2 22 4.2 57.8 44 4.6 53.9 111 

84 3.9 60.6 4.2 62.7 20 4.0 59.3 40 4.3 54.1 104 

88 4.0 60.3 4.2 63.1 20 4.1 59.7 41 4.5 52.2 113 

93 4.1 60.7 4.4 63.7 21 4.2 58.7 43 4.2 49.0 112 

97 4.0 61.6 4.0 64.7 18 4.0 58.7 41 4.5 47.6 122 

101 4.3 61.3 4.1 61.6 20 4.6 57.6 48 4.7 46.0 133 

Mean for weeks 

1-13 4.4 24.4 4.2 25.3 52 4.3 24.7 107 4.3 24.4 231 

14-52 3.9 45.6 3.6 48.1 23 3.6 45.1 49 3.5 43.6 106 

53-101 4.0 58.5 4.1 60.3 20 4.0 56.8 43 4.2 51.5 106 

a Grams of feed consumed per animal per day b Milligrams of primidone consumed per kilogram body weight per day 
256 Primidone, NTP TR 476 
257 

APPENDIX L 

INGREDIENTS, NUTRIENT COMPOSITION, 

AND CONTAMINANT LEVELS 

IN NIH-07 RAT AND MOUSE RATION 

TABLE L1 

TABLE L2 

TABLE L3 

TABLE L4 

Ingredients of NIH-07 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

Vitamins and Minerals in NIH-07 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . 

Nutrient Composition of NIH-07 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . 

Contaminant Levels in NIH-07 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . 

258 

258 

259 

260 
258 Primidone, NTP TR 476 

TABLE L1 

Ingredients of NIH-07 Rat and Mouse Rationa 

Ingredientsb Percent by Weight 

Ground #2 yellow shelled corn 24.50 

Ground hard winter wheat 23.00 

Soybean meal (49% protein) 12.00 

Fish meal (60% protein) 10.00 

Wheat middlings 10.00 

Dried skim milk 5.00 

Alfalfa meal (dehydrated, 17% protein) 4.00 

Corn gluten meal (60% protein) 3.00 

Soy oil 2.50 

Dried brewer's yeast 2.00 

Dry molasses 1.50 

Dicalcium phosphate 1.25 

Ground limestone 0.50 

Salt 0.50 

Premixes (vitamin and mineral) 0.25 

a NCI, 1976; NIH, 1978 b Ingredients were ground to pass through a U.S. Standard Screen No. 16 before being mixed. 

TABLE L2 

Vitamins and Minerals in NIH-07 Rat and Mouse Rationa 

Amount Source 

Vitamins 

A 5,500,000 IU Stabilized vitamin A palmitate or acetate 

D3 4,600,000 IU D-activated animal sterol 

K3 2.8 g Menadione 

d-"-Tocopheryl acetate 20,000 IU 

Choline 560.0 g Choline chloride 

Folic acid 2.2 g 

Niacin 30.0 g 

d-Pantothenic acid 18.0 g d-Calcium pantothenate 

Riboflavin 3.4 g 

Thiamine 10.0 g Thiamine mononitrate 

B12 4,000 μg 

Pyridoxine 1.7 g Pyridoxine hydrochloride 

Biotin 140.0 mg d-Biotin 

Minerals 

Iron 120.0 g Iron sulfate 

Manganese 60.0 g Manganous oxide 

Zinc 16.0 g Zinc oxide 

Copper 4.0 g Copper sulfate 

Iodine 1.4 g Calcium iodate 

Cobalt 0.4 g Cobalt carbonate 

a Per ton (2,000 lb) of finished product 
Primidone, NTP TR 476 259 

TABLE L3 

Nutrient Composition of NIH-07 Rat and Mouse Ration 

Mean ± Standard 

Nutrient Deviation Range Number of Samples 

Protein (% by weight) 23.10 ± 0.52 22.2 – 24.0 22 

Crude fat (% by weight) 5.34 ± 0.23 4.80 – 5.70 22 

Crude fiber (% by weight) 3.17 ± 0.33 2.50 – 3.80 22 

Ash (% by weight) 6.46 ± 0.27 6.08 – 7.03 22 

Amino Acids (% of total diet) 

Arginine 1.280 ± 0.083 1.110 – 1.390 11 

Cystine 0.308 ± 0.071 0.181 – 0.400 11 

Glycine 1.158 ± 0.048 1.060 – 1.220 11 

Histidine 0.584 ± 0.027 0.531 – 0.630 11 

Isoleucine 0.917 ± 0.033 0.867 – 0.965 11 

Leucine 1.975 ± 0.051 1.850 – 2.040 11 

Lysine 1.274 ± 0.049 1.200 – 1.370 11 

Methionine 0.437 ± 0.109 0.306 – 0.699 11 

Phenylalanine 0.999 ± 0.120 0.665 – 1.110 11 

Threonine 0.904 ± 0.058 0.824 – 0.985 11 

Tryptophan 0.218 ± 0.153 0.107 – 0.671 11 

Tyrosine 0.685 ± 0.094 0.564 – 0.794 11 

Valine 1.086 ± 0.055 0.962 – 1.170 11 

Essential Fatty Acids (% of total diet) 

Linoleic 2.407 ± 0.227 1.830 – 2.570 10 

Linolenic 0.259 ± 0.065 0.100 – 0.320 10 

Vitamins 

Vitamin A (IU/kg) 6,730 ± 529 5,940 – 8,580 22 

Vitamin D (IU/kg) 4,450 ± 1,382 3,000 – 6,300 4 

"-Tocopherol (ppm) 35.43 ± 8.98 22.5 – 48.9 11 

Thiamine (ppm) 16.27 ± 2.29 12.0 – 23.0 22 

Riboflavin (ppm) 7.83 ± 0.923 6.10 – 9.00 11 

Niacin (ppm) 99.22 ± 24.27 65.00 – 150.00 11 

Pantothenic acid (ppm) 30.55 ± 3.52 23.0 – 34.6 11 

Pyridoxine (ppm) 9.11 ± 2.53 5.60 – 14.0 11 

Folic acid (ppm) 2.46 ± 0.63 1.80 – 3.70 11 

Biotin (ppm) 0.268 ± 0.047 0.190 – 0.354 11 

Vitamin B12 (ppb) 40.5 ± 19.1 10.6 – 65.0 11 

Choline (ppm) 2,991 ± 382 2,300 – 3,430 10 

Minerals 

Calcium (%) 1.18 ± 0.08 1.04 – 1.32 22 

Phosphorus (%) 0.90 ± 0.06 0.770 – 1.00 22 

Potassium (%) 0.886 ± 0.063 0.772 – 0.971 9 

Chloride (%) 0.529 ± 0.087 0.380 – 0.635 9 

Sodium (%) 0.316 ± 0.033 0.258 – 0.371 11 

Magnesium (%) 0.166 ± 0.010 0.148 – 0.181 11 

Sulfur (%) 0.272 ± 0.059 0.208 – 0.420 10 

Iron (ppm) 350.5 ± 87.3 255.0 – 523.0 11 

Manganese (ppm) 92.48 ± 5.14 81.7 – 99.4 11 

Zinc (ppm) 59.33 ± 10.2 46.1 – 81.6 11 

Copper (ppm) 11.81 ± 2.50 8.09 – 15.4 11 

Iodine (ppm) 3.54 ± 1.19 1.52 – 5.83 10 

Chromium (ppm) 1.66 ± 0.46 0.85 – 2.09 11 

Cobalt (ppm) 0.76 ± 0.23 0.49 – 1.15 7 
260 Primidone, NTP TR 476 

TABLE L4 

Contaminant Levels in NIH-07 Rat and Mouse Rationa 

Mean ± Standard 

Deviationb Range Number of Samples 

Contaminants
 

Arsenic (ppm) 0.59 ± 0.16 0.10 – 0.80 22
 

Cadmium (ppm) 0.08 ± 0.07 0.04 – 0.20 

Mercury (ppm) <0.02
 

22
 

Lead (ppm) 0.30 ± 0.13 0.18 – 0.70 22
 

22
 

Selenium (ppm) 0.37 ± 0.08 0.10 – 0.40 22
 

Aflatoxins (ppb) <5.0 22
 

Nitrate nitrogen (ppm)c 7.10 ± 2.41 3.0 – 11.0 22
 

22
 

Nitrite nitrogen (ppm)c 0.79 ± 0.90 0.02 – 3.10 22
 

BHA (ppm)d 1.50 ± 1.92 1.00 – 10.0 

BHT (ppm)d 1.54 ± 0.96 1.00 – 5.00 22
 

Aerobic plate count (CFU/g) 172,545 ± 174,369 10,000 – 630,000 22
 

Coliform (MPN/g) 173 ± 331 3 – 1,100 22
 

Escherichia coli (MPN/g) <3 22
 

Salmonella (MPN/g) Negative 22
 

Total nitrosoamines (ppb)e 11.56 ± 4.53 4.80 – 19.70 22
 

N-Nitrosodimethylamine (ppb)e 9.21 ± 4.35 3.40 – 18.00 22
 

N-Nitrosopyrrolidine (ppb)e 2.39 ± 1.16 1.00 – 5.80 22
 

"-BHC <0.01 22
 

$-BHC <0.02 22
 

(-BHC <0.01 22
 

*-BHC <0.01 22
 

Heptachlor <0.01 22
 

Aldrin <0.01 22
 

Heptachlor epoxide <0.01 22
 

DDE <0.01 22
 

DDD <0.01 22
 

DDT <0.01 22
 

HCB <0.01 22
 

Mirex <0.01 22
 

Methoxychlor <0.05 22
 

Dieldrin <0.01 22
 

Endrin <0.01 22
 

Telodrin <0.01 22
 

Chlordane <0.05 22
 

Toxaphene <0.10 22
 

Estimated PCBs <0.20 22
 

Ronnel <0.01 22
 

Ethion <0.02 22
 

Trithion <0.05 22
 

Diazinon <0.10 22
 

Methyl parathion <0.02 22
 

Ethyl parathion <0.02 22
 

Malathion 0.09 ± 0.10 0.02 – 0.41 22
 

Endosulfan I <0.01 22
 

Endosulfan II <0.01 22
 

Pesticides (ppm)
 

Endosulfan sulfate <0.03 22
 

a CFU = colony forming units; MPN = most probable number; BHC = hexachlorocyclohexane or benzene hexachloride b For values less than the limit of detection, the detection limit is given as the mean. 

Sources of contamination: alfalfa, grains, and fish meal d Sources of contamination: soy oil and fish meal e All values were corrected for percent recovery. 

c 
261 

APPENDIX M
 

SENTINEL ANIMAL PROGRAM
 

METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262 
 

TABLE M1 Murine Virus Antibody Determinations for Rats and Mice
 

in the 14-Week and 2-Year Studies of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . 264 
 
262 Primidone, NTP TR 476 

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 14-week and 2-year studies 

of primidone. 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 

14-Week Study 

ELISA 

PVM (pneumonia virus of mice) 

RCV/SDA (rat coronavirus/

 sialodacryoadenitis virus) 

Sendai 

Study termination 

Study termination 

Study termination 

Hemagglutination Inhibition 

H-1 (Toolan’s H-1 virus) 

KRV (Kilham rat virus) 

Study termination 

Study termination 

2-Year Study 

ELISA 

Mycoplasma arthriditis Study termination 

Mycoplasma pulmonis Study termination 

PVM 6, 12, 13 and 18 months, study termination 

RCV/SDA 6, 12, 13 and 18 months, study termination 

Sendai 6, 12, 13 and 18 months, study termination 

Immunoflourescence Assay 

PVM 12 months 

RCV/SDA 12 and 13 months 

Sendai 12 months 

Hemagglutination Inhibition 

6, 12, 13 and 18 months, study termination 

KRV 6, 12, 13 and 18 months, study termination 

H-1 
Primidone, NTP TR 476 263 

MICE 

14-Week Study 

ELISA 

Ectromelia virus 

GDVII (mouse encephalomyelitis virus) 

LCM (lymphocytic choriomeningitis virus) 

Mouse adenoma virus-FL 

MHV (mouse hepatitis virus) 

PVM 

Reovirus 3 

Sendai 

Immunofluorescence Assay 

EDIM (epizootic diarrhea of infant mice) 

Reovirus 3 

Hemagglutination Inhibition 

MVM (minute virus of mice) 

K (papovavirus) 

Polyoma virus 

2-Year Study 

ELISA 

Ectromelia virus 

EDIM 

GDVII 

LCM 

Mouse adenoma virus-FL 

MHV 

M. arthritidis 

M. pulmonis 

PVM
 

Reovirus 3
 

Sendai
 

Immunofluorescence Assay 

MHV 

Hemagglutination Inhibition 

K 

MVM 

Polyoma virus 

Results of serology tests are presented in Table M1. 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

6, 12, and 18 months, study termination 

6, 12, and 18 months, study termination 

6, 12, and 18 months, study termination 

6, 12, and 18 months, study termination 

6, 12, and 18 months, study termination 

6, 12, and 18 months, study termination 

Study termination 

Study termination 

6, 12, and 18 months, study termination 

6, 12, and 18 months, study termination 

6, 12, and 18 months, study termination 

12 months 

6, 12, and 18 months, study termination 

6, 12, and 18 months, study termination 

6, 12, and 18 months, study termination 
264 Primidone, NTP TR 476 

TABLE M1 

Murine Virus Antibody Determinations for Rats and Mice in the 14-Week and 2-Year Studies 

of Primidone 

Interval Incidence of Antibody 

in Sentinel Animals 

Positive Serologic 

Reaction for 

14-Week Studies 

Rats 

Study termination 0/10 None positive 

Mice 

Study termination 0/10 None positive 

2-Year Studies 

Rats 

6 Months 

12 Months 

13 Months 

18 Months 

Study termination 

0/10 

1/10 

0/9 

0/9 

3/10a 

None positive 

PVM 

None positive 

None positive 

M. arthriditis 

Mice 

6 Months 

12 Months 

18 Months 

Study termination 

0/10 

0/10 

0/10 

0/10 

None positive 

None positive 

None positive 

None positive 

a Further evaluation of samples positive for M. arthritidis by immunoblot and Western blot procedures indicated that the positive titers 

may have been due to a cross reaction with antibodies of nonpathogenic Mycoplasma or other agents. Only sporadic samples were 

positive and there were no clinical findings or histopathologic changes of M. arthritidis infection in animals with positive titers. 

Accordingly, M. arthritidis-positive titers were considered false positives. 
265 

APPENDIX N 

SINGLE-DOSE TOXICOKINETIC STUDIES 

IN F344/N RATS AND B6C3F1 MICE 

INTRODUCTION ........................................................... 266 

MATERIALS AND METHODS ................................................... 266 

RESULTS ............................................................... 267 

TABLE N1 Plasma Concentrations of Primidone in F344/N Rats 

after a Single Gavage Dose of Primidone ................................ 269 

TABLE N2 Plasma Concentrations of Phenobarbital in F344/N Rats 

after a Single Gavage Dose of Primidone ................................ 270 

TABLE N3 Toxicokinetic Parameters in F344/N Rats after a Single Gavage Dose of Primidone: 

One-Compartment Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 

TABLE N4 Toxicokinetic Parameters in F344/N Rats after a Single Gavage Dose of Primidone . . . 271 

FIGURE N1 Plasma Concentrations of Primidone and Phenobarbital in Male F344/N Rats 

after a Single Gavage Dose of 30 mg/kg ................................. 272 

FIGURE N2 Plasma Concentrations of Primidone and Phenobarbital in Male F344/N Rats 

after a Single Gavage Dose of 80 mg/kg ................................. 272 

FIGURE N3 Plasma Concentrations of Primidone and Phenobarbital in Male F344/N Rats 

after a Single Gavage Dose of 130 mg/kg ................................ 272 

FIGURE N4 Plasma Concentrations of Primidone and Phenobarbital in Female F344/N Rats 

after a Single Gavage Dose of 30 mg/kg ................................. 273 

FIGURE N5 Plasma Concentrations of Primidone and Phenobarbital in Female F344/N Rats 

after a Single Gavage Dose of 80 mg/kg ................................. 273 

FIGURE N6 Plasma Concentrations of Primidone and Phenobarbital in Female F344/N Rats 

after a Single Gavage Dose of 130 mg/kg ................................ 273 

TABLE N5 Plasma Concentrations of Primidone in B6C3F1 Mice 

after a Single Gavage Dose of Primidone ................................ 274 

TABLE N6 Plasma Concentrations of Phenobarbital in B6C3F1 Mice 

after a Single Gavage Dose of Primidone ................................ 275 

FIGURE N7 Plasma Concentrations of Primidone (A) and Phenobarbital (B) 

in Male B6C3F1 Mice after a Single Gavage Dose of 30 mg/kg Primidone .......... 276 

FIGURE N8 Plasma Concentrations of Primidone (A) and Phenobarbital (B) 

in Male B6C3F1 Mice after a Single Gavage Dose of 80 mg/kg Primidone .......... 276 

FIGURE N9 Plasma Concentrations of Primidone (A) and Phenobarbital (B) 

in Male B6C3F1 Mice after a Single Gavage Dose of 200 mg/kg Primidone ......... 277 

FIGURE N10 Plasma Concentrations of Primidone (A) and Phenobarbital (B)

 in Female B6C3F1 Mice after a Single Gavage Dose of 30 mg/kg Primidone ........ 277 

FIGURE N11 Plasma Concentrations of Primidone (A) and Phenobarbital (B) 

in Female B6C3F1 Mice after a Single Gavage Dose of 80 mg/kg Primidone ......... 278 

FIGURE N12 Plasma Concentrations of Primidone (A) and Phenobarbital (B) 

in Female B6C3F1 Mice after a Single Gavage Dose of 200 mg/kg Primidone ........ 278 
266 Primidone, NTP TR 476 

SINGLE-DOSE TOXICOKINETIC STUDIES 

IN F344/N RATS AND B6C3F1 MICE 

INTRODUCTION 

Primidone is a congener of the barbiturate phenobarbital, and it is widely used in the management of 

epileptic seizures. Primidone is converted to two active metabolites, phenobarbital and 

phenylethylmalonamide. Primidone’s anticonvulsant effects are likely due to the combined action of the 

parent compound and its active metabolites. Primidone was nominated by the National Cancer Institute and 

the International Agency for Research on Cancer (IARC) for animal testing due to its high production 

volume and widespread human exposure, its mutagenicity in genotoxicity studies, and the carcinogenicity of 

the metabolite phenobarbital in rodent studies. The purpose of the present study is to characterize the 

plasma toxicokinetic profile of primidone and its major metabolite, phenobarbital, following a single oral 

dose in male and female F344/N rats and B6C3F1 mice. These data will be used to select dosages for the 

2-year studies, correlate toxic effects with systemic availability, establish toxicokinetic parameters, improve 

the usefulness of toxicology study results in risk assessment, and help determine the appropriate bleeding 

time for the toxicokinetic studies conducted at the end of the 2-year studies. 

MATERIALS AND METHODS 

Primidone was obtained from Siegfried Limited (Zofingen, Switzerland) in one lot (G041889), which was 

also used for the 2-year studies conducted at Battelle Columbus Laboratories (Columbus, OH). 

Methylcellulose was obtained by the National Toxicology Program (Research Triangle Park, NC) in one lot 

(876672). Results of purity and stability analyses of lot G041889 are presented in Appendix I. 

Additionally, the identity of the bulk chemical was confirmed by infrared spectroscopy. The relative purity 

was determined to be 100.4% by comparison to a frozen reference sample. Additional purity reanalyses 

were performed approximately every 2 to 4 months by comparing test article samples to reference samples; 

the relative purity ranged from 99% to 102%. Dose formulations administered by gavage were prepared as 

needed by suspending appropriate amounts of primidone in 0.5% aqueous methylcellulose. Analyses by the 

study laboratory using high-performance liquid chromatography (HPLC) indicated that all dose formulations 

administered to the animals were homogeneous and most were within 10% of the target concentrations. 

Stability studies of the 6, 16, and 26 mg/mL rat dose formulations and the 3, 8, and 20 mg/mL mouse dose 

formulations conducted by the study laboratory indicated that suspensions were stable for up to 7 days when 

stored at room temperature and protected from light in sealed containers. The 200 mg/kg dose formulation 

was also checked to ensure that the doses could be resuspended after storage and successfully administered 

by gavage. One 20 mg/mL dose formulation in the mouse study was not within 10% of the target 

concentration. 

F344/N rats and the first pool of B6C3F1 mice were obtained from Charles River Laboratories (Kingston, 

NY) for the rat study and from Charles River Laboratories (Portage, MI) for the mouse study. The second 

pool of B6C3F1 mice was obtained from Simonsen Laboratories (Gilroy, CA). Upon receipt, the rats and 

mice were observed for parasites and indications of disease. The rats were quarantined for approximately 

14 days and were 13 to 14 weeks old at the start of the studies. The first pool of mice were quarantined for 

approximately 11 to 14 days and were 12 to 13 weeks old at the start of the studies. The second pool of 

mice were quarantined for approximately 3 weeks and were 14 to 15 weeks old at the start of the studies. 

Filtered municipal water and NIH-07 open formula pelleted diet were available ad libitum. During the 

studies, all animals were housed individually. 
Primidone, NTP TR 476 267 

Doses and sampling time points for the single dose toxicokinetic studies were selected based on analyses of 

data from preliminary toxicokinetic studies and in preparation for the 2-year toxicokinetic studies. 

Groups of 18 male and 18 female rats were administered a single dose of 30, 80, or 130 mg primidone/kg 

body weight by gavage. Groups of 25 to 28 male and 24 to 28 female mice were administered 30, 80, or 

200 mg/kg by gavage. There were no vehicle controls in either study. The dosing volume in the rat study 

was approximately 5 mL/kg and the dosing volume in the mouse study was approximately 10 mL/kg. The 

animals were anesthetized with a mixture of carbon dioxide and oxygen, and blood samples were collected 

from the retroorbital sinus (rats) or by cardiac puncture (mice). In the rat study, blood samples were 

collected from two male and two female rats in the 30 and 80 mg/kg groups per time point at 0.25, 0.5, 

1.5, 3, 6, 9, 12, 18, and 22 hours after the administration of primidone. Two male and two female rats 

from the 200 mg/kg groups and two to four male and female mice from the 30 or 80 mg/kg groups were 

sampled at 0.25, 0.5, 1.5, 3, 6, 9, 12, 22, and 30 hours after the administration of primidone. Two to four 

male and female mice from the 200 mg/kg group were sampled at 0.25, 0.5, 1.5, 3, 6, 9, 12, 22, 30, and 

48 hours after the administration of primidone. Blood samples were collected only once from each animal. 

The samples were collected into heparinized tubes, and the plasma was separated and stored at !20E C or 

lower until analysis. 

All animals were observed twice daily for signs of morbidity and mortality. Individual body weights were 

recorded at randomization and on study day 1. Body weights from study day 1 were used for the 

calculation of dosing volumes. 

Plasma samples were analyzed using two HPLC systems which used a Supelco Supelcosil LC-18 column, 

ultraviolet detection (254 nm), and a gradient elution mobile phase of 0.18 M ammonium 

acetate:acetonitrile (75:25) with a 1.0 mL/minute flow rate. 

The average plasma concentrations of primidone and phenobarbital and standard deviations were calculated. 

The logarithms of these values were plotted as a function of time. The areas under the curve and standard 

errors for plasma concentration versus time were calculated using the trapezoid rule of the form 

AUCt

 = 3{(C +Cn-1)/2}×{t ) tn-1}, where AUCt n n is the cumulative area under the curve to time t and Cn-1 

and C are successive concentrations at tn-1 and t , respectively. The area under the curves to infinity n n

(AUC0 

4

) were calculated from AUC4

0 = AUCt

+Ct/8, where Ct is the last measured time point and 8 is the 

terminal rate constant determined from the slope of the terminal phase of the log plasma concentration-time 

profiles. The terminal elimination rate constants (k ) were determined from the slope of the terminal phases e

of the log plasma concentration-time profiles. Linear regression of the last two or three data points gave the 

slope (8). The half-lives (t1⁄2) were calculated as 0.693/8. The total body clearance (Cltot) was calculated as 

Dose/AUC0 

4

. The volume of distribution (Vd) was calculated as dose/estimated plasma concentration at t0. 

The maximum observed concentration (Cmax) and corresponding time (Tmax) were determined from the 

plasma concentration-time data as the maximum observed plasma concentration and corresponding time, 

respectively. 

RESULTS 

The plasma concentrations of primidone in rats are presented in Table N1. The plasma phenobarbital 

concentrations in rats are presented in Table N2. The semilogarithmic plots of plasma concentration-time 

data for primidone and phenobarbital for male and female rats administered 30, 80, or 130 mg/kg are 

shown in Figures N1 to N6. The k , k , and Vd values were calculated as toxicokinetic parameters for a a e

one compartment model and are presented in Table N3. The parameters were estimated by simultaneously 

fitting the model to the data from all three dose concentrations (30, 80, 130 mg/kg). The AUC0 

4

, t1⁄2, Cltot, 

Cmax max , and T values for rats are presented in Table N4. 
268 Primidone, NTP TR 476 

Plasma concentrations of primidone were dose- and time-dependent for male and female rats. Absorption 

of primidone administered in 0.5% methyl cellulose by gavage was rapid; for all dosed groups, plasma 

primidone concentrations were detectable 15 minutes after dosing. Although the time course and dose 

response profile were similar for male and female rats, primidone plasma concentrations were consistently 

higher for female rats than for male rats (approximately double at most dose concentrations and time points 

sampled). Elimination of primidone was rapid and sex-dependent, as plasma half-lives were 2- to 5-fold 

greater in female rats than in male rats. 

Plasma concentrations of phenobarbital were also dose- and time-dependent. However, for a given dose, 

plasma phenobarbital concentrations were consistently higher for male rats than for female rats. 

Metabolism of primidone in rats is thus indicated to be sex-dependent, with males metabolizing primidone 

more rapidly than females. Plasma phenobarbital was detected in male rats within 15 minutes of dosing, 

but was below the limits of quantitation at 15 and 30 minutes post-dose in female rats. Plasma 

phenobarbital concentrations had not fallen below the limits of quantitation by the last time points sampled 

(1,320 or 1,800 minutes) in either male or female rats. 

The plasma concentrations of primidone in mice are presented in Table N5. The plasma phenobarbital 

concentrations in mice are presented in Table N6. The semilogarithmic plots of plasma concentration-time 

data for primidone and phenobarbital for male and female mice administered 30, 80, or 200 mg/kg are 

shown in Figures N7 to N12. 

Plasma primidone concentrations were dose- and time-dependent for male and female mice. It was 

determined that the variability of a small sample size and a large number of samples below the limit of 

quantitation resulted from biological variation. Pharmacokinetics parameters could not be calculated for the 

mice due to inadequate data. Absorption of primidone administered by gavage was rapid; for all dose 

groups and males and females, plasma concentrations of primidone were detectable within 15 minutes after 

dosing. There was a slight trend toward greater plasma primidone concentrations in male mice than in 

female mice. Plasma primidone concentrations remained above the detection limit for at least 30 hours 

following a 30 or 80 mg/kg dose and for at least 48 hours after a 200 mg/kg dose. 

Plasma phenobarbital concentrations appeared dose- and time-dependent for males and females. Plasma 

phenobarbital concentrations were detected within 15 minutes after dosing. In agreement with the plasma 

primidone data, male mice had slightly higher plasma phenobarbital concentrations than the females. 

Primidone appeared to be more rapidly metabolized to phenobarbital in male mice than in female mice, as 

peak plasma phenobarbital concentrations were observed at earlier time points in males. 
269 

c 

Primidone, NTP TR 476 

TABLE N1 

Plasma Concentrations of Primidone in F344/N Rats after a Single Gavage Dose of Primidonea 

30 mg/kg 80 mg/kg 130 mg/kg 

n 2 2 2 

Male 

Time After Dosing (minutes) c 15 8.1b 11.2 —

30 — 25.1 24.4b 

90 12.0 34.9 51.7 

180 7.2 37.2 51.9 

360 0.3 13.5 23.7 

540 BLQ 3.5 9.7 

720 BLQ 1.5 4.3 

1,080 BLQ BLQ — 

1,320 BLQ BLQ BLQ 

1,800 — — BLQ 

Female 

Time After Dosing (minutes) 

15 15.8b 21.1 — 

30 — 40.2 51.5b 

90 22.3 59.6 73.5 

180 22.4 67.6 104 

360 12.0 40.2 66.4 

540 5.9 22.7 53.2 

720 3.9 14.2 20.1 

1,080 0.3 2.6 — 

1,320 BLQ 0.2 0.9 

1,800 — — BLQ 

a Data are given in μg/mL as the mean of two samples. BLQ=Below limit of quantitation b n=4
 

No samples were analyzed at this time point.
 
270 

c 

Primidone, NTP TR 476 

TABLE N2 

Plasma Concentrations of Phenobarbital in F344/N Rats after a Single Gavage Dose of Primidonea 

30 mg/kg 80 mg/kg 130 mg/kg 

n 2 2 2 

Male 

Time After Dosing (minutes) c 15 0.33b 0.37 —

30 — 1.21 0.53b 

90 1.99 3.22 4.12 

180 2.72 4.43 5.01 

360 3.31 5.93 8.69 

540 2.83 7.12 9.68 

720 2.55 5.75 8.62 

1,080 1.60 3.57 — 

1,320 1.05 2.87 4.56 

1,800 — — 2.02 

Female 

Time After Dosing (minutes) 

15 BLQb BLQ — 

30 — BLQ BLQb 

90 0.40 0.72 0.93 

180 0.59 1.05 1.03 

360 1.03 1.89 2.27 

540 0.95 2.28 2.75 

720 1.02 2.12 2.49 

1,080 0.96 1.93 — 

1,320 0.83 1.61 2.27 

1,800 — — 2.02 

a Data are given in μg/mL as the mean of two samples. BLQ=Below limit of quantitation b n=4
 

No samples were analyzed at this time point.
 
271 

c 

Primidone, NTP TR 476 

TABLE N3 

Toxicokinetic Parameters in F344/N Rats after a Single Gavage Dose of Primidone: 

One-Compartment Modela 

b c d ka ke Vd 

(hours-1) (hours-1) (L/kg) 

Male 0.433 0.556 0.1934
 

Female 0.7996 0.1706 0.1425
 

a The parameters were estimated by simultaneously fitting the model to the data from three dose levels (30, 80, and 130 mg/kg). b k = Absorption rate constant a

k = Terminal elimination rate constant e d Vd = Volume of distribution 

TABLE N4 

Toxicokinetic Parameters in F344/N Rats after a Single Gavage Dose of Primidonea 

4b c d e f Dose AUC0 t1⁄2 Cltot Cmax Tmax 

(mg/kg) (mg@hour/L) (hours) (L/hour@kg) (mg/L) (hours) 

Male 

30 39.663 0.70 0.756 12.0 1.50 

80 193.06 1.22 0.414 37.2 3.00 

130 318.42 1.22 0.408 51.9 3.00 

Female 

30 174.8 4.03 0.172 22.4 3.00 

80 533 3.72 0.15 67.6 3.00 

130 851.5 2.74 0.153 104 3.00 

a The data were calculated from the plasma concentration-time curves, where each point represents the mean of two male or two female 

rats. 

b AUC4

0 = Area under the curve to infinity c t1⁄2 = Elimination half-life 

d 4 Cltot = Dose/AUC0 e Cmax = Maximum mean concentration

f Tmax = Time of maximum mean concentration (estimated from last four time points)
 

 

272 Primidone, NTP TR 476 

Figure N1 Figure N2 

Plasma Concentrations of Primidone Plasma Concentrations of Primidone 

and Phenobarbital in Male F344/N Rats and Phenobarbital in Male F344/N Rats 

after a Single Gavage Dose of 30 mg/kg after a Single Gavage Dose of 80 mg/kg 

Figure N3 

Plasma Concentrations of Primidone and Phenobarbital 

in Male F344/N Rats after a Single Gavage Dose of 130 mg/kg 
Figure N4 

Plasma Concentrations of Primidone 

and Phenobarbital in Female F344/N Rats 

after a Single Gavage Dose of 30 mg/kg 

Figure N5 

Plasma Concentrations of Primidone 

and Phenobarbital in Female F344/N Rats 

after a Single Gavage Dose of 80 mg/kg 

Primidone, NTP TR 476 273 

Figure N6 

Plasma Concentrations of Primidone and Phenobarbital 

in Female F344/N Rats after a Single Gavage Dose of 130 mg/kg 
274 Primidone, NTP TR 476 

TABLE N5 

Plasma Concentrations of Primidone in B6C3F1 Mice after a Single Gavage Dose of Primidonea 

30 mg/kg 80 mg/kg 200 mg/kg 

n 2 2 2 

Male 

Time After Dosing (minutes) 

15 65.8b 72.2 68.6 

30 26.4 120 128c 

90 58.8 156b 157 

180 35.8b 64.8 123b 

360 35.9 29.4b 125b 

540 15.1 23.8b 30.4b 

720 21.4b 10.2 35.8 

1,320 

1,800 

32.0b 

19.6 

16.4 

16.5 

41.6 

—d 

2,880 — — 33.6 

Female 

Time After Dosing (minutes) 

15 23.3 32.9 70.2 

30 29.0 85.6 133b 

90 20.8 52.9 122 

180 19.2 52.3 130 

360 4.0b 21.5 75.4b 

540 10.9 8.8 15.7b 

720 8.7 2.8b 3.0 

1,320 

1,800 

18.6b 

12.1 

105 

9.9b 

5.4 

— 

2,880 — — 15.1 

a 

b Data are given in μg/mL as the mean of two samples. 

n=3 c n=4 

d No samples were analyzed at this time point. 
275 

c 

Primidone, NTP TR 476 

TABLE N6 

Plasma Concentrations of Phenobarbital in B6C3F1 Mice after a Single Gavage Dose of Primidonea 

30 mg/kg 80 mg/kg 200 mg/kg 

n 2 2 2 

Male 

Time After Dosing (minutes) 

15 0.37 0.46 0.63 

30 4.40 1.28 0.92b 

90 2.62 6.05c 2.92 

180 2.70c 3.34 4.44c 

360 2.62 4.63c 9.85c 

540 3.66 6.45c 11.1c 

720 1.93b 3.83 15.4 

1,320 0.87c 2.46 4.78 

d 1,800 0.75 BLQ —

2,880 — — BLQ
 

Female 

Time After Dosing (minutes) 

15 0.40 0.49 0.59 

30 0.65 1.13 1.10c 

90 1.60 1.69 2.75 

180 3.09 3.64 4.73 

360 3.45c 7.59 9.83c 

540 2.63 4.77 10.3c 

720 1.35 4.16b 5.29 

1,320 1.25e 0.70c 6.59 

1,800 BLQ 0.11e — 

2,880 — — BLQ 

a Data are given in μg/mL as the mean of two samples. BLQ=Below limit of quantitation b n=4 

n=3 

d No samples were analyzed at this time point. e n=1 
 

 

276 Primidone, NTP TR 476 

Figure N7 

Plasma Concentrations of Primidone (A) and Phenobarbital (B) 

in Male B6C3F1 Mice after a Single Gavage Dose of 30 mg/kg Primidone 

Figure N8 

Plasma Concentrations of Primidone (A) and Phenobarbital (B) 

in Male B6C3F1 Mice after a Single Gavage Dose of 80 mg/kg Primidone 
 

 

Primidone, NTP TR 476 277 

Figure N9 

Plasma Concentrations of Primidone (A) and Phenobarbital (B) 

in Male B6C3F1 Mice after a Single Gavage Dose of 200 mg/kg Primidone 

Figure N10 

Plasma Concentrations of Primidone (A) and Phenobarbital (B) 

in Female B6C3F1 Mice after a Single Gavage Dose of 30 mg/kg Primidone 
 

 

278 Primidone, NTP TR 476 

Figure N11 

Plasma Concentrations of Primidone (A) and Phenobarbital (B) 

in Female B6C3F1 Mice after a Single Gavage Dose of 80 mg/kg Primidone 

Figure N12 

Plasma Concentrations of Primidone (A) and Phenobarbital (B) 

in Female B6C3F1 Mice after a Single Gavage Dose of 200 mg/kg Primidone 
279 

APPENDIX O
 

CONTINUOUS BREEDING STUDY
 

IN SWISS (CD-1®) MICE
 

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280 

MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280 

RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282 

TABLE O1 Survival, Body Weights, and Feed Consumption of F0 Swiss (CD-1®) Mice 

in the Extended Dose-Setting Study of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . 283 

TABLE O2 Fertility, Reproductive Performance, and Body Weight Data 

for Swiss (CD-1®) Mice in the Extended Dose-Setting Study 

of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284 

TABLE O3 Fertility, Reproductive Performance, Length of Gestation, and Body Weight Data 

for F0 and F1 Swiss (CD-1®) Mice in the Continuous Breeding Study 

of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 

TABLE O4 Litter and Body Weight Data for F1 Swiss (CD-1®) Mouse Pups 

in the Continuous Breeding Study of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 

TABLE O5 Survival and Body Weights of F1 Swiss (CD-1®) Mouse Pups (Final Litter) 

in the Continuous Breeding Study of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 

TABLE O6 Fertility, Reproductive Performance, Length of Gestation, and Body Weight Data 

for F1 and F2 Swiss (CD-1®) Mice in the Offspring Assessment Phase 

of the Continuous Breeding Study of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . 288 

TABLE O7 Sperm Parameters and Estrous Cycle Characterization for F1 Swiss (CD-1®) Mice 

in the Offspring Assessment Phase of the Continuous Breeding Study 

of Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 

TABLE O8 Organ Weights and Organ-Weight-to-Body-Weight Ratios 

for F1 Swiss (CD-1®) Mice Administered Primidone in Feed . . . . . . . . . . . . . . . . . . . 290 
 
280 Primidone, NTP TR 476 

CONTINUOUS BREEDING STUDY 

IN SWISS (CD-1®) MICE 

INTRODUCTION 

The potential reproductive toxicity of primidone was evaluated in Swiss (CD-1®) mice because no 

information is available in the literature. The effects of exposure to primidone on reproduction were 

assessed with a continuous breeding study in Swiss (CD-1®) mice administered primidone in feed (NTP, 

1991). 

Reproductive assessment by the methods presented in Lamb (1985), Reel et al. (1985), and Heindel et al. 

(1989) consists of four phases: dose range finding, continuous breeding, identification of the affected 

gender (crossover mating trial), and offspring assessment. A 2-week dose-setting phase is conducted to 

determine exposure concentrations of the continuous breeding phase. The dose-setting phase of the current 

study was lengthened by introducing a mating and pup survival trial. During the continuous breeding 

phase, the effects of the maximum tolerated exposure concentration estimated in the dose-setting phase and 

two lower exposure concentrations on fertility and reproduction of first-generation (F0) 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 are affected. Offspring assessment includes 

evaluation of reproductive performance of second-generation (F1) animals from the final litters of the 

continuous breeding phase. The F1 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 (F2) offspring. 

In the primidone study, the exposure concentrations for the continuous breeding study were based on results 

of the extended dose-setting study. Because fertility was not significantly affected during the continuous 

breeding phase, crossover mating trials were not conducted. Pups from all exposure groups were 

maintained for offspring assessment until weening. Only controls and 1,500 ppm groups were assessed for 

F1 fertility measures. 

MATERIALS AND METHODS 

Primidone was obtained from Siegfried LTD (Zofingen, Switzerland) in one lot (G041889), which was also 

used for the 14-day, 14-week, and 2-year studies conducted at Battelle Columbus Laboratories (Columbus, 

OH). Results of purity and stability analyses of lot G041889 are presented in Appendix J. Dose 

formulations were prepared every 4 weeks, and analyses by Research Triangle Institute (Research Triangle 

Park, NC) (for the extended dose-setting phase and the continuous breeding phase), Radian Corporation 

(Austin, TX) (continuous breeding phase), or Midwest Research Institute (Kansas City, MO) (offspring 

assessment phase) indicated that the dose formulations were homogeneous and within 10% of the target 

concentration. 

Male and female VAF Crl:Swiss CD-1® (ICR)BR outbred albino mice were obtained from Charles River 

Breeding Laboratories, Inc. (Portage, MI), before the dose-setting phase and again for the continuous 

breeding phase. Upon receipt, serum samples were collected from sentinel males and females, and the 

serum samples were analyzed for antibody titers to rodent viruses. All serum samples were negative. Mice 

were quarantined for 2 weeks and were 11 weeks old at the start of the extended dose-setting phase and the 

continuous breeding phase. Mice were housed two per cage by gender during quarantine. NIH-07 open 

formula meal diet containing the appropriate concentrations of primidone was available ad libitum and 

deionized water was available ad libitum for all phases of the study. Sentinel animals were monitored for 
Primidone, NTP TR 476 281 

disease and viral titers throughout the study; sera collected at the end of the study were positive for minute 

virus of mice. 

During the extended dose-setting phase, the mice were housed two per cage by gender for 7 days while 

being exposed to 0, 125, 250, 500, 1,000, or 2,000 ppm; mice were then housed in breeding pairs for 

28 days while exposure continued. Females were allowed to deliver one litter, and pups remained with 

their dams. During the extended dose-setting phase, clinical findings, feed consumption, dam body 

weights, pregnancy index, live pups per litter, proportion of pups born alive, gender of live pups, and pup 

body weights were recorded (Tables O1 and O2). 

During the continuous breeding phase, the mice were housed two per cage by gender for 7 days while being 

exposed to 0, 150, 500, or 1,500 ppm; 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 

findings, feed consumption, pregnancy index, litters per pair, length of gestation, dam body weights, live 

pups per litter, proportion of pups born alive, gender of live pups, and pup body weights were recorded 

(Tables O3 and O4). For the last litter, pup survival and body weights were recorded on lactation days 0, 

4, 7, 14, and 21 (Table O5). 

To assess the offspring of exposed mice, the final litter of pups born to each F0 mouse dam in the 0 and 

1,500 ppm groups was raised to sexual maturity. After weaning, siblings were housed two per cage by 

gender 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 findings, feed consumption, 

mating index, pregnancy index, fertility index, dam body weights, length of gestation, live pups per litter, 

proportion of pups born alive, gender of live pups, and pup body weights were recorded (Table O6). 

Before necropsy of the F1 mice, vaginal cytology data were collected (Table O7). At necropsy, epididymal 

spermatozoal data were collected (Table O7) 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 O8). 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 or PAS and hematoxylin (testis only). 

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 was used to identify overall differences 

in fertility across exposure groups. 

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) was used 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). Multiple comparisons were made with Dunn’s test or Wilcoxon’s test (Conover, 1971). 
282 Primidone, NTP TR 476 

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. Least-squares 

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 (Table O7). 

RESULTS 

All mice in the extended dose-setting phase survived until the end of the study (Table O1). Final mean 

body weights of all groups of exposed mice were similar to those of the controls. Feed consumption by the 

250 ppm females was significantly greater than that by the controls during week 1. The exposure 

concentrations of 125, 250, 500, 1,000, or 2,000 ppm primidone administered in feed resulted in average 

daily doses of 20, 40, 90, 175, or 350 mg primidone/kg body weight for males and 20, 40, 80, 160, or 

320 mg/kg for females. Exposure to primidone had no adverse effect on reproductive parameters, live pups 

per litter, sex ratio, or pup weights (Table O2). Pup survival on day 4 was slightly decreased in the 

2,000 ppm groups (Table O2); therefore, the exposure concentrations selected for the continuous breeding 

phase were 150, 500, and 1,500 ppm. 

During the continuous breeding phase, exposure to primidone had no adverse effect on reproductive 

parameters (Table O3). The numbers of live pups per litter and the total and adjusted total pup weights of 

exposed groups were similar to those in the controls for individual litters and for the combined litters 1 

through 5 (Table O4). For the final litter of pups, the survival rates of male and female pups in exposed 

groups were similar to the control values at each time point (Table O5). The mean body weight of male 

pups in the 500 ppm group was significantly greater than that of the controls on lactation day 21; the mean 

body weights of female pups in all exposed groups were significantly greater than that of the controls on 

lactation day 21. 

During the offspring assessment phase of the continuous breeding study, exposure to 1,500 ppm had no 

adverse effect on reproductive parameters, live pups per litter, sex ratio, or pup body weights (Table O6). 

The estrous cycle length of 1,500 ppm females was significantly longer than that of the controls (Table O7). 

No significant differences in epididymal spermatozoal motility, percent abnormality, or concentration were 

observed between 1,500 ppm males and the controls. The absolute and relative liver weights of 1,500 ppm 

males and females were significantly greater than those of the controls (Table O8); similar effects were 

observed in the 14-week studies with F344/N rats and B6C3F1 mice. The seminal vesicle weights of 

1,500 ppm males were significantly less than those of the controls. These differences may be indicative of 

altered internal hormone states, perhaps secondary to changes in hepatic hormone clearance induced by the 

hepatomegalic effects of primidone. 

In summary, primidone at concentrations up to 1,500 ppm had only minimal effects on reproductive 

parameters in the F0 mice. The only reproductive effects were a significant increase in estrous cycle length 

and reduced seminal vesicle weight in F1 mice exposed to 1,500 ppm. 
283 

c 

Primidone, NTP TR 476 

TABLE O1 

Survival, Body Weights, and Feed Consumption of F0 Swiss (CD-1®) Mice 

in the Extended Dose-Setting Study of Primidone 

Dose 

(ppm) 

Survivala Initial 

Mean Body Weightb

 (g) 

Final Change 

Final Weight 

Relative 

to Controls 

(%) 

Feed 

Consumptionc 

Week 1 Week 3 

Male 

0 

125 

250 

500 

1,000 

2,000 

8/8 

8/8 

8/8 

8/8 

8/8 

8/8 

28.2 ± 0.7 

28.4 ± 0.6 

28.8 ± 0.6 

27.1 ± 0.4 

27.6 ± 0.9 

29.1 ± 0.9 

31.1 ± 0.8 

30.7 ± 0.6 

31.9 ± 0.8 

30.3 ± 0.8 

30.6 ± 1.1 

31.4 ± 0.9 

2.9 

2.3 

3.1 

3.2 

3.0 

2.3 

99 

103 

97 

98 

101 

4.9 

4.8 

4.7 

4.8 

5.2 

5.0 

5.3 

5.4 

5.3 

5.5 

5.1 

5.6 

Female 

0 

125 

250 

500 

1,000 

2,000 

8/8 

8/8 

8/8 

8/8 

8/8 

8/8 

25.1 ± 0.5 

25.1 ± 0.4 

25.4 ± 0.4 

25.0 ± 0.3 

25.9 ± 0.5 

25.7 ± 0.3 

41.0 ± 1.2 

43.1 ± 2.2 

42.8 ± 0.6 

41.0 ± 1.5 

40.3 ± 0.8 

41.8 ± 1.0 

15.9 

18.0 

17.4 

16.0 

14.4 

16.1 

105 

104 

100 

98 

102 

4.8 

5.2 

5.8* 

5.6 

5.3 

5.1 

5.3 

5.4 

5.3 

5.5 

5.1 

5.6 

*	 Significantly different (P#0.05) from the control group by Williams’ or Dunnett’s test a Number of animals surviving at day 28/number initially in group b	 Weights are given as mean ± standard error; weight changes are given as mean. 

Feed consumption is expressed as grams of feed consumed per animal per day. 
284 

c 

Primidone, NTP TR 476 

TABLE O2 

Fertility, Reproductive Performance, and Body Weight Data 

for Swiss (CD-1®) Mice in the Extended Dose-Setting Study of Primidonea 

0 ppm 125 ppm 250 ppm 500 ppm 1,000 ppm 2,000 ppm 

Adult Data 

Pregnancy indexb 8/8 (100%) 7/8 (88%) 8/8 (100%) 8/8 (100%) 8/8 (100%) 8/8 (100%) 

Dam weight at delivery (g) 34.2 ± 0.96 33.65 ± 0.99 34.3 ± 0.55 34.3 ± 0.62 34.3 ± 0.87c 35.9 ± 0.57 

Pup Data 

Day 0 

Number of litters 8 7 8 8 7d 8 

Live male pups/litter 5.3 ± 0.4 5.0 ± 0.4 5.4 ± 0.8 5.4 ± 0.7 4.6 ± 0.5 5.3 ± 0.5 

Live female pups/litter 6.8 ± 0.6 6.3 ± 0.7 5.5 ± 0.5 5.5 ± 0.5 5.6 ± 0.7 5.6 ± 0.7 

Total live pups/litter 12.0 ± 0.5 11.3 ± 0.8 10.9 ± 0.6 10.9 ± 0.7 10.1 ± 0.7* 10.9 ± 0.4 

Live pups/litter (%) 100 ± 0 99 ± 1 100 ± 0 100 ± 0 100 ± 0 98 ± 1 

Sex ratioe

 (%) 44 ± 3 45 ± 4 48 ± 5 49 ± 5 45 ± 5 49 ± 5 

Male pup weight (g) 1.55 ± 0.03 1.57 ± 0.03 1.61 ± 0.05 1.57 ± 0.03 1.60 ± 0.05 1.53 ± 0.04 

Female pup weight (g) 1.49 ± 0.03 1.46 ± 0.03 1.53 ± 0.05 1.47 ± 0.03 1.57 ± 0.02 1.45 ± 0.04 

Total live pup weight (g) 

Adjusted total pup weightf

 (g) 

1.52 ± 0.03 

1.55 ± 0.03 

1.51 ± 0.02 

1.52 ± 0.03 

1.57 ± 0.05 

1.56 ± 0.03 

1.52 ± 0.03 

1.51 ± 0.03 

1.58 ± 0.03 

1.55 ± 0.03 

1.49 ± 0.04 

1.49 ± 0.03 

Day 4 

Male survival (%) 98 ± 4 100 ± 0 100 ± 0 86 ± 12 98 ± 6 85 ± 6c 

Female survival (%) 96 ± 4 100 ± 0 95 ± 5 88 ± 13 98 ± 2 83 ± 13c 

Total survival (%) 97 ± 2 100 ± 0 97 ± 3 87 ± 12 98 ± 2 85 ± 8c 

Male pup weight (g) 3.10 ± 0.13 3.12 ± 0.10 3.04 ± 0.24 3.11 ± 0.15 3.25 ± 0.16 3.02 ± 0.15 

Female pup weight (g) 3.03 ± 0.14 2.90 ± 0.11 3.05 ± 0.23 2.93 ± 0.17 3.23 ± 0.16 2.97 ± 0.16c 

* Significantly different (P#0.05) from the control group by Dunn’s test a Data for body weights, live pups/litter, and sex ratio are given as mean ± standard error. Differences from the control group were not 

significant by a chi-square test (pregnancy indices), Dunn’s test (sex ratio and body weights), or Dunnett’s test (adjusted pup weights). b Fertile pairs/cohabiting pairs 

n=7 

d No data were collected for one of eight litters. e Live male pups/live pups f Least-squares estimate of the mean of the average pup weight adjusted for average litter size 
 

Primidone, NTP TR 476 285 

TABLE O3 

Fertility, Reproductive Performance, Length of Gestation, and Body Weight Data 

for F0 and F1 Swiss (CD-1®) Mice in the Continuous Breeding Study of Primidonea 

0 ppm 150 ppm 500 ppm 1,500 ppm 

F0 Adult Data 

Pregnancy indexb 

Litter 1 39/39 (100%) 17/17 (100%) 20/20 (100%) 17/17 (100%) 

Litter 2 39/39 (100%) 17/17 (100%) 20/20 (100%) 16/17 (94%) 

Litter 3 39/39 (100%) 17/17 (100%) 20/20 (100%) 16/17 (94%) 

Litter 4 39/39 (100%) 17/17 (100%) 20/20 (100%) 16/17 (94%) 

Litter 5 33/39 (85%) 16/17 (94%) 20/20 (100%) 14/17 (82%) 

Average litters/pair 4.8 ± 0.1 4.9 ± 0.1 5.0 ± 0.0 4.6 ± 0.2 

Cumulative days to litter 

Litter 1 21.5 ± 0.5 22.8 ± 1.0 20.6 ± 0.3 22.1 ± 1.4 

Litter 2 42.3 ± 1.0 42.8 ± 1.0 40.6 ± 0.4 43.7 ± 1.7 

Litter 3 63.5 ± 1.3 63.1 ± 1.0 61.1 ± 0.6 64.3 ± 1.8 

Litter 4 84.0 ± 1.4 84.7 ± 1.1 81.7 ± 0.7 84.9 ± 1.9 

Litter 5 101.8 ± 0.6 104.8 ± 1.2* 102.4 ± 0.9 102.9 ± 1.3 

Dam weight during lactation of litter 5 (g) 

n 37 16 20 15 

Lactation day 0 42.2 ± 0.7 42.5 ± 0.8 43.6 ± 0.9 41.4 ± 1.2 

Lactation day 4 44.0 ± 0.7 44.0 ± 0.8 45.9 ± 1.0 44.6 ± 1.2 

Lactation day 7 46.2 ± 0.9 46.1 ± 1.1 48.3 ± 1.1 47.5 ± 1.3 

Lactation day 14 47.6 ± 1.1 49.3 ± 1.0 50.4 ± 1.1 49.6 ± 1.5 

Lactation day 21 40.3 ± 0.7 38.9 ± 2.8 39.6 ± 2.2 40.5 ± 1.4 

F1 Pup Data (Litters 1 Through 5) 

Number of breeding pairs 39 17 20 17 

Live male pups/litter 5.7 ± 0.3 5.9 ± 0.2 6.1 ± 0.3 5.8 ± 0.2 

Live female pups/litter 5.7 ± 0.3 5.9 ± 0.4 6.2 ± 0.3 6.2 ± 0.2 

Total live pups/litter 11.5 ± 0.6 11.6 ± 0.4 12.2 ± 0.5 12.0 ± 0.3 

Average live pups/litter (%) 93 ± 3 99 ± 1 98 ± 1 99 ± 0 

Sex ratioc

 (%) 51 ± 1 51 ± 2 50 ± 1 48 ± 2 

Male pup weight (g) 1.61 ± 0.02 1.60 ± 0.02 1.62 ± 0.02 1.58 ± 0.02 

Female pup weight (g) 1.55 ± 0.02 1.53 ± 0.03 1.56 ± 0.02 1.52 ± 0.02 

Total live pup weight (g) 

Adjusted total pup weightd (g) 

1.58 ± 0.02 

1.58 ± 0.01 

1.57 ± 0.02 

1.56 ± 0.02 

1.59 ± 0.02 

1.60 ± 0.02 

1.55 ± 0.02 

1.54 ± 0.02 

*	 Significantly different (P#0.05) from the control group by Dunn’s test a Data for average litters/pair, cumulative days to litter, body weights, live pups/litter, and sex ratio are given as mean ± standard error. 

Differences from the control group were not significant by a chi-square test (pregnancy indices), Dunn’s test (average litters per pair, 

body weights, sex ratio, and live pups/litter), or Dunnett’s test (adjusted pup weights). b	 Females delivering litters/cohabiting pairs 

Live male pups/live pups d	 Least-squares estimate of the mean of the average pup weight adjusted for average litter size 

c 
286 

c 

Primidone, NTP TR 476 

TABLE O4 

Litter and Body Weight Data for F1 Swiss (CD-1®) Mouse Pups in the Continuous Breeding Study 

of Primidonea 

0 ppm 150 ppm 500 ppm 1,500 ppm 

Litter 1 

Number of pairs delivering 39e 17 20 17 

Live pups/litterb 11.3 ± 0.5 11.3 ± 0.8 11.1± 0.6 10.5 ± 0.6 

Total live pup weightc

 (g) 1.58 ± 0.02 1.60 ± 0.05 1.59 ± 0.02 1.57 ± 0.02 

Adjusted total live pup weightd (g) 1.59 ± 0.02 1.60 ± 0.02 1.59 ± 0.02 1.54 ± 0.02 

Litter 2 

Number of pairs delivering 39e 17 20 16 

Live pups/litter 11.6 ± 0.6 12.6 ± 0.6 13.1 ± 0.6 12.7± 0.4 

Total live pup weight (g) 1.59 ± 0.04 1.58 ± 0.03 1.57 ± 0.02 1.53 ± 0.03 

Adjusted total live pup weight (g) 1.58 ± 0.02 1.58 ± 0.03 1.59 ± 0.03 1.53 ± 0.03 

Litter 3 

Number of pairs delivering 39f 17 20 16 

Live pups/litter 11.0 ± 0.8 11.6 ± 1.0 13.0 ± 0.6 13.0 ± 0.9 

Total live pup weight (g) 1.62 ± 0.02 1.65 ± 0.05 1.59 ± 0.02 1.56 ± 0.04 

Adjusted total live pup weight (g) 1.61 ± 0.02 1.62 ± 0.02 1.61 ± 0.02 1.57 ± 0.02 

Litter 4 

Number of pairs delivering 39f 17 20 16 

Live pups/litter 12.0 ± 0.9 11.8 ± 0.7 12.6 ± 0.7 12.1 ± 0.7 

Total live pup weight (g) 1.60 ± 0.03 1.53 ± 0.08 1.63 ± 0.02 1.61 ± 0.04 

Adjusted total live pup weight (g) 1.62 ± 0.03 1.51 ± 0.04 1.63 ± 0.04 1.60 ± 0.04 

Litter 5 

Number of pairs delivering 33g 16e 20 14 

Live pups/litter 11.8 ± 0.8 11.0 ± 0.9 11.5 ± 0.9 11.3 ± 0.5 

Total live pup weight (g) 1.56 ± 0.02 1.57 ± 0.02 1.61 ± 0.03 1.59 ± 0.03 

Adjusted total live pup weight (g) 1.58 ± 0.02 1.57 ± 0.03 1.60 ± 0.02 1.56 ± 0.03 

Litters 1 Through 5 

Number of pairs delivering 39 17 20 17 

Live pups/litter 11.5 ± 0.6 11.6 ± 0.4 12.2 ± 0.5 12.0 ± 0.3 

Total live pup weight (g) 1.58 ± 0.02 1.57 ± 0.02 1.59 ± 0.02 1.55 ± 0.02 

Adjusted total live pup weight (g) 1.58 ± 0.01 1.56 ± 0.02 1.60 ± 0.02 1.54 ± 0.02 

a Data are given as mean ± standard error. Differences from the control group were not significant by Dunn’s (live pups/litter and total 

live pup weights) or Dunnett’s test (adjusted pup weights). b Mean of average number of live pups per litter for each fertile pair 

Mean of average live pup weight for each fertile pair d Least-squares estimate of the mean of the average pup weight adjusted for average litter size for each fertile pair e No live pups were born in one litter. f No live pups were born in four litters. 

g No live pups were born in two litters. 
287 

c 

Primidone, NTP TR 476 

TABLE O5 

Survival and Body Weights of F1 Swiss (CD-1®) Mouse Pups (Final Litter) 

in the Continuous Breeding Study of Primidonea 

0 ppm 150 ppm 500 ppm 1,500 ppm 

Day 0 

Number of litters 33b 16c 20d 14 

Male pup weight (g) 1.61 ± 0.02 1.59 ± 0.02 1.65 ± 0.03 1.62 ± 0.04 

Female pup weight (g) 1.54 ± 0.02 1.54 ± 0.03 1.57 ± 0.04 1.56 ± 0.03 

Day 4 

Male survival (%) 87 ± 6 90 ± 7 89 ± 7 99 ± 1 

Female survival (%) 87 ± 6 93 ± 7 92 ± 5 99 ± 1 

Total survival (%) 87 ± 6 91 ± 7 88 ± 7 98 ± 1 

Male pup weight (g) 3.05 ± 0.06 3.20 ± 0.10 3.21 ± 0.12 2.99 ± 0.14 

Female pup weight (g) 2.97 ± 0.07 3.12 ± 0.11 3.05 ± 0.11 2.90 ± 0.12 

Day 7 

Male survival (%) 86 ± 6 89 ± 7 89 ± 7 99 ± 1 

Female survival (%) 86 ± 6 93 ± 7 92 ± 5 99 ± 1 

Total survival (%) 86 ± 6 90 ± 7 88 ± 7 98 ± 1 

Male pup weight (g) 4.59 ± 0.11 4.80 ± 0.13 4.76 ± 0.17 4.50 ± 0.20 

Female pup weight (g) 4.49 ± 0.11 4.68 ± 0.13 4.58 ± 0.16 4.37 ± 0.17 

Day 14 

Male survival (%) 85 ± 6 89 ± 7 89 ± 7 99 ± 1 

Female survival (%) 86 ± 6 93 ± 7 92 ± 5 99 ± 1 

Total survival (%) 85 ± 6 90 ± 7 88 ± 7 98 ± 1 

Male pup weight (g) 7.17 ± 0.18 7.78 ± 0.21 7.73 ± 0.29 7.21 ± 0.31 

Female pup weight (g) 6.98 ± 0.18 7.67 ± 0.20* 7.48 ± 0.25 7.13 ± 0.29 

Day 21 

Male survival (%) 83 ± 6 87 ± 7 89 ± 7 92 ± 7 

Female survival (%) 86 ± 6 91 ± 7 92 ± 5 92 ± 7 

Total survival (%) 84 ± 6 89 ± 7 88 ± 7 92 ± 1 

Male pup weight (g) 10.82 ± 0.38 11.84 ± 0.55 12.78 ± 0.58* 12.23 ± 0.51 

Female pup weight (g) 10.26 ± 0.36 11.62 ± 0.49* 11.85 ± 0.45* 11.68 ± 0.38* 

*	 Significantly different (P#0.05) from the control group by Dunn’s or Shirley’s test. a Data are given as mean ± standard error. Differences from the control group in survival were not significant by Dunn’s test. b	 No live pups were born in two litters. Because only female pups were born in one litter, n=30 for male pup survival and body weights. 

No live pups were born in one litter. Because only female pups were born in one litter, n=14 for male pup survival and body weights. d	 Because only male pups were born in one litter, n=19 for female pup weights at day 0. Thereafter, n=18 for male and female body 

weights. 
288 

c 

Primidone, NTP TR 476 

TABLE O6 

Fertility, Reproductive Performance, Length of Gestation, and Body Weight Data 

for F1 and F2 Swiss (CD-1®) Mice in the Offspring Assessment Phase 

of the Continuous Breeding Study of Primidonea 

0 ppm	 1,500 ppm 

F1 Adult Data 

Mating indexb 20/20 (100%) 20/20 (100%) 

Pregnancy indexc 18/20 (90%) 20/20 (100%) 

Fertility indexd 18/20 (90%) 20/20 (100%) 

Dam weight at delivery (g) 36.13 ± 0.86 35.66 ± 0.50 

Days to litter 19.1 ± 0.1 19.0 ± 0.1 

F2 Pup Data 

Number of litters 18 20 

Live male pups/litter 5.8 ± 0.4 5.0 ± 0.4 

Live female pups/litter 5.8 ± 0.4 5.9 ± 0.4 

Total live pups/litter 11.6 ± 0.5 10.9 ± 0.3 

Total live pups/litter (%) 100 ± 0 99 ± 1 

Sex ratioe

 (%) 50 ± 4 45 ± 3 

Male pup weight (g) 1.64 ± 0.04 1.61 ± 0.03 

Female pup weight (g) 1.57 ± 0.03 1.53 ± 0.03 

Total live pup weight (g) 1.60 ± 0.03 1.57 ± 0.03 

Adjusted total live pup weightf

 (g) 1.62 ± 0.02 1.55 ± 0.02 

a Data for body weights, days to litter, live pups/litter, and sex ratio are given as mean ± standard error. Differences from the control 

group were not significant by a chi-square test (mating indices, pregnancy indices, or fertility indices), Wilcoxon’s test (average 

litters/pair, cumulative days to litter, dam weights, sex ratio, and live pups/litter), Dunn’s test (nonadjusted pup weights), or Dunnett’s 

test (adjusted pup weights). b	 Females with sperm plug/cohabiting pairs 

Fertile pairs/cohabiting pairs d	 Fertile pairs/females with sperm plug e Live male pups/live pups f Least-squares estimate of the mean for all litters of the average (per litter) pup weight adjusted for average litter size 
Primidone, NTP TR 476 289 

TABLE O7 

Sperm Parameters and Estrous Cycle Characterization for F1 Swiss (CD-1®) Mice 

in the Offspring Assessment Phase of the Continuous Breeding Study of Primidonea 

0 ppm 1,500 ppm 

n 20 20 

Male 

Epididymal spermatozoal parameters 

Motility (%) 

Abnormal (%) 

80.2 ± 1.6 

5.1 ± 0.56b 

76.7 ± 1.4 

3.7 ± 0.40 

Concentration (106

/g cauda epididymal tissue) 1,019 ± 38 1,011 ± 67 

Female 

Estrous cycle length (days) 4.57 ± 0.11 4.92 ± 0.10* 

Estrous stages (% of cycle) 

Diestrus 25.8 26.7 

Proestrus 22.9 20.0 

Estrus 32.1 35.4 

Metestrus 19.2 17.9 

* Significantly different (P#0.05) from the control group by Shirley’s test a Epididymal spermatozoal parameters and estrous cycle lengths are given as mean ± standard error. Differences from the control group 

for epididymal spermatozoal motility, abnormality, and concentration are not significant by Wilcoxon’s test. By multivariate analysis of 

variance, exposed females do not differ significantly from control females in the relative length of time spent in the estrous stages. b n=19 
290 Primidone, NTP TR 476 

TABLE O8 

Organ Weights and Organ-Weight-to-Body-Weight Ratios 

for F1 Swiss (CD-1®) Mice Administered Primidone in Feeda 

0 ppm 1,500 ppm 

n 20 20 

Male 

Necropsy body wt 35.8 ± 0.86 35.3 ± 0.60 

R. Cauda Epididymis 

Absolute 0.02 ± 0.00 0.02 ± 0.00 

Relative 0.51 ± 0.01 0.49 ± 0.03 

R. Epididymis 

Absolute 50.2 ± 1.2 49.5 ± 1.1 

Relative 1.4 ± 0.03 1.4 ± 0.03 

Kidneys and Adrenal Glands 

Absolute 815.7 ± 40.9 699.1 ± 20.2 

Relative 22.7 ± 0.96 19.9 ± 0.55* 

Liver 

Absolute 1.9 ± 0.06 2.7 ± 0.09* 

Relative 53.6 ± 1.3 76.7 ± 1.4* 

Prostate Gland 

Absolute 23.4 ± 1.9 21.7 ± 1.3 

Relative 0.65 ± 0.05 0.62 ± 0.03 

Seminal Vesicles 

Absolute 424.8 ± 19.4 360.2 ± 10.9* 

Relative 11.8 ± 0.39 10.2 ± 0.27* 

R. Testis 

Absolute 125.1 ± 2.8 125.6 ± 3.1 

Relative 3.5 ± 0.09 3.6 ± 0.11 

Female 

Necropsy body wt 31.1 ± 0.63 31.1 ± 0.45 

Kidneys and Adrenal Glands 

Absolute 516.5 ± 11.6 510.3 ± 8.3 

Relative 16.7 ± 0.30 16.4 ± 0.19 

Liver 

Absolute 1.8 ± 0.04 2.4 ± 0.07* 

Relative 57.0 ± 0.90 75.6 ± 1.5* 

R. Ovary 

Absolute 9.7 ± 0.53 9.3± 0.81 

Relative 0.31 ± 0.02 0.30 ± 0.03 

* Significantly different (P#0.05) 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 ± standard error).