NATIONAL TOXICOLOGY PROGRAM 

Technical Report Series 

No. 443 

TOXICOLOGY AND CARCINOGENESIS 

STUDIES OF OXAZEPAM 

(CAS NO. 604-75-1) 

IN SWISSWEBSTER AND B6C3F, MICE 

(FEED STUDIES) 

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES 

PublicHealthService 

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. 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. Selectionper se is not an indicator of a 

chemical's carcinogenic potential. 

These NTP Technical Reports are available for sale from the National Technical Information Service, 

U.S. Department of Commerce, 5285 Port Royal Road, Springfield, VA 22161 (703-487-4650). Single 

copies of this Technical Report are available without charge while supplies last from NTP Central Data 

Management, NIEHS, P.O. Box 12233, MD AO-01, Research Triangle Park, NC 27709 (919-541-1371). 
NTP TECHNICAL REPORT 

ON THE 

TOXICOLOGY AND CARCINOGENESIS 

STUDIES OF OXAZEPAM 

(CAS NO. 604-75-1) 

IN SWISS-WEBSTER AND B6C3Fl MICE 

(FEED STUDIES) 

NATIONAL TOXICOLOGY PROGRAM 

P.O. Box 12233 

ResearchTrianglePark, NC 27709 

August 1993 

NTP TR 443 

NIH Publication No. 93-3359 

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES 

Public Health Service 

National Institutes of Health 
2 Oxazepam, NTP TR 443 

CONTRIBUTORS 

National Toxicology Program 

Evaluated and interpreted results and reported findings 

C.J. Alden, Ph.D. 

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

D.A. Bridge, B.So 

J.R. Bucher, Ph.D. 

S.L. Eustis, D.V.M., Ph.D. 

T.J. Goehl, Ph.D. 

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

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

J.K. Haseman, Ph.D. 

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

B.A. Schwetz, D.V.M., Ph.D. 

C.C. Shackelford, D.V.M., M.S., Ph.D. 

D.B. Waiters, Ph.D. 

K.L. Witt, M.S., Oak Ridge Associated Universities 

Battelle Columbus Laboratories 

Conducted studies, evaluated pathology findings 

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

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

Experimental Pathology Laboratories, Inc. 

Provided pathology qualio~ assurance 

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

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

Dynamac Corporation 

Prepared qualio~ assurance audits 

S. Brecher, Ph.D., Principal Investigator 

NTP Pathology Working Group 

Evaluated slides, prepared pathology report on Swiss-Webster mice 

(3 March 1992) 

L.H. Brennecke, D.V.M., Chair 

Pathology Associates, Inc. 

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

North Carolina State University 

S.L. Eustis, D.V.M., Ph.D. 

National Toxicology Program 

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

Experimental Pathology Laboratories, Inc. 

J.R. Harley, D.V.M. 

National Toxicology Program 

J. Mahler, D.V.M. 

National Toxicology Program 

C.C. Shackelford, D.V.M., M.S., Ph.D. 

National Toxicology Program 

Evaluated slides, prepared pathology report on B6C3F 1 mice 

(8 September 1992) 

L.H. Brennecke, D.V.M., Chair 

Pathology Associates, Inc. 

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

National Toxicology Program 

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

National Toxicology Program 

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

Experimental Pathology Laboratories, Inc. 

D.S. Marsman, D.V.M., Ph.D. 

National Toxicology Program 

J.A. Popp, D.V.M., Ph.D. 

Chemical Industry Institute of Toxicology 

C.C. Shackelford, D.V.M., M.S., Ph.D. 

National Toxicology Program 

Biotechnical Services, Inc. 

Prepared Technical Report 

D.D. Lambright, Ph.D., Principal Investigator 

G.F. Corley, D.V.M. 

P.R. Dennis, M.C.M. 

L.M. Harper, B.A. 
CONTENTS 

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

APPENDIX A Summary of Lesions in Male Swiss-Webster Mice in the 57.Week Feed Study 

APPENDIX B Summary of Lesions in Female Swiss-Webster Mice in the 57-Week Feed Study 

APPENDIX C Summary of Lesions in Male B6C3F~ Mice in the 2-Year Feed Study 

APPENDIX D Summary of Lesions in Female B6C3Ft Mice in the 2-Year Feed Study 

of Oxazepam ...................................................... I73 

APPENDIX J Ingredients, Nutrient Composition, and Contaminant Levels 

EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY ............... 10 

TECHNICAL REPORTS REVIEW SUBCOMMITTEE ................................... 11 

SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS ........... 12 

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

MATERIALS AND METHODS .................................................... 21 

RESULTS .................................................................... 29 

DISCUSSION AND CONCLUSIONS ................................................ 59 

REFERENCES ................................................................ 65 

of Oxazepam ...................................................... 73 

of Oxazepam ...................................................... 105 

of Oxazepam ...................................................... 135 

APPENDIX E Genetic Toxicology ................................................. 211 

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

APPENDIX G Neurobehavioral Studies ............................................. 223 

APPENDIX ~-I Chemical Characterization and Dose Formulation Studies ................... 247 

APPENDIX I Feed and Compound Consumption in the 57-Week Feed Studies ............... 259 

in NIH-07 Rat and Mouse Ration ...................................... 265 

APPENDIX K Sentinel Animal Program ............................................ 271 
4 Oxazepam, NTP TR 443 

APPENDIXL Ras Proto-Oncogene Activation of LiverNeoplasmsfromB6C3FlMice 

Administered Oxazepam in Feed for 2 Years . . . . . . . . . l . . . .. l l .. l . . ... . . . l . 275 

APPENDIXM Measures of Replicative DNA Synthesis in Liver in a 90-Day Feed Study 

of Oxazepam in Male B6C3Fl Mice . . . . ... . . . . . . . . . . . . . . . . . . . .. . . . . .. . . . 281 

APPENDIXN Clinical Pathology AnalysesinMaleB6C3FlMice in a 90-Day Feed Study 

ofoxazepam ...................................................... 291 

APPENDIX 0 Toxicokinetics of Oxazepam in B6C3F, and Swiss-Webster Mice . . .. . . .. . . . .. . 295 

APPENDIX P Comparative Metabolism Studies in B6C3Fl and Swiss-Webster Mice 

Administered Oxazepam by Oral Gavage . . . . . . . . . . . . . . . . . . . .. . . .. . . .. . . . . 307 
5 

ABSTRACT 

H 

C6H5 

OXAZEPAM 

CAS No. 604-75-1 

Chemical Formula: C,,H,,CIN,O, Molecular Weight: 286.74 

Synonym: 7-Chloro-l,3-dihydro-3-hydroxy-5-phenyl-W-l,4-benzodiazepin-2-one 

Trade Names: Tazepam, Wy-3498,Sera 

Oxazepam is one of anumber of benzodiazepines 

used therapeutically as a sedative-hypnotic and antianxiety agent. Toxicology and carcinogenesis studies 

were performed by administering oxazepam (greater 

than 99% pure)in feed tomaleand female 

Swiss-Webster and B6C3Fl mice for 14 weeks, 

57 weeks (Swiss-Webster), or 2 years (B6C3Fl). 

Neurobehavioral assessments were performed during 

the studies. Genetic toxicology studies wereconductedin Salmonella typhinturiunl andcultured 

Chinesehamster ovary cells, and peripheralblood 

sampleswere analyzed for frequency of micronucleated normochromatic erythrocytes. Supplemental 

studies were performed to compare the metabolism 

and toxicokinetics of oxazepam in the two mouse 

strains, to evaluate the effect on liver cell replication 

rates, to perform clinical pathology assessments, and 

to examine the mutation spectrum and frequency of 

activated H-ras oncogenes in liver neoplasms from 

the 2-year study with B6C3Fl mice. 

14-WEEK STUDY IN 

SWISS-WEBSTERMICE 

Groups of 10male and10female Swiss-Webster mice 

received oxazepam in feed at concentrations of 0, 

625, 1,250,2,500, 5,000, or 10,OOOppm for 14 weeks. 

One 625 ppm male and one 10,000ppm female were 

killed moribund before the end of the study, and the 

condition of the female mouse was attributed to 

oxazepam exposure. Mean body weight gains of 

exposed groups were similarto those of the controls. 

Exposed mice displayed chemical-related sedation and 

lethargy during the first study week, but appeared 

normalthereafter. In the neurobehavioralstudies, 

reductions in grip strength were evident in both male 

andfemale mice at week 2 andpersistedin males 

through week 11. An antianxiety effect was detected 

in exposed mice in measures of motor activity, startle 

response, and reactions to thermal stimulus. 

At necropsy, absolute and relative liver weights were 

increased in an exposure-related mannerandwere 

approximately two-fold greater in 10,000 ppm mice 

than in controls. Centrilobular hepatocellular hypertrophy was present only in exposed mice, and the 

severity increased with dose. 

14-WEEK STUDY IN B6C3F, MICE 

Groups of 10 male and 10 female B6C3Fl mice 

received oxazepam in feed atconcentrations of 0, 

625, l,250,2,500,5,OOO, or 10,OOO ppm for 14 weeks. 

There were no deathsthat were clearly related to 

oxazepam exposure. Mean body weight gains of 

exposed groups were similarto those of the controls. 
6 Oxazepam, NTP TR 443 

Exposed mice displayed chemical-related sedation and 

lethargy during only the first study week. In neurobehavioral studies, reductions in grip strength were 

evidentin males at week 2 butwere no longer 

observed at week 12. An antianxiety effect was noted 

in exposed mice in measuresof motor activity, startle 

response, and reactions to thermal a stimulus 

(females). 

57-Week Study in Swiss-Webster Mice 

Survival, Body Weights, Feed and Compound 

Consumption, and Clinical Findings 

At57 weeks, survival of exposed mice was significantly lower than that of controls (males: 0 ppm, 

45/60, 2,500 ppm, 19/60, 5,000 ppm, 10/60, females: 

47/60, 28/59, 17/59), causing the study to be terminated. Mean body weights of exposed males were 

CHRONICSTUDIES 

Groups of 60 male and 60 female Swiss-Webster and 

B6C3Fl mice received oxazepam in feed at concentrations of 0,2,500, or 5,OOO ppm. Additional groups 

of 60 maleand60 female B6C3Fl mice received 

125 ppm in feed to allow for study of a group with 

projected serum concentrations of oxazepam similar 

to those achieved in humanstakingatherapeutic 

dose. Tenmaleand10 female B6C3Fl mice per 

groupwereevaluatedat 15 months. Average daily 

oxazepam consumption varied throughout the studies, 

and the overall daily average ranged from 10 to 

29 mgkg body weight for the 125 ppm groups, 234 to 

512 mgkg for the 2,500 ppm groups,and 444 to 

1,085 mgkg for the 5,OOO ppm groups. Serum 

oxazepam concentrations determined at 57 weeks in 

Swiss-Webster mice and at the 15-monthinterim 

evaluation of B6C3Fl mice were approximately 

1 pg/mL in the 125 ppm groups, 4 to 7 pg/mL in the 

2,500 ppmgroups, and7to10pg/mLin the 

5,OOO ppm groups. 

Neurobehavioralassessmentsduring thechronic 

studies of each strainof mice were confounded by the 

poor survival and deterioratingcondition of mice 

with hepatic neoplasia. However, within the

limitations of the studies,there were nonotable 

changes in the types of behaviors observed compared 

to those observed in the 14-week studies, nor was 

there an enhancement in the degree to which they 

were exhibited. 

had a mean body weight greater thanthat of the 

controls early in the study; after week 29, the mean 

body weight of this group was similar to that of the 

controls. Feed consumption by exposed males and 

females was slightly lower than that by the controls, 

and females in allgroups,includingcontrols,consumed slightly more feed than males throughout the 

study. Dietary levels of 2,500 and 5,OOO ppm oxazepam resulted in averagedaily compound consumption 

levels of 270 and 570 mg/kg for males and 320 and 

670 mgkg for females. Hypoactivity and sedation 

were observed in exposed mice during the first week 

of the study. There were no other clinical findings 

associated with oxazepam exposure. 

Pathology Findings 

Systemic amyloidosis was the principal causeof death 

in mice dying before the study was terminated. The 

lower survival of mice receiving oxazepam was attributed toan increasein the extent and severity of 

amyloid deposits in many organs, including the heart 

and kidney. Atrial thrombosis and pulmonary lesions 

consistent with chronic heartfailureoccurredat 

higher incidences and with greater severity in exposed 

mice. 

The incidence of hepatocellularadenomas (males: 

1/60, 35/60, 50/60; females: 0/60, 22/59, 47/59) and 

carcinomas (males: 0/60, 5/60, 19/60; females: 1/60, 

1/59, 11/59) were increasedin exposed mice. The 

incidences of eosinophilic foci were also increased in 

exposed mice (males: 0/60, 22/60, 22/60, females: 

0/60, 20/59, 14/59), and there was evidence of 
Oxazepam, NTP TR 443 7 

increased hepatocyte centrilobular hypertrophy 

(males: 12/60, 46/60, 47/60; females: 3/60, 51/59, 

53/59). 

2-Year Study in B6C3Fl Mice 

Survival, Body Weights, Feed and Compound 

Consumption, and Clinical Findings 

Survival of mice receiving 2,500 and 5,OOO ppm was 

significantly lower thanthat of controls (males: 

0 ppm, 45/50; 125 ppm, 44/50; 2,500 ppm, 15/50; 

5,OOO ppm, 0/50; females: 39/50, 41/50, 2/50, 0/50). 

Mean body weight gains of exposed male and female 

mice were similar to controls untilabout week 15 

when weight gains for mice exposed to 2,500 or 

5,OOO ppm slowed in relation to controls, resulting in 

weight gains approximately 30% to 40% lower than 

those of the controls throughoutthe remainder of the 

study. Mean body weight gain of male mice exposed 

to 125 ppm was similar to that of the controls, while 

that of female mice receiving 125 ppm was 10% to 

15% lower than that of the controlsafterabout 

week 45. Feed consumption by exposed males and 

females was similar to that by controls.Dietary 

levels of 125, 2,500, and 5,OOO ppm resultedin 

average daily oxazepam consumption levels of 12, 

310, and 690 mgkg body weight for males and 15, 

350, and 780 mgkg for females. In the 5,000 ppm 

groups, lethargy and sedation were observed in a few 

mice during the first week of study. 

Pathology Findings 

The early deaths of many of the B6C3Fl mice exposed to oxazepam wereattributed to a marked 

increase in the incidences of hepatoblastoma (males: 

0/49, 2/50, 21/50,13/50; females: 0/50, 1/50, 8/50, 

8/50), hepatocellular adenoma (males: 17/49, 18/50, 

34/50,32/50; females: 25/50,35/50,35/50,36/50),and 

hepatocellularcarcinoma (males: 9/49, 5/50, 45/50, 

50/50; females: 9/50, 5/50, 49/50, 44/50). Moderate 

hypertrophy of centrilobular hepatocytes occurred in 

mice receiving 2,500 and 5,000 ppm (males: 0/49, 

2/50, 26/50, 43/50; females: 0/50, 2/50, 11/50, 29/50). 

An increase in the incidence of follicular cell hyperplasia of the thyroid gland occurred in all exposed 

groups of mice (males: 4/49,22/50,49/50,47/50; 

females: 16/50, 34/50, 49/50, 44/50), and thyroid 

gland follicular cell adenoma was increased in exposed females (0/50, 4/50, 5/50, 6/50). Testicular 

atrophy occurred in the 2,500 and 5,OOO ppm groups 

(1/50, 0/50, 25/50, 38/50), and the incidence of 

epididymal lymphocyte infiltration was increased in 

all exposed groups (2/50, 14/50, 33/50, 21/50). 

The frequency of hepatocellular neoplasms with an 

activated H-rus oncogene inthe B6C3Fl mice and the 

mutationspectrum of the H-rus geneweredetermined. The mutation spectrumof the H-rus genes in 

the relatively few neoplasms from exposed mice that 

did have an activated H-rus did not differ from the 

spectrum of mutations observed in neoplasms from 

controls,but the proportion of neoplasms with an 

activated H-rus genedecreased with increasing 

oxazepam dose. While 11 of 19 (58%)neoplasms 

from control mice had an activated H-rus gene, only 

1of 40 neoplasms from mice receiving 2,500 or 

5,000 ppm oxazepam exhibited a similarmolecular 

lesion. Thirteen of 37 (35%) neoplasms from mice in 

the 125 ppm group had an activated H-rus oncogene, 

suggesting that, although the incidence of all liver 

neoplasms was not statistically increased compared to 

controls, there was an increase in a similar subset of 

neoplasms (lacking an activated H-rus) that occurred 

with increased incidence at higher doses. 

SUPPLEMENTAL STUDIES 

Because exposure to oxazepam causedincreased 

incidences of liver neoplasms, supplementalshorttermstudieswereperformed. Oxazepam given in 

feed to male B6C3Fl mice at 25, 125, 2,500, or 

5,OOO ppm for up to 13weeks was found to cause a 

dose-relatedincreaseinnuclearlabeling index in 

studies measuring the incorporation of bromodeoxyuridine into replicating liver cells. This increase was 

statistically significant at all but the 25 ppm exposure 

level and was limited to mice evaluated at 15 days. 

Cell replicationrates in most groupsevaluatedat 

30 days and after were similarto control rates. There 

was minimal evidence suggestive of hepatocyte 

necrosis either by light microscopy or in clinical 

chemistry measures. There was, however, evidence of 

cholestasis, likely due to physical obstruction of bile 

canaliculi by swollen hepatocytes. 

The metabolic fate and toxicokinetics of oxazepam 

wereevaluated in each strain of mice and were 

compared to published data from humanstudies. 
8 Oxazepam, NTP TR 443 

Both mice andhumans formglucuronides of 

oxazepam and form 3- and 4-hydroxy and methoxy 

derivatives of the phenyl group. Oxidative metabolism of the phenyl groupappearstobemore 

prevalent in mice than is reported for humans. 

Elimination half-lives of parentcompound do not 

differ between Swiss-Webster and B6C3Fl mice and 

are similar to values reported for humans. 

GENETIC TOXICOLOGY 

Oxazepam was not mutagenic in any of several strains 

of Salmonella typhimurium, nor did it induce sister 

chromatid exchanges or chromosomalaberrations 

inculturedChinesehamster ovary cells. These 

in vino testswereperformed with and without S9 

metabolic activation. Results from an in vivo mouse 

peripheral blood micronucleustest performed on the 

B6C3Fl mice used in the 14-week study werealso 

negative. 

~~ 

CONCLUSIONS 

Under the conditions of these feed studies, there was 

clear evidence of carcinogenic activity* of oxazepam in 

maleandfemale Swiss-Webster micebased on 

increased incidences of hepatocellular adenoma and 

carcinoma. There was clear evidence of carcinogenic 

activity of oxazepam in male and female B6C3Fl mice 

based on increased incidencesof hepatoblastoma and 

hepatocellularadenoma and carcinoma. Increased 

incidences of hyperplasia of thyroid gland follicular 

cells in male and female B6C3Fl mice and of follicular cell adenomas in female B6C3Fl mice were also 

related to oxazepam exposure. 

Administration of oxazepam to Swiss-Webster mice 

resulted in centrilobular hepatocellular hypertrophy 

and increased incidences and severity of systemic 

amyloidosis. Administration of oxazepam to B6C3Fl 

mice also resulted in centrilobular hepatocellular 

hypertrophy.' 

l Explanation of Levels of Evidence of Carcinogenic Activity is onpage 10. A summary of the Technical Reports Review 

Subcommittee commentsand the public discussion on this report appear on page 12. 
Oxazepam, NTP TR 443 9 

Summary of the Chronic Carcinogenesis and Genetic Toxicology Studiesof Oxazepam 

Male Female Male Female 

Swiss-Webster Swiss-Webster B6C3F, B6C3F, 

Mice Mice Mice Mice 

0,2,500, or 0, 2,500, or 0, 125, 2,500, or 0, 125, 2,500, or 

5,OOO PPm 5,OOO ppm (approxi- 5,000 ppm (approxi- 5,000 ppm (approxi-

(approximately 270 or mately 320 or 670 mately 12, 310, or mately 15, 350, or 

570 m&g in feed) mgkg in feed) 690 mgAg in feed) 780 m&g in feed) 

Body weights Exposed groups lower 2,500 ppm group 2,500 and 5,OOO ppm Exposed groups lower 

than controls higher than controls groups lowerthan than controls 

controls 

47/60, 28/59, 17/59 45/50, 44/50, 15/50, 39/50, 41/50, 2/50, 

0/50 0/50 

Nonneoplastic ef- Multiple organs: Multiple organs: Liver:centrilobular Liver:centrilobular 

fects increased incidence increased incidence hypertrophy (OW, hypertrophy (0/50, 

and severity of sys- and severity of sys- 2/50, 26/50, 43/50); 2/50, 11/50, 29/50) 

temic amyloid deposi- temic amyloid deposi- Thyroid gland: follic- Thyroidgland:folliction tion ular cell hyperplasia ular cell hyperplasia 

Liver:centrilobular Liver:centrilobular (4/49, 22/50, 49/50, (16/50, 34/50, 49/50, 

hypertrophy (12/60, hypertrophy (3/60, 47/50) 44/50) 

46/60, 47/60) 51/59, 53/59) 

Neoplastic effects Liver:hepatocellular Liver:hepatocellular Liver: Liver: 

adenoma (1/60,35/60, adenoma (0/60,22/59, hepatoblastoma (0/49, hepatoblastoma (0/50, 

50/60); carcinoma 47/59); carcinoma 2/50, 21/50, 13/50); 1/50, 8/50, 8/50); 

(0/60,5/60, 19/60) (1/60, 1/59, 11/59) hepatocellular adeno- hepatocellular adenoma (17/49, 18/50, ma (25/50, 35/50, 

34/50, 32/50); carci- 35/50, 36/50); carcinoma (9/49, 5/50, noma (9t50, 5/50, 

45/50, 50/50) 49/50, 44/50) Thyroidgland:follicular 

cell adenoma (0/50, 

4/50, 5/50, 6/50) 

Level of evidence 

of carcinogenic 

Clear evidence Clear evidence Clear evidence Clear evidence activity 

Genetic toxicology 

Salmonella yphimm'um gene mutation: Negative in strains TA97, TA98, TA100, TA102, and TA1535 with and without S9 

Sister chromatid exchanges 

Chinese hamster ovary cellsin vim: Negative with and without S9 

Chromosomal aberrations 

Chinese hamster ovary cellsin vim: Negative with and without S9 

Micronucleated nornochromatic 

erythrocytes in B6C3Fl mice: Negative at 14 weeks 

" Survival of Swiss-Webster mice based on a 57-week study; survival of B6C3Fl mice based on 2-year study. 
10 Oxazepam, NTP TR 443 

EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTMTY 

The National Taxicology Program describes the resultsof 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 examinationof all 

available evidence, including animal studies such as those conductedby 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 categoriesof 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 conceptof actual weight of 

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

five categories is selected to describe the findings. These categories refer to the strength of the experimental evidence and not to 

potency or mechanism. 

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

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

increase of benign neoplasms if there is an indication from this or other studies of the ability of such neoplasms to 

progress to malignancy. 

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

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

that required for clear evidence. 

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

increase of neoplasms that may be chemical related. 

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

increases in malignant or benign neoplasms. 

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

limitations, cannotbe 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 understandingof long-term carcinogenesis studies in laboratory animals, especiallyfor those evaluations that may be on the 

borderline between two adjacent levels. These considerations should include: 

l adequacy of the experimental design and conduct; l Occurrence of common versus uncommon neoplasia; l progression (or lack there00 from benign to malignant neoplasia as well as from preneoplastic to neoplastic lesions; 

l some benign tumors 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 

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

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

organ or tissue; 

l latency in tumorinduction; 

l multiplicity in site-specificneoplasia; 

l metastases; 

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

(same lesion in another sex or species); 

l presence or absence of dose relationships; 

l statisticalsignificanceoftheobservedtumorincrease; 

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

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

l structure-activitycorrelations; and 

l in somecases,genetictoxicology. 
Oxazepam, NTP TR 443 11 

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 oxazepam on 

December 1, 1992,are listed below. Subcommittee members serve as independent scientists, not as representatives of any institution, 

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

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

l to determine if the design and conditions of the NTP studies were appropriate, l to ensure that the Technical Report presents the experimental resultsand 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. 

Curtis D. Klaassen, Ph.D., Chair 

Department of Pharmacology and Toxicology 

University of Kansas Medical Center 

Kansas City, KS 

Paul T. Bailey, Ph.D. 

Environmental and Health Sciences Laboratory 

Mobil Oil Corporation 

Princeton, NJ 

Louis S. Beliczky, M.S., M.P.H. 

Department of Industrial Hygiene 

United Rubber Workers International Union 

Akron, OH 

Arnold L. Brown, M.D. 

University of Wisconsin Medical School 

Madison, WI 

Gary P. Carlson, Ph.D. 

Department of Pharmacology and Toxicology 

Purdue University 

West Lafayette, IN 

Kowetha A Davidson, Ph.D. 

Health and Safety Research Division 

Oak Ridge National Laboratory 

Oak Ridge, TN 

Harold Davis, D.V.M., Ph.D. 

Medical Research Division 

American Cyanamid 

Pearl River, NY 

* Didnot attend 

Daniel S. Longnecker, M.D.* 

Department of Pathology 

Dartmouth Medical School 

Lebanon, NH 

Louise Ryan, Ph.D., PrincipalReviewer 

Division of Biostatistics 

Dana-Farber Cancer Institute 

Boston, MA 

Ellen K. Silbergeld, Ph.D.* 

University of Maryland Medical School 

Baltimore, MD 

Robert E.Taylor, Ph.D., Principal Reviewer 

Department of Pharmacology 

Howard University College of Medicine 

Washington, DC 

Matthew J. van Zwieten, D.V.M., Ph.D. 

Department of Safety Assessment 

Merck, Sharp & Dohme Research Laboratories 

WestPoint,PA 

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

National Cancer Institute 

Frederick, MD 

Lauren Zeise, Ph.D.* 

Reproductive and Cancer Hazard Assessment Section 

California Environmental Protection Agency 

Berkeley, CA 
12 Oxazepam, NTP TR 443 

SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMI'ITEE COMMENTS 

On December1,1992, the draft Technical Reporton 

the toxicology and carcinogenesis studiesof oxazepam 

received public review by the National Toxicology 

ProgramBoard of ScientificCounselorsTechnical 

Reports Review Subcommittee. The review meeting 

was held at the National Instituteof Environmental 

Health Sciences, Research Triangle Park, NC. 

Dr. J.R. Bucher,NIEHS,introducedthe toxicology 

and carcinogenesis studies of oxazepamby discussing 

the usesandrationale for study,describing the 

experimentaldesigninSwiss-WebsterandB6C3Fl 

mice, reporting on survival and body weight effects, 

and commentingon compound-related neoplasms and 

nonneoplasticlesionsin both mouse strains. 

Dr. Bucherreportedthatdue to the marked 

enhancement of liverneoplasiainbothstrains,a 

number of supplemental studies were performed at 

NIEHSincludingstudy a to evaluate rates o f

replicative DNA synthesis in the liver, metabolic fate 

and studies, toxicokinetic and analysis of the

frequencyofoccurrence of an activated H-ras 

oncogeneinhepatocellularneoplasmsinB6C3Fl 

mice. The proposed conclusions were clear evidence 

of carcinogenic ncriviry of oxazepaminmaleand 

female Swiss-Webster mice and in male and female 

B6C3Fl mice. 

Dr.Ward,aprincipal reviewer, agreed with the 

proposedconclusions. He said it should be noted 

thatin the B6C3F1mousestudy,thetwohighest 

exposure levels exceeded maximumtolerated dose 

guidelines, but despite the severe depression in body 

weightgain, liver neoplasms wereassociatedwith 

earlymortalityandincreased feed consumption. 

Dr. Bucher thought this was a reasonable point for 

furtherdiscussion by the Subcommittee.Dr.Ward 

said it was importanto establish whether the thyroid 

follicular cell hyperplasia was goiter (diffuse)or focal 

(not diffuse) in B6C3Fl mice. Dr. Bucher responded 

that at the two highest exposure levels, hyperplasia 

was a diffusegoiter type. Dr.Wardaskedthat the 

appendixes associated with the supplemental studies 

be discussed in the Results section. 

Dr. Taylor, the second principal reviewer, agreed with 

theproposedconclusions. He complimented the 

inclusion of the mechanistic studies and also urged 

thatheappendixes be discussed in the Results 

section. Dr. Taylor thoughtthe detailed discussionof 

chlordiazepoxide genotoxicity was notnecessary since 

little of this agentmetabolized to oxazepam,while 

genotoxicitynformationmight be useful on 

temazepam, which is metabolically converted largely 

to oxazepam. Dr. Bucher agreed to add genotoxicity 

information on temazepamif available. (Genotoxicity 

information was not available in the literature.) 

Dr.Ryan, the thirdprincipalreviewer,agreedwith 

the proposedconclusions. She said the different 

patterns of weight gain betweenmaleandfemale 

Swiss-Webstermicewere of someconcern,and 

wondered if these patternscould be explained 

through the varying incidences of toxicity and 

neoplasia. Dr. Bucher said there was not a clear-cut 

cause and effect relationship that would explain the 

differences. Dr.Ryanasked why no studieswere 

conducted to assess reproductive toxicity since oneof 

the rationales forthe study was to examine the use of 

the drug by pregnant Dr. women.Bucher

commented that adequate reproductive and developmental toxicology studies hadbeenconductedasa 

part of the FDA drugapproval process. Dr. Ryan 

notedthatsince the 125ppmexposure level in 

B6C3F, mice was included in an attempt to produce 

ablood level in the therapeuticrangeforhumans, 

interpretation of the findings for humans should be 

addressedin the Conclusions. Dr. Buchersaid he 

would add a phrase that there were indications inthe 

study that the amount of oxazepam was sufficient at 

that level to influenceexpression of the neoplastic 

process (page 62). 

Dr. Davidson asked that some of the nonneoplastic 

lesions, notably heart lesions (amyloidosis) in SwissWebster mice and testicular lesions in B6C3Fl mice, 

be summarized in the text along with the appropriate 

statistical analysis. Dr. Bucherexplained that since 

the amyloidosis was a systemic effect, such a focuson 

the heart lesions couldbe misleading. With regard to 

the testicular lesions, he said it was likely that this 

was atreatment-relatedeffectbutcouldalso be 

secondary to debilitation of the animal. There was 

no evidence fromthe14-weekstudythatthetestis 

was a target organ. Dr. Davis thought there needed 

to be clear a presentation in the text of the

toxicokinetic studies including area under the curve 

(AUC)information,noting that the extensive 
Oxazepam, NTP TR 443 13 

amyloidosis inonestrain of mice could affect 

chemical disposition depending on the organs

involved. Dr. Bucher said AUC data were included, 

and noted that young Swiss-Webster mice were used 

for the toxicokinetic studiesso amyloidosis would not 

have been present. 

In comments from the public, Dr. Michael McClain, 

Hoffman-LaRoche, stated that the existence of the 

thyroid follicular cell hypertrophy along with hyperplasia of a diffuse type provided fairly clear evidence 

that the thyroid gland effects were probably secondary 

to hormone imbalance. Dr. Klaassen asked whether 

serum thyroid-stimulating hormone(TSH) levels had 

been measured. 

Dr. Bucher replied that thyroid hormone status was 

not determined inthe studies doneto date, but there 

wereplans to measureTSHandother thyroid 

hormonesinfurtherstudies. In response to a 

question by Dr. Klaassen about measurement of 

P-450isoforms, Dr. Julian Leakey, NCTR, reported 

that his laboratory was going to be doing studies in 

ratsand mice treated with oxazepam, looking at 

induction of specific isoforms of P-450. Dr. Joseph 

Contrera, Center for Drug Evaluation and Research, 

FDA, praised the interaction between the FDA and 

the NTP inthe design and conduct of oxazepam 

studies. 

Dr.Ward moved that the Technical Report on 

oxazepam be accepted with the revisions discussed 

and with the conclusionsaswrittenformale and 

female Swiss-Webster mice and maleand female 

B6C3Fl mice, clear evidence of carcinogenic activity. 

Dr. Taylor seconded the motion, which was accepted 

by nine yes votesone with abstention 

(Dr. van Zwieten). 
14 Oxazepam, NTP TR 443 
15 

INTRODUCTION 

H 

OXAZEPAM 

CAS NO. 604-7.5-1 

Chemical Formula: C,,H,,CIN202 Molecular Weight: 286.74 

Synonym: 7-Chloro-l,3-dihydro-3-hydroxy-5-phenyl-2H-l,4-benzodiazepin-2-one 

Trade Names: Tazepam, Wy-3498,Sera 

CHEMICALANDPHYSICAL No definiteproductiondata are availablefor

oxazepam or for the benzodiazepine drugs; however, PROPERTIES 

the use of benzodiazepine by the general population Oxazepam is a bitter tasting, whitecrystalline powder, has been reported as 8% in the United Kingdom, 7% insoluble in water, but soluble in alcohol, chloroform, 

andether (Remington'sPharmaceuticalSciences, 

1980). The material is nonhygroscopic and stable in 

light. It has a melting point range of 205" to 206" C 

(Merck, 1983). 

USE AND HUMANEXPOSURE 

Oxazepam and related benzodiazepine drugs are used 

in the treatment of anxiety. Most clinically useful 

drugsthispurpose for are variants of the

1,Cbenzodiazepine structure (Figure 1) consisting of 

two aromatic rings and a 7-membered heterocycle. 

One of the aromatic rings is fused to the 7-membered 

ring and contains a chlorine or otherelectronegative 

groupassubstituent. a All clinically important 

derivatives contain a 5-aryl or 5-cyclohexenyl group. 

Most of the drugs vary in substituent groups at the 

1-3 positions (Goodman and Gifman's, 1990). 

Oxazepam, known under the trade name Sera@, is 

producedandsold by Wyeth Laboratories and has 

been on the market since 1965. 

in the UnitedStates, and8%to10% in Norway 

(Pedersen and Lavik, 1991). In 1983, 2.6 million 

prescriptionsfor oxazepam werewrittenin the 

United States, and oxazepam ranked 132nd and 125th 

in overall frequency of prescriptions written for all 

drugsin 1984 and 1985, respectively (Anonymous, 

1986, Baum, 1986). Oxazepam is also acommon 

metabolite of several other benzodiazepines, someof 

which are more widely prescribed, including diazepam 

(Valium@). In 1983, 25.5 million U.S. prescriptions 

were written for diazepam, making it the fourth most 

prescribed drug. 

REGULATORYSTATUS 

Benzodiazepines are prescriptiondrugsregulated 

under the Federal Food, Drug, and Cosmetic Act of 

1938 and are on Schedule IV of the Drug EnforcementAdministrationControlledSubstancesCode 

(Tocus et al., 1983). Although productionworkers 

andispensers are exposed to benzodiazepines, 
16 Oxazepam, NTP TR 443 

FIGURE1 

1,CBenzodiazepine Structure 

no workplaceexposurelimits have been recommended for these types of chemicals (ACGIH, 1987). 

Environmental contamination has not been shown. 

PHARMACOLOGY 

AI1 benzodiazepines currently in use share a number 

of effects including sedation, hypnosis, decreased 

anxiety, muscle relaxation, amnesia, and anticonvulsant activity. They are consideredcentral 

nervous system (CNS) depressants but arenot 

generaldepressantsand,withintherapeuticdose 

ranges, all effects are related to specific CNS events 

(Goodman and Gifntan’s, 1990). Eachdrug differs 

slightly withinspectrum this of actions (e.g., 

flurazepam has a strong hypnotic effect in humans) 

(Randall et af., 1969). Other drugs are marketed 

specifically for use in obstetrics, for epilepsy, or for 

insomnia (CRM, 1980). These differences may reflect 

the different intrinsicaffinities of the drugs for 

benzodiazepinereceptors. In addition, the various 

drugshavedifferentpharmacokinetics(Greenblatt 

andShader, 1978; Eadie, 1984), and differences in 

disposition and ratesof biotransformation may affect 

the spectrum of effects. Oxazepam is a relatively 

short-actingagent typically prescribed for relief of 

anxiety and given orally, 10 to 15 mg, three or four 

times per day (PDR, 1991). 

The therapeutic effects of the benzodiazepines are 

thought to be due to a receptor-mediated response 

that increases the efficiency of submaximal 

GABAergictransmissionmediated by a variety of 

long-fiber neuronsandinterneurons in the CNS 

(Richards et af., 1986). AGABAergicreceptor 

protein complex has been isolated from brain tissue. 

This complex is associated with achlorideion 

channel and has associated proteins that are separate 

binding sites for barbituratesand the benzodiazepines 

(Barnard et a)., 1984). The benzodiazepine binding 

site is on the alphasubunit(Levitan et af., 1988). 

The complex is subject to a complicated pattern of 

allosteric interactions which ultimately affect chloride 

conductancein the neuron. The clinically useful 

benzodiazepines all act to increase the permeability 

of the GABA receptor complex to chloride (Richards 

et af., 1986). GABAergicneurons are in highest 

concentration in the substantia nigra, globus pallidus, 

and hypothalamus in the human brain (Cooperet af., 

1978). However, the density of CNS-type benzodiazepine receptors is highest in the cortical regions 

of the cerebrum and cerebellum, suggesting other 

functions for the CNS-type receptors (Saano, 1988). 

The anxiety-reducing effect of benzodiazepines inthe 

ratbrainhasbeenassociatedwith GAEiAergic circuits in the mammillary body (Kataoka et af.,1982). 

At least one other benzodiazepine receptor type has 

beenidentified in the brain, specifically in glial 

tissues in the pineal gland and olfactory bulb, and is 

alsofoundinheart, liver, lung, testis,and other 

tissues. The role of this receptor is not clear, but it 

appears to be a mitochondrial protein that may use 

porphyrins as endogenous ligands (Snyder et af.,1987; 

Verma and Snyder, 1989; Calvo et af.,1991), and may 

be involved in the regulation of steroid biosynthesis 

(Krueger and Papadopoulos, 1992). 
Introduction 17 

ABSORPTION, DISPOSITION,

METABOLISM, AND EXCRETION 

Experimental Animals 

In the mouse, 27.3% of anoral gavage dose of 

22 mgkg was recovered in urine and 57.8% in feces 

during 5 days following administration of 

14C oxazepam (labeled at the2 carbon). The majority 

of urinary radioactivity was found to represent 

oxazepam glucuronide and 4'-hydroxyoxazepam

glucuronide.Lesser amounts of 6-chloro-4-phenyl2-(1H)-quinazoline carboxylic acid and 4' -hydroxy3'-methoxyoxazepam were also identified, the latter 

as a sulfate.Fecalmetaboliteswere not identified 

(Sisenwine et al., 1987). 

In the rat, following a single oral dose of 2 mgkg 

with similarly labeled material as was given to mice, 

the radiolabel was foundinmost tissues within 

30minutes. Liver radiolabel peaked early and 

clearedwithin 24 hours (Walkenstein et al., 1964). 

During the first 48 hours, 65%of the label appeared 

in the feces asunidentifiedmetabolites. Seven 

labeled metabolites were found in the urine. 

Sisenwine et al. (1972) identified number a o f

oxazepam metabolites collected in the urine over a 

5-day dosing period in which 40 mg/kg per day was 

given orally to rats. The major peak appeared to be 

the 4'-hydroxyoxazepam glucuronide. Other metabolites included oxazepam substituted with a hydroxyl 

and a methoxy group on the phenyl ring, a metabolite 

in which the diazepine ring was condensed to a sixmembered quinazolinone, and unchanged drug. 

Labeled drug was administered in 0.5% Tween 80 to 

rats,andurine,bile, and feces were collected over 

48 hours; 66% of the dose was recovered from the 

bile primarily within 12 hours (Sisenwine and Tio, 

1986). Additional metabolites identified in the urine 

were 3'-hydroxyoxazepam and an unidentified compound thought to be a dihydrodiol. Major biliary and 

fecal metabolites included 4'-hydroxyoxazepam, the 

tentative dihydrodiol, and unchanged drug. 

Humans 

Oxazepam is readily absorbed following oral administration,andpeak blood levels in humans are 

achieved in 0.75 to 8 hours when given in tablet 

form, with an average of 2.7 hours (Shaderand 

Greenblatt, 1981). The half-life of oxazepam in the 

blood of humans is 6.8 -c 1.3hours. It has a volume 

of distribution of 0.6 +. 0.2 L/kg and a clearance of 

1.05 k 0.36 mL/min/kg. Approximately 98% of the 

drug is bound to plasma proteins (Goodman and 

Gilman's, 1990). About 95% is converted to the 

C3 glucuronide conjugate by UDPglucuronyl transferase 2 (Rajaonarison et al., 1991) and excreted in 

the urine;minoramounts of six other metabolites 

have been identified (Sisenwine et al., 1972). Only 

the parent compound is thought to have antianxiety 

activity. 

TOXICITY 

Experimental Animals 

Oral LDS, values have been reported to range from 

about 1,500 mgkg to greaterthan 5,OOO mgkg in 

various strainsof mice (Marcucci et al., 1968; Randall 

et al., 1970; Scrollini et al., 1975; Petrescu et al., 

1981) and were greater than 5,000 mag in Wistar 

andCharlesRiver CD rats (Owen et al., 1970; 

Scrollini et af., 1975). 

Owen et al. (1970) administered oxazepam in feed to 

CharlesRiver CD rats at concentrations of OM%, 

0.125%, 0.25%, and 0.5%. After 6 weeks, 2/20 highdose rats had died, and weight gain was decreased in 

the 0.25% males. Liver, adrenal gland,and kidney 

weights were greater in dosed rats than in controls. 

The only histopathologic finding was an increase in 

liver parenchymal fat. 

Groups of 30 male and 30 female rats were fed diets 

containing 0, 0.015%, 0.03%,or 0.06%, 

0.12% oxazepam for 55 weeks. Deathswerenot 

clearly chemical related, and otherthan increased 

liver weights, no effects on body weight gain or hematology parameters or significant chemical-related 

gross or histopathologic lesions wereobserved (Owen 

et al., 1970). 

The increased liver weights observed in these and 

other studies with benzodiazepines suggest 

stimulation of proliferation of smooth endoplasmic 

reticulum(Orlandi et af., 1975). However, the 

benzodiazepines do not appearto stimulate their own 

metabolism and have been found to inhibit metabolism of other drugs such as morphine or aminopyrine 

in Wistar rats (Vega et al., 1984) and to stimulate 

metabolism of certain chemicals (i.e., benzene and 

aniline) (Jablonska et al., 1975). This coincides with 

theirreputation of not producing significant 

toleranceduringlong-termtherapy (Goodman and 

Gilman's, 1990). Physical dependence hasbeen 
18 Oxazepam, NTP TR 443 

demonstratedinrats with several of the drugs 

including diazepam (Martin et al., 1982). 

Humans 

The benzodiazepines are a poor choiceforsuicide 

purposesand,despite many attempts, deaths by 

overdose are rare (Finkleet al., 1979). Overdoses of 

oxazepam commonly result in drowsiness, blurred 

vision, and ataxia. As inrats,timulation of 

proliferation of smooth endoplasmic reticulum has 

beenshown in liver biopsies fromhumans taking 

diazepam (Orlandi et al., 1975). Physical dependence 

is produced in humans given benzodiazepines. 

CARCINOGENICITY 

Experimental Animals 

A number of long-termrodentstudies have been 

performed with the benzodiazepines. Fox and 

Lahcen (1974) observed liver neoplasms in oxazepamtreated Swiss-Webster mice during the course of 

reproductive toxicity studies. Micewere housed as 

breeding pairs from 3 to 12 months of age and were 

fed an oxazepam-supplementeddiet at doses of 

0.05% and 0.15%. They were killed at 14 months of 

age. The incidences of liver neoplasms increased in 

males (0/13, 3/12, 8/13) and females (0/10, 0/10, 5/8) 

with dose. The neoplasms were generally multiple 

and gave the livers a massively nodular appearance. 

Histopathologically, the neoplasms were diagnosed as 

hepatocellular adenomas, which showed peliosis and 

extramedullary hematopoiesis. 

De la Iglesia et al. (1981) fed diazepam or prazepam 

in the dietatconcentrations sufficient to result in 

doses up to 75 m@g per day to male and female CF, 

mice and Wistar rats for 80 and 104 weeks, respectively. The incidence of malignant liver neoplasms 

was increasedinmale mice receiving diazepam. 

Temazepam, which is metabolized to oxazepam in the 

mouse, was administered in the diet to CRCD rats 

for 2 years and to CRCD-1 mice for 18 months at 

doses of 10 to 160m@g per day. Female mice had 

a slightly increased incidence of liver adenomas 

(Robinson et al., 1984). 

PROMOTIONSTUDIES 

The benzodiazepines have been testedinvarious 

promotion assays because of reports, primarily from 

one laboratory, that diazepam treatment accelerated 

the growth of intrarenally implanted neoplasm cells 

(Walker 256) (Horrobin et al., 1979) and that it was 

positive in an in vino metabolic cooperation assay for 

neoplasmpromoters(TroskoandHorrobin, 1980). 

These reports appeared following publication of an 

epidemiological study that suggested an association 

between increased incidences of breastcancerand 

benzodiazepine use in women (Stoll, 1976). This 

association was later discounted (Kleinerman et al., 

1984), butfurtheranimalexperimentation has 

provided mixed results. 

Remandet et al. (1984) fed F344 rats N-2-

fluorenylacetamide for 8weeks and followed this for 

12 weeks with diets containing one of six benzodiazepines. They reported no increased incidences of 

liver neoplasms or enzyme-altered foci. Preat et al. 

(1987) reported positive promotional activity with 

oxazepam in Wistar ratsin two different assays for 

hepatocarcinogenesis. In one, animals were initiated 

with diethylnitrosamine (DEN) and were treated with 

2-acetylaminofluorenecarbon and tetrachloride 

during the next 2 weeks; they then received oxazepam 

in the dietfor 30 weeks. In the otherprotocol, 

initiation with DEN was preceded by partial 

hepatectomy, and promotion was effected by dietary 

administration for 1year. Diwan et al. (1986) found 

diazepamand oxazepam to be promoters of DENinitiated liver neoplasms in mice. In this tudy, 

groups of B6C3Fl mice received injections of DEN at 

5 weeks of age; at 7 weeks they were fed diets 

containing diazepam or oxazepam at 0.05% or 0.15%, 

or given phenobarbital inwater at 500 ppm. Mice 

were killed periodically through 60 weeks of age. 

The incidence of neoplasms was increased in mice 

receiving diazepam and in those receiving 0.15% 

oxazepam. A few adenomas werealsoobservedin 

uninitiated mice receiving 0.15% diazepam (3/15)or 

0.05% oxazepam (2/16), and none were observed in 

mice receiving only phenobarbital. Diazepamand 

oxazepam were alsofound to induce hepatic P-450 

contentandincreaseaminopyrineN-demethylase 

activity. Diwan et al. (1986) have proposed that 

promotion of hepatocellular carcinogenesis is associated with induction of N-demethylase activity and 

appearstobequite speciesandstrain specific. 

Diazepam did not induce cytochrome P-450 in the 

liver of Sprague-Dawley rats (Vorne and IdanpaanHeikkila, 1975), and this was considered consistent 

with the negative promotional findings of Remandet 

et al. (1984) in their study with F344 rats. 
19 Introduction 

REPRODUCTIVE AND 

DEVELOPMENTAL TOXICITY 

Experimental Animals 

Although the benzodiazepines have been used in 

treating toxemia and preeclampsia as well as the 

psychiatric complications of pregnancy (Shannon 

et al., 1972; Kanto, 1982), there are many reports of 

fetotoxic and teratogenic effects of these and other 

minor tranquilizers when given to pregnant animals. 

Tucker (1985) provided a critical review of studies of 

developmental toxicity of benzodiazepines in the rat. 

Saito et al. (1984) found increases in fetal toxicity 

(resorptions,dead fetuses, and malformations) in 

pregnant rats given doses of diazepam or chlordiazepoxide of 100 mgkg per os during days 7 to 14. 

Miller and Becker (1975) first found diazepam produced cleft palate following oral administration of 

87.5 or 125 mgkg to Swiss-Webster mice on days 11 

to 13. This has since received considerable study and 

is now attributed to potentiation of the GABAergic 

inhibition of the palate shelf reorientation (Wee and 

Zimmerman, 1983). In general, exposures to high 

doses in utero produce litter decreased sizes,

decreased pup weights, and increases in malformations. Exposures to lower doses (5 to 20 mgkg per 

day) during criticalperiods(after day 14 in rats) 

produce no immediately obvious effects at birth but 

result in various behavioral deficits during later life 

anda variety of poorly understood changes in the 

concentration of neurotransmitters in various brain 

areas (Livezey et al., 1986a; Ryan and Pappas, 1986, 

Shibuya et al., 1986). Central to these studies have 

been attemptsto correlate changes in benzodiazepine 

receptor concentration with altered behavior. Livezey 

et al. (1986b) have argued that in utero exposure to 

benzodiazepinesduring the period of receptor 

development (after gestational day 14 in rats) results 

in the unborn because higher levels accumulate due 

to the slower elimination from the fetus. There have 

been reports of increases in severe congenital anomalies in infants whose mothers took chlordiazepoxide 

andother benzodiazepines(including oxazepam) 

during pregnancy (Milkovich and van den Berg, 

1974); there have also been reports to the contrary 

(Hartz et al., 1975). 

GENETICTOXICITY 

Oxazepam has not been tested extensively for mutagenicity, but the data reported for oxazepam and its 

structural analogues indicate that thisclass of chemicals is probably not genotoxic. Positive responses in 

a Salmonella gene mutation assay were reported only 

by Batzinger et al. (1978). They described an increase 

in revertants for strains TAl00 andTA98when exposure was carried out in the presence of rat liver S9 

activation enzymes. Insufficient data werereported 

to allow an evaluation of the results. In a brief 

abstract that presented littleexperimentaldetail, 

Matula and Downie (1983) reported negative results 

in strains TAlOO and TA98, with and without S9. 

Balbi et al. (1980) detected no mutagenic activity with 

oxazepam in four strains of Salmonella, with or 

without S9, but their report did not include complete 

data tables for those tests that gave negative results. 

No evidence of chromosomenondisjunction was 

observed in Aspergillus nidulans treated with an 

unspecified concentration of oxazepam in the absence 

of S9 (Bignami etal., 1974). Unscheduled DNA 

synthesis was not detected in rat liver cells in vitro 

(Swierenga et al., 1983), and no inductionof chromosomal aberrations was observed in bone marrow cells 

of mice administered oxazepam in doses of 

in a decreased benzodiazepine receptor concentration 0.85 mgkg body weight by intraperitoneal injection, 

andresultsinarat that suffers chronic anxiety five times weekly for 8 weeks (Degraeve et al., 1985). 

demonstrated by hyperarousal, inability of the 

animals to habituate to a novel environment, and a A variety of genotoxicity tests have been performed

largereductionin theamount of deep slow-wave with two of the widely used structural analogues of 

sleep. oxazepam, diazepam, and chlordiazepoxide. Diazepam was nonmutagenicin Salmonella (Batzinger 

Humans et al., 1978; Waskell, 1978; Preiss et al., 1982; Zeiger 

Exposure of the human fetus to diazepam results in et al., 1992). There was no evidence of diazepama set of symptoms collectively known as the "floppy induced chromosome loss or nondisjunction in yeast 

infant syndrome," which includes hypothermia, hyper- (Bignami et al., 1974; Matula andDownie, 1983; 

bilirubinemia, hypotonia, asphyxia, respiratory Crebelli et al., 1989; Parry et al., 1989; Whittaker 

complications,andpoor sucking response. This is et al., 1990; Crebelli et al., 1991). The effects 

likely due to the ready transfer of the drugs across reported for diazepam in cultured mammalian cells 

the placenta. Pharmacologic effects are exaggerated varied. Two laboratories(Ishidate et al., 1978; 
20 Oxazepam, NTP TR 443 

Matsuoka et af.,1979) found no induction of chromosomal aberrations in cultured Chinese hamster ovary 

cells with or without S9. However, a positive study 

for inductionof chromosomal aberrations in cultured 

Chinese hamster ovary cells without S9 was reported 

(Lafi and Parry, 1988), and disruptionof mitosis with 

concomitantchromosome loss was observed in 

culturedChinesehamster ovary cells following 

treatmentwithdiazepam,withoutS9(Hsu et af., 

1983;Parry et af.,1986; Lafi et af.,1987). Results of 

tests for induction of chromosomal aberrations and 

sisterchromatid exchanges in human lymphocytes 

(Staiger, 1970; Zhurkov, 1975) or fibroblasts (Staiger, 

1969;Kawachi et af.,1980; Sasaki et af.,1980) treated 

in vitro withdiazepamwere uniformly negative. 

Unscheduled DNA synthesis was not detected in rat 

liver cells treated in vitro with diazepam (Swierenga 

et af.,1983; Williams et af.,1989). 

In vivo tests with diazepam showed little indicationof 

genotoxic activity. No evidence of mitotic disruption 

or induction of chromosomalberrations was

observed in mousebone marrow cells following 

administration of 100 to 150 mgkg diazepam (Miller 

and Adler, 1989;Xu and Adler, 1990). Diazepam did 

not induce chromosomal aberrations in bone marrow 

cells of hamsters (Schmid and Staiger, 1969) or rats 

(Ishimura et af., 1975; Kawachi et af., 1980). In 

addition, no increases in chromosomal aberrations 

(Stenchever et af.,1970a; White et af.,1974) or sister 

chromatid exchanges (Torigoe, 1979; Husum et al., 

1985) wereobservedinperipheral lymphocytes 

obtained from patients treated with diazepam either 

chronically, as a management for anxiety or muscle 

spasm, or acutely, as part of a surgical routine. 

Fewer genotoxicity testresults are available for 

chlordiazepoxide, but indications are that it, too, is 

not genetically active. Chlordiazepoxide did not 

induce nondisjunction in A. nidufans (Bignami et af., 

1974), or chromosomalaberrationsincultured 

Chinesehamster ovary cells (Sasaki et af., 1980), 

humanfibroblasts(Staiger, 1969), or leukocytes 

(Bregman, 1970; Stenchever et af., 1970b). In vitro 

micronucleustests with hamsterandhuman cells 

were negative (Sasaki et af.,1980). Results of in vivo 

investigations indicate that chlordiazepoxide does not 

induce chromosomal aberrations in mouse (Peterson 

et af., 1978; Degraeve et af., 1985) or hamster 

(Schmid and Staiger, 1969) bone marrow cells. 

Finally, no induction of chromosomal aberrations was 

observed in lymphocytes obtained frompatients 

administeredchlordiazepoxide (upto 200 mg/day) 

(Stenchever et af.,1970b). 

STUDYRATIONALE 

Oxazepam and fourother benzodiazepines(chlordiazepoxide HC1, chlorazepate, and diazepam,

flurazepam) were nominated for study by the Food 

andDrugAdministration(FDA)and by NIEHS 

based on their high usevolume, use by pregnant 

women,and the lack of adequaterodent carcinogenicity studies. An agreement was reached with 

Hoffman-LaRoche, Inc., the manufacturer of chlordiazepoxide HCl, diazepam, and flurazepam, for 

studies to be carried out on these drugs under their 

auspices in cooperation with the NTP. These studies 

are currently underway. No studies were performed 

on chlorazepate because of the very similar 

metabolite profile between this drug and diazepam. 

Oxazepam was evaluatedin 14-week and chronic 

studies by the NTP, andthisTechnical Report 

contains the results of studiesperformed with the 

Swiss-Webster and B6C3Flstrains of mice. Studies 

with rats were not initiated at the same time as the 

mousestudiesbecauseadequate carcinogenicity 

studies of oxazepam with the Sprague-Dawley rat 

strainhadbeen submitted FDA to by the

manufacturer, Wyeth Laboratories. Subsequently, 

because of the marked neoplastic responses found in 

the two mousestrains reviewed in this report, the 

NTP initiated further 2-year studies of oxazepam with 

the Fischer 344/N rat. 

Swiss-Webster mice were used in addition to the 

B6C3Fl strain because of the evidence of oxazepaminduced hepatocellularneoplasianthisstrain 

reported by Fox and 'Lahcen (1974). Thecurrent 

studiesneurobehavioral include assessments, 

measures of serum oxazepam concentrations,and 

histopathologic evaluation of tissues. Because

evidence of hepatocellular neoplasia was found in the 

2-year studies, an additionalset of studies was 

performed to evaluate the comparative metabolismof 

oxazepam in the Swiss-Webster and B6C3Fl mouse, 

the mitogenic properties of oxazepam on the liver, 

and the incidence of liver neoplasms with activated 

H-rasoncogenesandtheirmutationalspectrum. 

Studies also wereconducted to establish the

pharmacokinetics of oxazepam administered to 

animals in feed. 
21 

MATERIALS AND METHODS 

PROCUREMENT AND 

CHARACTERIZATION 

OF OXAZEPAM 

Oxazepam was obtained from Roussel Corporation 

(Englewood Cliffs, NJ) in one lot (86017.01), which 

was used throughout the studies. Identity, purity, and 

stability analyses were conducted by the analytical 

chemistry laboratory, Midwest Research Institute 

(Kansas City, MO). The reports on analyses 

performed in supportof the oxazepam studies are on 

file at the National Instituteof Environmental Health 

Sciences (NIEHS). 

The chemical, a white, powdered solid, was identified 

as oxazepam by infrared, ultravioletbisible,and 

nuclear magnetic resonance spectroscopy. The purity 

of oxazepam was determined by elemental analyses, 

KarlFischerwater analysis, functional group 

titration, thin-layer chromatography, and highperformance liquid chromatography. 

Elemental analyses for carbon, hydrogen, nitrogen, 

and chlorine were in agreement with the theoretical 

values for oxazepam. Karl Fischer analysis indicated 

less than 0.03% water. Functionalgrouptitration 

indicatedapurity of 101%. Thin-layer chromatography was performed using two systems: one indicated a major spot and one trace impurity, and the 

other indicated amajorspot.High-performance 

liquid chromatography resolved a major peak with no 

impurity peaks with areas 0.1% or greater relative to 

the major peak. Majorpeakcomparison between 

this lot and a United States Pharmacopeia XXI standard indicated a relative purityof 103%. The overall 

purity was determined to be greater than 99%. 

Stability studies performed by the analytical chemistry 

laboratory using high-performance liquid chromatography indicated that oxazepam was stable for 2 weeks 

when stored protected from light at temperatures up 

to 60" C. The stability of the bulk chemical was 

monitored periodically at the study laboratory using 

infraredspectroscopyandhigh-performance liquid 

chromatography. No degradation of the bulk 

chemical was observed throughout the studies. 

PREPARATION AND ANALYSIS 

OF DOSEFORMULATIONS 

The dose formulations were prepared monthly for the 

14-week studies and every 2 weeks for the chronic 

studies by mixing oxazepam and feed in a PattersonKelley twin-shell blender (Table Hl). The mixture 

was stored in sealed, labeled, plastic buckets forto up 

3 weeks at 5" C. 

Homogeneity and dose formulation stability analyses 

of the 500 ppmconcentrationwereperformedat 

the analytical chemistry laboratory using highperformance liquid chromatography. Homogeneity 

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

protected from light at 5" C. 

Periodic analyses of the doseformulationswere 

conducted at the study laboratory and at the analytical chemistry laboratory using high-performance 

liquid chromatography. During the 14-week studies, 

all dose formulations for Swiss-Webster and B6C3Fl 

mice were within 10% of the target concentrations 

(Table H2). During the chronic studies, dose

formulationswere analyzed approximately every 

8 weeks; all dose formulations for Swiss-Webster and 

B6C3Fl mice were within 10% of the target concentrations except two 125 ppm formulations for B6C3Fl 

mice. Theseformulations dose were remixed.

Results of the doseformulation analyses for the 

chronicstudies are presented in Table H3. Results 

of periodicreferee analyses performed by the 

analytical chemistry laboratory indicated good 

agreement with the results obtained by the study 

laboratories (Table H4). 

14-WEEK STUDIES 

The 14-week studies were conducted to evaluate the 

cumulative toxic effects of repeatedexposure to 

oxazepam and to determine the appropriate doses to 

be used in the chronic studies. 

Male and female Swiss-Webster mice were obtained 

from CharlesBreeding River Laboratories 
22 Oxazepam, NTP TR 443 

(Portage,MI) andmaleand female B6C3Fl mice 

wereobtainedfromSimonsenLaboratories, Inc. 

(Gilroy, CA). At receipt, the animalswere34 to 

40days old. The mice were quarantinedfor 13 or 

14 days before dosing began. Before the beginning of 

the studies, five males and five females of each strain 

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 males and five females of each strain using the 

protocols of the NTP SentinelAnimalProgram 

(Appendix K). 

Groups of 10male and 10female Swiss-Webster and 

10male and 10 female B6C3Fl mice were assigned to 

the corestudy and received 0,625,1,250,2,500,5,000, 

or 10,OOO ppmoxazepamin feed for 14 weeks. A 

second group of 10 maleand10 female SwissWebsterand 10 maleand10 female B6C3Fl mice 

were assigned to the- special studyandwere maintained on dosed feed until scheduledterminations 

during weeks 2 and 12. Animals were housed individually; water and feed were available ad libitum. 

Clinical findings were recordedonce weekly. The 

animals were weighed at the beginning of the studies, 

weekly, and at the endof the studies. Further details 

of study design and maintenance animal are

summarized in Table 1. 

The core study mice were subjected to a series of 

neurobehavioral tests prior to the beginning of the 

14-week studies and during weeks 2 and 12 of the 

studies. The neurobehavioral tests included undifferentiated motor activity, forelimb and hindlimb grip 

strengths,thermal sensitivity, and acoustic startle 

responsiveness (Appendix G). 

Ten mice per exposure group inthe special study 

were anesthetized with CO, and blood samples were 

collected by cardiac puncture during weeks 2 and 12 

for serum oxazepam determinations. At the end of 

the 14-week study, a necropsy was performed on all 

remaining animals. Theheart, right kidney, liver, 

lung, right testis, and thymus of mice 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 6 pm, and stained with hematoxylin 

and eosin. A complete histopathologic examination 

was performed on allcontroland 10,000 ppm animals, and on all core study animals dying before the 

end of the study. Table 1lists the tissues and organs 

routinely examined. 

CHRONICSTUDIES 

Study Design 

Groups of 60 male and 60female Swiss-Webster mice 

received 0,2,500, or 5,000 ppm oxazepam in feed for 

57 weeks; groups of 60 male and 60 female B6C3Fl 

mice received 0, 125, 2,500, or 5,000 ppm oxazepam 

in feed for104 to 105 weeks. Ten male and 10 

female B6C3Fl mice per exposure group were evaluated after 15 months of chemical exposure. 

Source and Specification of Animals 

Male and female Swiss-Webster mice were obtained 

from Charles River Breeding Laboratories and male 

andfemale B6C3Fl mice wereobtainedfrom 

Simonsen Laboratories, Inc., for use in the chronic 

studies. The mice were quarantined for 13to 15 days 

before the beginning of the studies. Fivemaleand 

five female Swiss-Webster and B6C3F, mice were 

selected for parasite evaluation and gross observation 

of disease. Serology samples were collected for viral 

screening. Swiss-Webster mice were approximately 

46 days old and B6C3Fl mice were approximately 

44 days old at the beginning of the chronic studies. 

The health of the animals was monitored during the 

studiesaccordingto the protocols of theNTP 

Sentinel Animal Program (Appendix K). 

Animal Maintenance 

All animals werehoused individually. Feed and 

waterwereavailable ad libitum. Feed consumption 

was recorded every 4 weeks for. a 7-day period 

(Appendix I). Cages and racks were rotated every 

2 weeks. Further details of animal maintenance are 

given in Table 1. Information on feed composition is 

provided in Appendix J. 

Clinical Examinations and Pathology 

All animals wereobserved twice daily. Clinical 

findings were recorded every 4 weeks. Animals were 

weighed weekly for the first 13 weeks and every 

4 weeks thereafter. Ten male and 10 female SwissWebster mice from each exposure group were anesthetized with a mixture of CO, and oxygen at the end 

of the study, and blood was drawn by cardiac puncture to determineserumoxazepamconcentrations. 

Ten male and 10 female B6C3F, mice from each 

groupwereselectedforinterimevaluationsafter 
Materials and Methods 23 

15 months. All B6C3Fl mice selectedfor the 

15-monthinterimevaluationand10maleand 

10 female B6C3Fl mice in the 0, 125, and 2,500 ppm 

groups (excluding animals for selectedneurobehavioralevaluation)wereanesthetized with a 

mixture of CO, and oxygen and blood was collected 

by cardiacpuncturefordetermination of serum 

oxazepam concentrations. 

A necropsy was performed on all animals. The 

kidneys and livers of B6C3Fl mice were weighed. At 

necropsy, all organs and tissues were examined for 

gross lesions, andallmajor tissues were fixed and 

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

to a thickness of 4 to 6 pm, and stained with hematoxylin and eosin for microscopic examination. A 

complete histopathologic examinationwas performed 

on all animals andon tissues with grossly visible 

lesions. Tissues examined are listed in Table 1. 

Groups of 10male and 10 female Swiss-Webster mice 

fromeachgroupwereevaluated by noninvasive 

proceduresforneurobehavioral toxicity during the 

prestudyperiodandafter 6 and 12 months of 

exposure. Similarly, groups of 10 male and 10 female 

B6C3Fl mice were evaluatedforneurobehavioral 

toxicity during the prestudy period and after 6,12,18, 

and 24 months of exposure. The same animals were 

tested at eachtimepoint,but animals dying early 

werereplaced by mice randomly selected from the 

survivors. The tests included motor activity, startle 

responsiveness, forelimb and hindlimb grip strength, 

andthermal sensitivity. Further details of these 

studies are outlined in Appendix G. 

Microscopic evaluations were completed by the study 

laboratory pathologist, and the pathology data were 

enteredintothe Toxicology DataManagement 

System. The microscopic slides, paraffin blocks, and 

residual wet tissues were sent to the NTP Archive for 

inventory, slideblock match, and wet tissue audit. 

The slides, individual animaldata 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 by the quality assessment 

laboratory. The quality assessment pathologist 

microscopically reviewed selectedneoplasmsand 

nonneoplastic lesions. 

The quality assessment reportand slideswere 

submitted to the NTP PathologyWorking Group 

(PWG) chair, who reviewed the selected tissues for 

which disagreement a in diagnosis between the 

laboratory and quality assessment pathologist existed. 

Representative slides histopathology containing 

examples of lesionsrelated to chemical administration, examples of disagreementsin diagnoses 

between the laboratoryandquality assessment 

pathologist, or lesions of generalinterestwere 

presented by the chair to the PWG for review. The 

PWG consisted of the quality assessment pathologist 

and other pathologists experienced in rodent toxicologic pathology. Thisgroup examined the tissues 

without any knowledge of exposure levels or 

previously rendered diagnoses. Forthechronic 

studies, tissues examined included heart, right kidney, 

liver, lung, pancreas (males), skeletal muscle (males), 

and spleen. When the PWG consensus differed from 

the opinion of the laboratory pathologist, the diagnosis was changed. Thus, the final diagnoses 

represent a consensus of contractor pathologists and 

the PWG. Details of these review procedures have 

been described, in part, by Maronpot and Boorman 

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

analyses of the pathology data, the diagnosed lesions 

foreachtissuetypewereevaluatedseparately or 

combined according to the guidelines of McConnell 

el al. (1986). 

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 

accidentally killed 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) 

methodfortesting two groupsforequalityand 

Tarone’s (1975) life table test to identify dose-related 

trends. AllreportedP values for the survival 

analyses are two sided. 

Calculation of Incidence 

The incidences of neoplasms or nonneoplastic lesions 

as presented in Tables Al, A4, B1, B4, C1, C5, Dl, 

and D5 are given as the number of animals bearing 

such lesions at a specific anatomic siteand the 

number of animals with that site examined microscopically. For calculation of statistical significance, 
24 Oxazepam, NTP TR443 

the incidences of most neoplasms (Tables A3, B3, C3, 

and D3) andof all nonneoplastic lesions are given as 

the number of affected animals and the number of 

animals with thesite examined microscopically. 

However, when examination macroscopic was 

required to detect neoplasms in certain tissues (e.g., 

skin, intestine, harderian gland, and mammary gland) 

beforemicroscopicevaluation or when neoplasms 

hadmultiplepotentialsites of occurrence (e.g., 

leukemia or lymphoma), the denominators consist of 

thenumber of animals on which a necropsy was 

performed. 

Analysis of Neoplasm Incidences 

With the exception of malignant liver neoplasms, the 

neoplasms in these studieswereconsidered 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 

neoplasmswere 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 asa 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 groupswerecompared on the 

basis of the likelihood score test for the regression 

coefficient of dose.Thismethod of adjusting for 

intercurrentmortality is the prevalence analysis of 

Dimeand Lagakos (1983), further described and 

illustrated by Dime and Haseman (1986). When 

neoplasms are incidental, thiscomparison of the 

time-specific neoplasm prevalences also provides a 

comparison of the time-specific neoplasm incidences 

(“bight 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 

methodsinclude the life table test (Cox, 1972; 

Tarone, 1975), appropriate for rapidly lethal neoplasms and used in the evaluation of hepatocellular 

carcinomas and hepatoblastomas in this Technical 

Report,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 alsoused 

to evaluateselectednonneoplastic lesions. For 

further discussion of these statistical methods, refer 

to Haseman (1984). 

Analysis of Nonneoplastic Lesion Incidences 

Amyloid deposition in the heartwas a major causeof 

death in Swiss-Webster mice. Because the remaining 

nonneoplastic lesions in these studies 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 nonneoplastic 

lesion prevalence was modeled as a logistic function 

of chemical exposure and time. For lesions detected 

at the interim evaluation, the Fisher exact test was 

used, a procedure based on the overall proportion of 

affected animals. 

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. 

Organand body weight data, which have approximately normal distributions, were analyzed using the 

parametric comparison multiple procedures o f

Dunnett (1955) and Williams (1971, 1972). Serum 

oxazepam concentration data, which have typically 

skewed distributions, were analyzed using the

nonparametric comparison multiple method o f

Shirley (1977). 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 thanatesthatdoesnot 

assumeamonotonicdose-relatedtrend(Dunnett’s 

test). Neurobehavioral data were analyzed using 

Dunnett’s test. Average severity values were analyzed 

for significance using the Mann-Whitney U test 

(Hollander and Wolfe, 1973). 

Historical Control Data 

Although the concurrent control group is always the 

firstandmost appropriatecontrolgroup used for 

evaluation, historical control data can be helpful in 

the overallassessment of neoplasmincidence in 

certain instances. Consequently, neoplasm incidences 

from the NTP historical control database (Haseman 

et al., 1984,1985) are included in the NTF’ reports for 
Materials and Methods 25 

neoplasmsappearing to show compound-related 

effects. 

Quality Assurance Methods 

The 14-week and chronic studies were conducted in 

compliancewith Food andDrugAdministration 

GoodLaboratory PracticeRegulations(21 CFR, 

Part 58). In addition, asrecords from the chronic 

studies were submitted to the NTP Archives, these 

studies were audited retrospectively by an independent quality assurancecontractor.Separateaudits 

covering completeness and accuracy of the pathology 

data, pathology specimens,final pathology tables, and 

preliminary review draft of thisNTPTechnical 

Report wereconducted.Auditprocedures and 

findings are presented in the reports and are on file 

at NIEHS. The audit findings were reviewed and 

assessed by NTP staff, so allcomments had been 

resolved or wereotherwise addressed during the 

preparation of this Technical Report. 

GENETICTOXICOLOGY 

The genetic toxicity of oxazepam was assessed by 

testing the ability of the chemical to inducemutations in various strains of Sufnzoneffu typhinturiunt, 

sister chromatid exchanges and chromosomal aberrations in cultured Chinesehamster ovary cells, and 

increases in micronucleatedB6C3Flmouse peripheral 

blood erythrocytes. The protocols forthesestudies 

and the results are given in Appendix E. 

The genetic toxicity studies of oxazepam are part of 

a larger effortby the NTP to develop a database that 

would permit the evaluation of carcinogenicity in 

experimentalanimalsfrom the and structure

responses of the chemical in short-term in vino and 

in vivo genetic toxicity tests. These genetic toxicity 

tests were originally developed to study mechanisms 

of chemical-induced DNA damage and to predict 

carcinogenicity in animals, based on the electrophilic 

theory of chemical carcinogenesis and the somatic 

mutation theory (Miller and Miller, 1977; Straw, 

1981; Crawford, 1985). 

There is a strongcorrelationbetweena chemical’s 

potentialelectrophilicity(structuralalert toDNA 

reactivity), mutagenicity in Sufrnonelfu,and carcinogenicity in rodents. The combination of electrophilicity and Sulntonelfu mutagenicity is highly 

correlated with the induction of carcinogenicity in 

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

andTennant, 1991). Other in vino genetic toxicity 

tests do not correlate well with rodent carcinogenicity 

(Tennant et af., 1987; Zeiger et aL, 1990), although 

theseother testscanprovideinformation on the 

types of DNA and chromosome effects that can be 

induced by the chemical being investigated. Data 

from NTP studiesshowthata positive response in 

Sufnzoneffuis currently the mostpredictive in vitro 

testfor rodent carcinogenicity (89% o f the 

Sufnzoneffumutagens were rodent carcinogens), and 

that there is no complimentarity among the in vino 

genetic toxicity tests. That is, no battery of tests that 

included the Sufnzoneffutest improved the predictivity 

of the Salmonella testalone. The predictivity for 

carcinogenicity of a positive response in bonemarrow 

chromosome aberration or micronucleus tests is not 

yet defined. 
26 Oxazepam, NTP TR 443 

TABLE1 

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

14-Week Studies 

Study Laboratory 

Battelle Columbus Laboratories (Columbus, OH) 

Strain and Species 

Swiss-Webster and B6C3F1 mice 

Animal Source 

Swiss-Webster mice: Charles River Breeding Laboratories 

(Portage,MI) 

B6C3Fl mice: SimonsenLaboratories,Inc.(Gilroy,CA) 

Size of Study Groups 

Core study: 10 males and 10 females 

Special study: 10 males and 10 females 

Doses 

0, 625, 1,250, 2,500, 5,OOO, or 10,OOOppm in feed 

Time Held Before Studies 

13-14 days 

AverageAgeWhen Studies Began 

Swiss-Webstermice:48days 

B6C3Flmice: 53 days 

Date of First Dose 

Swiss-Webster mice: 8 June 1988 (males)or 9 June 1988 

(females) 

B6C3Flmice: 18 May 1988(males) or 19 May 1988 

(females) 

Duration of Dosing 

14 weeks 

Date of Last Dose 

Swiss-Webster mice: 8 September 1988 (males)or 

9 September 1988 (females) 

B6C3Fl mice: -l8-&gust 1988 (males)or 19 August 1988 

(females) 

Necropsy Dates 

Swiss-Webster mice: 8-9 September 1988 

B6C3Flmice: 18August1988(males) or 19 August 1988 

(females) 

Chronic Studies 

Battelle Columbus Laboratories (Columbus, OH) 

Swiss-Webster and B6C3F1 mice 

Swiss-Webstermice: Charles RiverBreeding Laboratories 

(Portage, MI) 

B6C3Flmice:SimonsenLaboratories,Inc.(Gilroy,CA) 

60 males and 60 females 

Swiss-Webster mice: 0, 2,500, or 5,000ppm in feed 

B6C3Flmice: 0, 125, 2,500, or 5,000ppm in feed 

13-15 days 

Swiss-Webstermice:46days 

B6C3Flmice:44days 

Swiss-Webster mice: 13 July 1989 (males) or 14 July 1989 

(females) 

B6C3Flmice:22 June 1989 (males) or 23 June 1989 

(females) 

Swiss-Webstermice: 57weeks 

B6C3Flmice:104-105weeks 

Swiss-Webstermice: 13 August 1990 (males) or 14 August 

1990 (females) 

B6C3Flmice:21 June 1991 (males) or 28 June 1991 

(females) 

Swiss-Webstermice: 13 August 1990 (males) or 14August 

1990 (females) 

B6C3Flmice:20-21 June 1991 (males);27-28June1991 

(females) 
Materials and Methods 27 

TABLE1 

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

14-Week Studies 

Average Age at Necropsy 

Swiss-Webstermice:140days 

B6C3Flmice:145days 

Method of Sacrifice 

CO, asphyxiation 

Method of Animal Distribution 

Animals were randomized by weight with a computer 

randomization program. 

Animals per Cage 

1 

Method of Animal Identification 

Tail tattoo and ear tag 

Qiet 

Zeigler NIH-07 open formula meal diet (Zeigler Brothers, 

Inc., Gardners, PA), available ad libitum, changed weekly or 

as necessary 

Water 

Tap water (City of Columbus) via automatic watering system 

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

ad libitum 

cages 

Polycarbonate (Lab Products, Inc., Maywood, NJ), changed 

weekly. There were three racks of cages. Vertical columns 

containing five cages of like exposure group were randomly 

assigned to positions on the racks. Every 2 weeks, cages were 

rotated vertically within each column and racks were rotated 

clockwise. 

Bedding 

Sani-Chip@ heat-treated hardwood chips (P.J. Murphy Forest 

Products Corp., Rochelle Park, NJ), changed weekly 

Cage Filters 

Spun-bonded polyester (Snow Filtration Co., Cincinnati, OH), 

changed once every 2 weeks 

Rack 

Stainless steel (Lab Products, Inc., Maywood, NJ), changed 

every 2 weeks 

Chronic Studies 

Swiss-Webstermice: 63 weeks 

B6C3Flmice:110-111weeks 

Same as 14-week studies 

Same as 14-week studies 

Same as 14-week studies 

Tail tattoo 

Same as 14-week studies. Feed consumption recorded every 

4 weeks for a 7day period. 

Same as 14-week studies 

Cages and rotation same as 14-week studies. There were 

three (Swiss-Webster) or four (B6C3Fl) racks of cages. 

Vertical columns containingsix or seven cages of like 

exposure group or five cages of sentinel mice were randomly 

assigned to positions on the racks. Initial cage placements are 

on file at NIEHS. 

Same as 14-week studies but supplied by PJ. Murphy Forest 

Products Corp., Montville, NJ 

Same as 14-week studies 

Same as 14-week studies 
28 Oxazepam, NTP TR 443 

TABLE1 

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

14-Week Studies 

Animal Room Environment 

Average temperature: 21"-24" C 

Relative humidity: 35%-65% 

Fluorescent light: 12 hours/day 

Room air changes: 10 changeshow 

Type and Frequency of Observation 

Animals were observed and clinical observations were 

recorded weekly; animals were weighed initially, weekly, and 

at the end of the studies. 

Necropsy 

Necropsy was performed on all animals. Organs weighed 

were heart, right kidney, liver, lung, right testis, and thymus. 

Clinical Pathology 

Blood was collected by cardiac puncture during weeks 2 and 

12 for serum oxazepam determinations. 

Histopathology 

Complete histopathology was performed on all control 

animals, all mice receiving 10,000 ppm, and all mice dying 

before the end of the study. In addition to gross lesions, the 

tissues examined included: adrenal gland, brain, clitoral 

gland, epididymis, esophagus, femur and marrow, gallbladder, 

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

lung, lymph nodes (mandibular and mesenteric), mammary 

gland, nose, ovary, pancreas, parathyroid gland, pituitary 

gland, preputial gland, prostate gland, salivary gland, seminal 

vesicle, skin, small intestine (duodenum, jejunum, ileum), 

spleen, stomach (forestomach and glandular), testis, thigh 

muscle, thymus, thyroid gland, trachea, urinary bladder, and 

uterus. In addition, the liver of all male mice, of all female 

Swiss-Webster mice, and of all female B6C3Fl mice except 

those receiving 625 ppm and the adrenal gland of all female 

Swiss-Webster mice were examined. 

Neurobehavioral Studies 

Core study animals were administered neurobehavioraltests 

prior to the study and during weeks 2 and 12. These tests 

included: undifferentiated motor activity, forelimb and 

hindlimb grip strength, thermal sensitivity, and startle 

responsiveness. 

Chronic Studies 

Average temperature: 20"-26" C 

Relative humidity: 25%-70% (Swiss-Webster); 30%-70% 

(B6C3Fl) 

Fluorescent light: 12 hours/day 

Room air changes: 10 changeshour 

Animals were observed twice daily and clinical observations 

were recorded every 4 weeks; animals were weighed weekly 

during first 13 weeks and at 4-week intervals thereafter. 

Necropsy was performed on all animals. Organs weighed 

were kidney and liver (B6C3Fl mice). 

Blood was collected by cardiac puncture at the 15-month 

interim evaluation (B6C3Fl mice only) and at the end of the 

studies (Swiss-Webster and B6C3Fl). Mice were allowed free 

access to dosed feed until immediately before blood 

collection. All blood samples were drawn between 9 a.m. and 

11 a.m. 

Complete histopathology was performed on all mice. In 

addition to gross lesions and tissue masses, the tissues 

examined included: adrenal gland, brain, clitoral gland, 

epididymis, esophagus, femur and marrow, gallbladder, heart, 

kidney, large intestine (cecum, colon, rectum), liver, lung, 

lymph nodes (mandibular and mesenteric), mammary gland, 

nose, ovary, pancreas, parathyroid gland, pituitary gland, 

preputial gland, prostate gland, salivary gland, seminal vesicle, 

skin, small intestine (duodenum, jejunum, ileum), spleen, 

stomach (forestomach and glandular), testis, thymus, thyroid 

gland, trachea, urinary bladder, and uterus. 

Ten male and 10 female mice per strain per exposure group 

were administered neurobehavioral tests prior to the study 

and after 6, 12, 18 (B6C3Fl), and 24 (B6C3Fl) months of 

exposure. These tests included: motor activity, startle 

responsiveness, forelimb and hindlimb grip strength, and 

thermal sensitivity. 
29 

RESULTS 

SWISS-WEBSTERMICE 

14-WEEK STUDY 

One male mouse in the 625 ppm group and one 

female in the 10,OOO ppm group were killed moribund during the study. One female receiving

1,250 ppm was killed accidentally. The 625 ppm 

malemousethat died early was found to have 

lymphoma, and this death was not considered related 

TABLE2 

to oxazepam exposure. No remarkable lesions were 

found inthe 10,000ppm female mouse, and this early 

death was considered related to oxazepam exposure. 

All other mice survived until the end of the study 

(Table 2). The final mean body weights of all 

exposed female groups were greater than that of the 

control group, and those of the 625 and 2,500 ppm 

groups were significantly greater. Mean body

Survival, Mean Body Weights, and Feed Consumption of Swiss-Webster Mice in the 14-Week Feed Study 

of Oxazepam 

Final Weight 

Mean Bodv Weightb (g) Relative Feed 

Dose Survivala Initial Final Change toControls Consumption' 

(PPW (W Week 2 Week 14 

Male 

0 lono 26.1 35.3 f 0.3 9.3 f 0.9 f 0.8 4.8 4.3 

625 9nod 25.9 f 0.3 12.0 38.0 f 0.8 f 0.9 108 5.0 4.0 

10/10 1,25026.1 f4.6 0.6

11.7 f 4.6 0.8

f4.30.9 

f 3.8 0.6

29.9 21.5 f 0.4 8.4 f 0.7 4.8 4.4 f 0.8 

f 0.4 34.4 8.4 & 0.7 4.0 97 

108 

99 

99 

2,500 26.5 lono f 0.3 38.1 & 1.0 4.3 

5,ooo 25.4 10/10 f 0.4 35.1 9.7 f 1.0 4.2 

10,ooo 1ono 

Female 

0 lono 

26.0 35.1f 0.4 9.1 f 0.7 4.2 

625 1o n0 21.9 f 0.2 32.3 f 0.7' 10.4 f 0.5 108 4.8 4.7 

9.5 4.3 f 0.5 

10/1022.1 2,500 32.5 ? 0.3 10.4 f 0.8. 109 f 0.8 

f 0.5 4.7 

2 4.5 04

1,250 22.3 31.9 f 0.3 f 0.5 107 4.8 

104 3.7 

woe 

4.5 5 .O 

5,m lono 21.4 31.6 f 0.3 4.0 10.2 106 f 0.3 

10,ooo 9/10f 21.7 31.2 f 0.4 9.5 f 0.4 

Significantly different (PSO.05) from the control group by Williams' or Dunnett's test 

Number of animals survivinghumberinitially in group 

Weights and weight changes are given as mean f standard error. Subsequent calculations are based on animals surviving to the 

end of the study. 

Feed consumption is expressed as grams per animal per day; data for week 2 are based on 20 animals; data for week 14 are based 

on 10 animals. 

Week of death: 10 

Week of death: 6 (accidental death) 

Week of death 13 
30 Oxazepam, NTP TR 443 

weight gains of exposed males and females were 

similar to those of the controls (Table 2). Feed 

consumption by 10,OOO ppm groups was somewhat 

lower than that by the control groups throughout the 

study (Table 2). Dietary levels of 625,1,250,2,500, 

5,OOO, and 10,OOO ppmresulted in average daily 

consumption levels of 80,170,330, 680, and 

1,400 mgkg body weight in males and 100, 220,440, 

830, and 1,620 mgkg in females. Chemical-related 

clinical findings in allmaleand femaleexposure 

groupsincluded hypoactivity, drowsiness, lethargy, 

and decreased exploratoryandspontaneouslocomotor activity. These findings were observed primarily during the first week of the study,and the 

short durationof these findings was attributed to the 

development of tolerance. Serum oxazepam concentrations increased with exposure level in males and 

females, but the increases were not proportionate to 

dose. Except in the 5,OOO and 10,OOO ppm groups at 

week 2, serum oxazepam concentrations in eachexposure group were similar between males and females 

(Table 3). 

Short-term administration(1 week) of oxazepam produced deficits in grip strength at high exposure levels 

in both malesand females (Tables G1 and G2). 

Thesedeficitsweretemporaryinfemale mice but 

persisted in male mice with continued administration 

(11 weeks). Decreased paw lick latencies in response 

TABLE3 

tothermalstimulationwere observed in exposed 

males and females at 12 weeks (Table G3). Increases 

in motor activity were observed at all exposure levels 

at study week 2 (Table G4). This disinhibitory effect 

may be indicative of an anxiety-reducing effect of 

oxazepam. Increased motor activity abated in males, 

but was stillevident in females at week 12. 

Oxazepam produced ageneralreductionin startle 

response in males at weeks 2 and 12 and in females 

at 12 weeks (Table G5). Sensitivity to an auditory 

prepulse as part of the startle response assessment 

was not affected by oxazepam administration (Table 

G6). Oxazepam effects on the sensory system in 

general may be involved in changes observed in 

startle behavior as well as altered thermal sensibility 

measurements. 

Except for relative liver weights of mice receiving 

625 ppm, the absolute and relative liver weights of all 

exposed males and females were significantly greater 

than those of the controls (Tables 4 and Fl). These 

increases were marked and were clearly dose related. 

In males, absolute and relative heart weights of the 

1,250 and 10,000 ppm groups and the relative heart 

weight of the 2,500 ppm group were significantly 

lower than those of the controls.This finding was 

not clearly dose related, and no cause for this change 

could be determined. The absolute kidney weights of 

females exposed to l,250,2,500,5,OOO,or 10,OOOppm 

were significantly greater than that of the controls. 

of Oxazepama 

Serum Oxazepam Concentrations in Swiss-Webster Mice in the 14-Week Feed Study 

Dose (ppm) 0 625 1,250 2,500 5,000 10,000 

Male 

2 weeks 

12weeks 

0.00 f 0.00 

0.00 f 0.00 

5.91 f 0.66 

6.21 f 0.53 

9.96 f 0.72 

9.30 ? 1.14 

13.6 f 1.23 

12.1 f0.94 

18.6 f 1.3 

20.3f 2.15 

29.3 f 3.18 

22.0 f 1.06 

Female 

2 weeks 

12weeks 

0.00 2 0.00 

0.00f 0.00 

6.05 f 0.62 

6.52 f 0.93 

9.08 & 0.77 

9.22 f 0.43 

15.2 f 1.61 

12.1 f 1.60 

29.3 f 4.04 

21.9 f 1.37 

36.9 f 4.49 

21.6 f 1.23 

a Mean k standard error for five animals; values are given as &mL. 
Results 31 

Centrilobularhepatocellularhypertrophy was observed in exposed animals, and the severity generally 

increased with dose (Table 4). This lesion was 

characterized by minimal to mild enlargement (hypertrophy) of hepatocytes that were centrilobularin 

distribution.Hypertrophichepatocytes had homogeneous or slightly granular eosinophilic cytoplasm. 

The nuclei were often enlarged and contained prominent basophilic chromatin clumps. A low incidence 

of focal hepatocellular necrosis occurred in several 

groups of female mice receiving oxazepam, but not in 

the control group (Table 4). The incidence and the 

severity of the lesion were not chemical related, and 

the highest incidence occurred in the females exposed 

to 2,500 ppm. Foci of hepatocellular necrosis also 

occurred in one control and several exposed male 

mice. Because of the generally low incidences and 

lack of dose-related increase in incidence or severity, 

the hepatocellular necrosis was not attributed to the 

ingestion of oxazepam. 

There was adose-relateddecreasedincidence of 

cytoplasmic vacuolation of cells within the x-zone of 

the adrenalcortex in female mice (Table 4). The 

vacuolated cells adjacent to the medulla (x-zone) are 

transitory and normally disappear gradually in virgin 

female mice. The decreased number of vacuolated 

cells in females receiving oxazepam indicates an 

accelerated regression of the x-zone and maturation 

of the adrenal gland. 

Dose Selection Rationale 

Because the degreeof increase in liver weight in mice 

atthe 10,OOO ppmconcentration was considered 

potentially life threatening during a 2-year study, the 

exposure levels selected were 0,2,500, and5,OOO ppm. 

TABLE4 

Liver Weights and Incidences of Selected Nonneoplastic Lesions in Swiss-Webster Mice 

in the 14-Week Feed Study of Oxazepam 

Dose (PPrm) 

Male 

10,000 0 5,000 625 1,250 2,500

Live? 10 10 10 10 10 10 

Liver Weights 

Absolute 1.808 f 0.080 2.354 f 0.074" 2.275 k 0.112. 2.761 k 0.134'. 3.035 f 0.115** 3.528f 0.137..b 

Relative 50.43 f 1.35 61.23 f 1.88 64.60 f 2.75;. 72.61 f 3.92;. 85.52 f 2.61.. 99.98 f 4.27+.b 

CentrilobularHypertrophy 0 (1.2)' 9AA 1oAA (1.3)(1.1) 10AA 10~~ (1.7) (1.6) 1011

Hepatocellular Necrosis 1 (1.0) 1 (1.0) 0 1 (1.0) 0 1 (1.0) 

Female 

Liver 10 10 10 10 10 10 

Liver Weights 

Absolute 1.405 f 0.028 1.731 f 0.049.; 1,9102 0.052.. 2.328 f 0.109'. 2.610 f 0.064.. 3.084 f 0.070.; 

Relative 47.32 k 1.14 54.28 f 1.47 61.16 f 2.12.' 72.42 f 2.77.; 84.16 f 1.93.. 100.63 k 1.95.. 

Centrilobular Hypertrophy 

Hepatocellular Necrosis 

0 

0 

1011(1.2)1011(1.1)1011(1.4) 

2 (1.0) 0 4 (1.0) 

~OAA(1.4)1011(1.9) 

3 (1.0) 2 (1.0) 

Adrenal Gland 

CytoplasmicVacuolization 10(2.5) 2AA (1.5) 4AA (1.2) IAA (1.0) IAA (1.0) OAA 

* Significantly different (PSO.05) fromthecontrolgroupbyWilliams' or Dunnett's test 

** PSO.01 

AA Significantly different(PSO.01)from the control group by Fisher's exact test 

a Number of mice with organ examined microscopically; organ weights and body weights are given in grams; organ-weight-to-bodyweight ratios are given as mg organ weight/g body weight (mean f standard error) 

n=9 ' Averageseverityoflesions in affectedmice: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked; 5 = severe 
32 Oxazepam, NTP TR 443 

57-WEEK STUDY 

Survival 

Estimates of survival probabilities formaleand 

female Swiss-Webster mice are shown in Table5 and 

in the Kaplan-Meier curves in Figure2. The original 

design of this study provided for administration of 

dosed feed to male mice for 103 weeks and to 

females for 104 weeks, followed by a 1-week observation period. The original study design also

included an interim evaluation at week 66. However, 

there were large numbers of moribund animals and 

TABLE5 

deathsafter 40 weeks of exposure. These deaths 

wereconsidered due to heartfailuresecondary to 

pulmonary hypertension and edema, which resulted 

from systemic amyloidosis, a conditioncommon in 

Swiss-Webster mice and apparentlyenhanced by 

oxazepam exposure. At 57 weeks, 19 males and 

28 females receiving 2,500 ppm, and 10 males and 

17 females receiving 5,OOO ppm were surviving, and 

the study was terminated. No interimevaluations 

were conducted. 

Survival of Swiss-Webster Mice in the 57-Week Feed Studyof Oxazepam 

Do= (PPm) 

Male 

Animals initially in study 

Moribund 

Natural deaths 

Animals surviving to study termination 

Percent probability of survival at end of stud9 

Mean survival (days)b 

Survival analyses' 

Female 

Animals initially in study 

Moribund 

Natural deaths 

Animals surviving to study termination 

Missingd 

Percent probability of survival at end of study 

Mean survival (days) 

Survival analyses 

0 2,500 5,000 

60 60 60 

10 13 24 

5 28 26 

45 19 10 

75 32 17 

373 338 335 

P<O.001 P<O.001 P<O.001 

60 60 60 

9 13 11 

4 18 31 

47 28e 17 

1 1 

78 48 29 

382 351 353 

P<0.001 PCO.001 P<O.001 

a Kaplan-Meier determinations based on the number of animals alive on first day of terminal sacrifice 

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 dosed columns. 

Censored from survival analyses 

e Includes one animal that died during the last week of the study. 
Results 33 

FIGURE2 

Kaplan-Meier Survival Curvesfor Swiss-Webster Mice Administered Oxazepam in Feed for 

57 weeks 
34 Oxazepam, NTP TR 443 

Body Weights, Feed Consumption, Compound part of the study. After week 29, the mean body

Consumption, and Clinical Findings weights of 5,OOO ppm females were similarto those of 

Mean body weights of exposed male mice were controls, but those of 2,500 ppm females remained 

similar to those of the controls in the early weeks of slightly greater than thoseof controls until the endof 

the study. Beginning at week 17, however, mean the study (Table 7 and Figure 3). Feed consumption 

body weights of exposed male mice were lower than by exposed males and females was slightly lower than 

those of the controls (Table 6 and Figure 3). Except that by the controls,and females in allgroups, 

for week 1, mean body weights of exposed females consumed slightly more feed than males throughout 

were greater than those of controls during the early the study (Tables I1 and 12). Dietary levels of 2,500 

TABLE6 

Mean Body Weights and Survival of Male Swiss-Webster Mice in the 57-Week Feed Studyof 

Oxazepam 

Weeks 0 ppm 2,500 ppm 5,000 ppm 

on Av. Wt. Number of Av. Wt. Wt. (% of Number of Av. Wt. Wt. (% of Number of 

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

1 26.7 60 26.5 99 60 26.5 99 60 

2 27.9 60 28.6 103 60 28.7. 103 60 

3 29.4 60 30.6 104 59 31.1 106 60 

4 30.0 60 31.5 105 59 31.9 106 60 

5 30.8 60 31.8 103 59 32.4 105 60 

6 31.9 60 32.4 102 59 33.4 105 60 

7 32.8 60 33.2 101 59 34.0 104 60 

8 33.4 60 33.2 99 59 34.0 102 60 

9 33.2 60 33.7 102 59 34.2 103 60 

10 34.3 60 34.5 101 59 35.2 103 59 

11 35.2 60 35.1 100 59 35.8 102 59 

12 35.1 60 34.8 99 59 35.4 101 59 

13 36.0 60 36.0 100 59 36.2 101 59 

17 38.4 60 37.5 98 58 37.4 97 59 

21 40.3 60 38.7 96 58 38.2 95 59 

25 41.6 59 39.8 96 56 39.2 94 58 

29 42.2 59 40.8 97 55 39.4 93 58 

33 42.8 59 40.7 95 54 39.2 92 58 

37 43.0 58 41.O 95 53 39.4 92 55 

41 42.2 55 40.3 96 52 39.2 93 50 

45 41.7 52 39.4 95 47 37.8 91 42 

49 41.5 49 38.6 93 40 37.4 90 36 

53 41.7 46 37.9 91 28 36.4 87 24 

57 41.1 45 37.4 91 20 37.2 91 11 

Mean for weeks 

1-13 32.1 32.5 101 33.0 103 

14-52 41.5 39.6 95 38.6 93 

53-57 41.4 37.7 91 36.8 89 
l 

Results 35 

40-.......... +.......... :........... :.......... 

i. 

:a 

1 iaIi 

35- .................... A*................ : p i 

e. i

30 - ....... a.: .................... ......... ..;. 

8 

8: 

; i 

8: 

R i 15- ........... :.......... :........... :.......... 

20 1 

0 5 IO IS 

................................. -....................................................... 

......................................................... : :I MALE MICE .....

0 PQY 

0 2500 PPU 

A 5000 PQY 

I I I 1 1 I 

25 30 35 40 S5 50 55 1 3 

WEEKS ON STUDY 

._.......... .................................. ............ 

0: 

io 6 0; 0 ; 

. p..... ...... f ...... ..... :o .... 

........................... _..................... 

i.......... i........... FEMALE MICE I 
36 Oxazepam, NTP TR 443 

TABLE7 

Mean Body Weights and Survival of Female Swiss-Webster Mice in the 57-Week Feed Study 

of Oxazepam 

Weeks 0 ppm 2,500 PPm 5,000 DPm 

on Av. Wt. Number of Av. Wt. Wt.(% of Number of Av. Wt.Wt. (% of Number of 

Study (g) Survivors (g) controls) Survivors (8) controls) Survivors 

1 21.4 60 21.0 98 60 21.0 98 60 

2 22.8 60 25.4 111 60 24.8 109 60 

3 23.7 60 26.8 113 60 26.6 112 60 

4 24.7 60 27.8 113 59 27.8 113 60 

5 25.4 60 28.5 112 59 28.4 112 60 

6 26.3 60 29.6 113 59 29.3 111 60 

7 26.9 60 30.2 112 59 30.4 113 60 

8 27.6 60 30.6 111 59 30.6 111 60 

9 27.3 60 30.4 111 59 30.7 113 60 

10 28.3 60 31.1 110 59 31.2 110 60 

11 28.4 60 31.5 111 59 31.6 111 60 

12 29.1 60 31.7 109 59 31.8 109 60 

13 29.4 60 31.8 108 59 31.9 109 60 

17 31.4 60 33.8 108 58 33.6 107 59 

21 32.4 60 34.9 108 58 33.9 105 59 

25 33.3 60 35.9 108 57 34.7 104 59 

29 34.5 58 36.3 105 57 34.9 101 59 

33 34.8 58 36.8 106 57 35.2 101 58 

37 35.0 58 37.4 107 56 35.5 101 58 

41 34.8 57 36.6 105 55 34.6 99 58 

45 34.3 56 36.4 106 51 34.7 101 53 

49 34.8 55 36.7 106 40 34.3 99 40 

53 35.0 52 36.3 104 36 34.4 98 28 

57 34.8 48 35.9 103 29 33.9 97 18 

Mean for weeks 

1-13 26.3 29.0 110 28.9 110 

14-52 33.9 36.1 106 34.6 102 

53-57 34.9 36.1 103 34.2 98 

and 5,OOO ppmoxazepamresulted in average daily (females) and 9 (males). By days 15and 16, the 

compound consumption levels of 270 and 570 mgkg appearanceand behavior of exposed animalswere 

for males and 320 and 670 m@g for females. similar to controls. 

On study days 4 (females) and 5 (males), clinical Serum Oxazepam Concentrations 

findings of hypoactivity, slow respiration, partially Serum oxazepam concentrations were similar in the 

closed eyelids, lethargy, and decreased spontaneous males and females receiving 2,500 and 5,OOO ppm 

exploratorybehaviorwerenoted in most exposed (Table 8). No dose- or sex-relateddifferencesin 

animals. Exceptfor hypoactivity, the incidences of serum oxazepam concentrations after occurred 

these clinical findings hadecreased by days 8 57 weeks of exposure. 
Results 37 

TABLE8 

Serum Oxazepam Concentrations in Swiss-Webster Mice in the 57-Week Feed Study 

of Oxazepama 

Male 

Serum oxazepam (pg/mL) 

n 

o+o 

10 

6.65 k 2.87 

10 

7.73 + 4.76 

10 

Female 

n 9b 6' 10 

Serumoxazepam(Bg/mL) o*o 7.25 + 1.44 6.89 + 3.04 

a Mean k standarddeviation 

An aliquot of serum sample from one animal was contaminated. The remaining volume of serum sample was insufficient for a 

repeat analysis. ' Analysisresultsfrom four animals were deleted due to instrument 

Pathology and Statistical Evaluation 

This section describes the statistically significant or 

biologically noteworthy changes in the incidences of 

Swiss-Webster mice with neoplasms of the liver and 

nonneoplasticlesions of the liver, heart, lung,and 

otherorgans.Summariesoftheincidences of 

neoplasmsandnonneoplasticlesions,individual 

animaltumordiagnoses,andstatistical analyses of 

primary neoplasms that occurred with an incidence of 

atleast 5% inatleastoneanimalgroupare 

presentedinAppendix A formaleSwiss-Webster 

mice and AppendixB for female Swiss-Webster mice. 

Liver: The principal toxic effects associated with the 

ingestionofoxazepaminthefeedoccurredinthe 

liver. The incidences of centrilobular hepatocellular 

hypertrophyinexposedmalesandfemaleswere 

significantly greaterthanthose of thecontrols 

(Tables 9, 10, A4, and B4). Inexposed mice, the 

hypertrophy was generally mild in severity while in 

the few affected controlmice the severitywas 

minimal. The hypertrophy was similar to that 

described in the 14-week study. 

The incidences of eosinophilic foci, a putative preneoplasticlesion,andofhepatocellularadenoma 

were significantly greater than those of the controls 

error. Insufficient specimen remained to repeat the analysis. 

in 2,500 and 5,000 ppmmalesandfemales. The 

incidence of hepatocellular carcinoma was 

significantly greater in2,500 ppm andales

5,000 ppmmalesand females. The incidence of 

hepatocellularneoplasmsand the number of mice 

withmultiplehepatocellularneoplasmsincreased 

withincreasingexposure level in bothmalesand 

females (Tables A1 and Bl). 

The eosinophilic foci, hepatocellular adenomas, and 

hepatocellular carcinomas constitute a morphologic 

continuum of increasingsize,progressive loss of 

normal hepatic architecture, increasing disorganization of hepatic plates, and increasing cellular pleomorphismand atypia. Theeosinophilicfociwere 

relatively discreteaggregates of oftenenlarged 

hepatocyteswithhomogeneouseosinophilic cytoplasm. Whilesome foci were largerthanasingle 

hepatic lobule, the lobular pattern was retained and 

the organization of the hepaticplates was only 

minimally altered. Hepatocellularadenomaswere 

discretenoduleslargereosinophilic than foci. 

Normal hepatic lobulation was not apparent and the 

hepaticplatesweredistorted to varying degrees 

within the adenomas. While the hepatocytes within 

the adenomas were often enlarged and eosinophilic, 

there was little or no pleomorphismor atypia. In 
38 Oxazepam, NTP TR 443 

TABLE9 

Incidences of Neoplasms and Nonneoplastic Lesions of the Liver in Male Swiss-Webster Mice 

in the 57-Week Feed Study of Oxazepam 

Dose (PPm) 

Live? 

Centrilobular Hypertrophy 

Basophilic Focus 

Eosinophilic Focus 

Focus (anytype) 

Hepatocellular Adenoma 

Overall rate' 

Adjusted rated 

Terminal ratee 

First incidence (days) 

Logistic regression testf 

Hepatocellular Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Hepatocellular Adenoma or Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

0 2,500 5,000 

60 60 60 

12 (1.3) 46.. (l.8)b 4791 (1.8) 

1 0 1 

0 22. * 2299 

1 2299 2299 

1/60 (2%) 35/60 (58%) 50160 (83%) 

2.2% 88.7% 98.0% 

1/45 (2%) 

P <0.001 

397 (T) 

Pc0.001 

268 

15/19 (79%) 

P<O.001 

231 

9/10 (90%) 

0/60(0%) 5/60 (8%) 19/60 (32%) 

0.0% 21.7% 72.0% 

2

0/45 (0%) 

356 

3/19 (16%) 

302 

5/10 (50%) 

P<0.001 P=O.003 P<O.001 

P<0.001 P=O.OlO P<0.001 

1/60 (2%) 35/60 (58%) 52/60 (87%) 

2.2% 88.7% 98.1% 

1/45 (2%) 15/19 (79%) 9/10 (90%) 

397 (T) 268 231 

P<O.001 P<O.001 P<0.001 

P<O.001 P<0.001 P<0.001 

** Significantly different (PSO.01) from the control group by the logistic regression test 

(T)Terminal sacrifice 

a Numberofanimals with liverexaminedmicroscopically 

Averageseverityoflesions in affected mice: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked 

Number of lesion-bearing animalshumber of animals necropsied or examined microscopically 

Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustmentfor intercurrent mortality. 

e Observed incidence in animals surviving until the end of the study 

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

to pairwise comparisons between the controls and that dosed group. The logistic regression analysis regards these lesions as 

nonfatal. 

g Not applicable; noneoplasms in animal group 
Results 39 

TABLE10 

Incidences of Neoplasms and Nonneoplastic Lesions of the Liver in Female Swiss-Webster Mice 

in the 57-Week Feed Study of Oxazepam 

Live? 60 59 59 

Centrilobular Hypertrophy 3 (1.3) 51.' (1.6)b 53.. (1.8) 

Basophilic Focus 0 0 0 

Eosinophilic Focus 0 20** 14.. 

Focus (any type) 0 20'1 14.. 

Hepatocellular Adenoma 

Overall rate' 0/60 (0%) 22/59 (37%) 47/59 (80%) 

Adjusted rated 0.0% 52.6% 95.7% 

Terminal ratee 0/47 (0%) lO/28 (36%) 15/17 (88%) 

First incidence (days) 2 291 2&4 

Logistic regression testf P<O.ool P<O.ool P<O.ool 

Hepatocellular Carcinoma 

Overall rate 1/60 (2%) 1/59 (2%) 11/59 (19%) 

Adjusted rate 2.1% 3.6% 51.6% 

Terminal rate 1/47 (2%) 1/28 (4%) 8/17 (47%) 

First incidence (days) 397 (TI 397 (q 337 

Life table test P<0.001 P=O.642 P<O.ool 

Logistic regression test P<O.ool P=O.642 P<O.ool 

Hepatocellular Adenoma or Carcinoma 

Overall rate 1/60 (2%) 23/59 (39%) 47/59 (80%) 

Adjusted rate 2.1% 55.2% 95.7% 

Terminal rate 1/47 (2%) 11/28 (39%) 15/17 (88%) 

First incidence (days) 397 (TI 291 284 

Life table test P<0.001 P<O.ool P<O.ool 

Logistic regression test P<0.001 P<0.001 P<O.OOl 

** Significantly different (PSO.01) from the control group by the logistic regression test 

(T)Terminal sacrifice 

a Number of animals with liver examined microscopically 

Averageseverityoflesions in affectedmice: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked 

Number of lesion-bearing animaldnumber of animals necropsied or examined microscopically 

Kaplan-Meier estimated neoplasm incidenceat the end of the study after adjustment for intercurrent mortality. 

e Observed incidence in animals surviving until the end of the study 

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

to pairwise comparisons between the controls and that dosed group. The logistic regression analysis regards these lesions as 

nonfatal. 

g Notapplicable;noneoplasms in animalgroup 
40 Oxazepam, NTP TR 443 

contrast,hepatocellularcarcinomas had heterogeneousgrowthpatternswithareas of distinct 

trabecular or adenoid arrangements. The neoplastic 

hepatocytes usually exhibited agreaterdegree of 

pleomorphismcharacterized by variation in size, 

staining qualityof the cytoplasms, and size and shape 

of the nuclei. 

Mufrefe Organs: Amyloid deposition is a common 

spontaneouscondition in Swiss-Webster mice, and 

exposure to oxazepam appears to have exacerbated 

thiscondition,resultingin significant dose-related 

increasesin the incidence and severity in multiple 

organscompared to those of the controls. The 

organsmostcommonly affected in bothmaleand 

female mice in this study were the heart, glandular 

stomach, intestine, spleen, lymph nodes, thyroid and 

parathyroid glands, adrenal cortex, and uterus

(Tables A4 and B4). Except in the heart, the severity 

of these lesions in these organs averaged minimal to 

mild in the controlsand mild to moderate in the 

5,OOO ppm group. 

In the heart, the severity of amyloid deposition in the 

myocardium ranged frommoderate to marked in 

exposed mice and minimal to mild in controls. Doserelatedincreasedincidences in myocardial amyloid 

deposition occurred in exposed males (43/60, 52/60, 

52/60; Table A4). Additionally, the incidences of 

atrial thrombosis increased in a dose-related manner 

(males: 1/60, 34/60, 35/60; females: 2/60,23/59, 

31/59; Tables A4 and B4). These thrombi were quite 

large and distended or occluded the atria. The 

thrombiwere usually associated with moderate to 

marked myocardial amyloid deposition. 

In the lung, oxazepam exposure was also associated 

with increased incidences of inflammatory fibrosis in 

males (0 ppm, 0/60; 2,500 ppm, 27/60, 5,OOO ppm, 

25/60; Tableand A4)females (0 ppm, 1/60,

2,500 ppm, 14/59; 5,OOO ppm, 8/59; Table B4) and 

dose-related increased incidences of mononuclear cell 

infiltrates (males: 1/60, 31/60, 37/60; females: 2/60, 

16/59,26/59; Tables A4 and B4). Lung inflammation 

was primarily multifocal; however, the fibrosis and 

mononuclear cell infiltration were usually widely 

disseminated or diffuse. The severity of inflammatory 

fibrosis in affected animals was similaramong 

exposed and controlgroups (males: 0, 2.3,2.2; 

females: 2.0, 1.9, 2.3). The lungchangeswere 

consistent with pulmonary hypertension and were 

most likely due to heart failure secondary to amyloid 

deposition and secondary to pulmonary edema. 

Uterus: The incidence of cystic endometrial hyperplasia decreased with increasing exposure level (13/60, 

2/59, 0/57; Table B4). 

Neurobehavioral Evaluation 

During the 6-month neurobehavioral evaluation, forelimb grip strength was significantly decreased in male 

mice exposed to 5,000 ppm. Hindlimb grip strength 

at 6 months and forelimb and hindlimb grip strengths 

at 12 monthswere not affected by oxazepam exposure 

(Tables G8 and G9).Femalesin the 2,500 ppm 

groupand males and females in the 5,OOO ppm 

groups exhibited significantly decreased pawlick 

latencies in the thermal sensitivity tests at 6 months 

(Table G10). No significant differences in paw lick 

latencies were observed at 12 months in any exposed 

group. Motor activity was not affected by oxazepam 

exposure at 6 months but was reduced by 53% in the 

5,000 ppm females at12months(Table G11). 

Startleresponse was not affected by oxazepam 

exposure. 
Results 41 

B6C3Fl MICE 

14-WEEK STUDY 

One male mouseinthe 10,OOO ppm group died 

during the study withaurinarytractinfection 

(Table 11). Mean body weight gains of exposed 

groupsweresimilar to those of thecontrols 

(Table 11). Feed consumption by all maleand 

female exposed groups was lower than that by controls early in the study, but was similar during the 

latter part of the study (Table 11). Dietary levels of 

625, 1,250, 2,500, 5,OOO, and 10,OOO ppm resulted in 

average daily consumption values of 100, 200, 390, 

TABLE11 

890, and 1,810 mgkg body weight in males and 130, 

260,450, 920, and 2,050 mgkg in females. Serum 

oxazepam concentrations increased with exposure 

level in both males and females; however, as in the 

Swiss-Webster study, these increases were not proportional to the increaseindose(Table 12). Serum 

oxazepam concentrations were similar in males and 

females at each exposure level. Chemical-related 

clinical findings of drowsiness, lethargy, and 

decreasedspontaneouslocomotor activity were 

observed in all exposed groups. These findings 

occurred for only a few days beginning about day 2 of 

the study. 

Survival, Mean Body Weights, and Feed Consumption of B6C3FL Mice in the 14-Week Feed Study 

of Oxazepam 

Final Weight 

Mean Body Weightb (9) Relative Feed 

Dose Survivala initial Final Change to Controls Consumption' 

(PPm) (%) Week 2 Week 14 

Male 

0 1ono 23.9 f 0.3 33.6 f 0.7 9.7 f 0.6 5.1 5.5 

625 10/10 24.2 f 0.5 35.3 f 0.7 11.1 f 0.4 105 3.9 4.9 

1,250 1ono 24.0 f 0.5 34.3 f 0.6 10.3 f 0.8 102 3.8 4.9 

2,500 lono 24.6 f 0.3 34.1 f 0.4 9.5 f 0.3 102 3.8 4.6 

5,Ooo 10/10 23.5 f 0.5 33.1 f 0.5 9.5 f 0.4 98 3.6 5.0 

10,Ooo 9/l Od 23.6 f 0.6 33.5 f 0.5 9.5 f 0.5 100 3.2 5.7 

Female 

0 lono 19.5 f 0.2 29.7 f 0.7 10.2 f 0.7 5.7 6.8 

625 1ono 20.2 2 0.5 31.7 f 0.7 11.5 f 0.7 107 3.4 6.5 

1,250 lono 19.1 f 0.4 30.5 f 0.6 11.4 f 0.7 103 3.3 6.3 

2,500 lono 19.8 f 0.3 29.4 f 0.6 9.6 f 0.5 99 3.2 4.5 

5,ooo lO/lO 19.6 & 0.4 30.3 f 0.6 10.7 f 0.6 102 3.3 5.1 

10,ooo 10/10 19.9 f 0.2 30.0 f 0.5 10.1 f 0.4 101 3.0 6.1 

a Number of animals survivinghumber initially in group 

Weights and weight changes are given as mean f standard error. Subsequent calculations are based on animals surviving to the 

end of the study. 

Feed consumption is expressed as grams per animal per day; data for week 2 are based on 20 animals; data for week 14 are based 

on 10 animals. 

Weekof death: 13 
42 Oxazepam, NTP TR443 

TABLE12 

Serum Oxazepam Concentrations in B6C3F, Mice in the 14-Week Feed Study of Oxazepama 

Do= (PPW 0 

Male 

10,000 625 5,000 1,250 2,500 

12 weeks 

2 weeks 

0.00 f 0.00 

0.00 f 0.00 

6.06 f 0.31846 

5.19 k OSb 

f 0.5711.1 

8.30f 0.85'10.9 

f 0.3316.5 

f 0.5716.9 

f 1.1819.9 

f 1.0822.5 

f 1.18 

k 1.92 

Female 

12 weeks 

2 weeks 

0.00 f 0.00 

0.00 f 0.00 

5.38 k 0.53 

5.75 f OSb 

8.04 f 0.66 

7.08 f 0.7611.8 

11.3 f 1.0717.3 

k 1.57 

k 1.67 

15.1 & 1.1722.7 

23.8 f 0.30 

f 1.36 

a

' Mean k standard error for five animals; values are given as pg/mL. 

n=4 ' n=3 

Oxazepam produced a deficit in grip strength, which 

was more marked inmales than ifemales

(Tables G12 and G13). This deficit was only temporary, however, because it was observed at 2 weeks but 

notat12 weeks. Increasesin motor activity were 

observed at all exposure levels at both 2 weeks and 

12 weeks (Table G15). This disinhibitory effect may 

be indicative of an anxiety-reducing effect of 

oxazepam. Somatosensory integrity was measured by 

the tactile startle response. Increases in initial startle 

reactivity were observed in 625 and 1,250 ppm males 

andin 2,500 ppm females at 2 weeks. Decreased 

reactivity was seen at 12 weeks and was particularly 

evident in the 2,500, 5,OOO, and 10,OOO ppm females. 

An overall increased sensitivity of exposed mice to an 

auditoryprepulseaspart of the tactile startle response was noted (TablesG16,G17,and G18). In 

addition, decreased paw lick latencies were observed 

in the 625, 1,250, and 10,OOO ppm females at 

12 weeks (Table G14). Changes in startle response 

and thermal sensitivity may be due to effect an on the

sensorycomponent of thestartle reflex circuit. 

Oxazepam effects on the sensory system in general, 

and specifically on arousal mechanisms, may have 

accounted for these changes. 

Absoluteand relative liver weights of all exposed 

males and females were notably greater than thoseof 

the controls (Tables 13 and F2). These increases 

were exposure related. Absolute thymus weights of 

625, 1,250, 2,500, and 10,OOO ppm males and relative 

thymus weights of 2,500 and 10,OOO ppm males were 

significantly greater than those of the controls, and 

absoluteand relative kidney weights of exposed 

females were variable,butwerealso significantly 

greaterthanthose of the controls. There were no 

histopathologic differences in the thymus or kidney 

that could account for these increases. 

Centrilobular hepatocellular hypertrophy occurred in 

exposed male and female B6C3Fl mice (Table 13). 

Exposure-relatedincreasesin the severity of this 

lesionoccurred in both males and females. These 

lesions were similarto those previously described for 

the Swiss-Webster mice. In both strains of mice, the 

presence of centrilobular hypertrophy correlatedwith 

exposure-relatedincreasesin the absoluteand 

relative liver weights. 

Dose Selection Rationale 

Because the degree of increase in liver weight in mice 

at the 10,000 ppm concentration was considered 

potentially life threatening during a 2-year study, the 

doses of oxazepam selected for the 2-year study were 

0, 2,500, and 5,000 ppm. An additionalexposure 

level of 125 ppm was selected in anattempt to 

produce a group of mice with serum oxazepam levels 

in the 1pg/mLrange,similar to that produced in 

humans by a therapeutic dose of oxazepam. 
Results 43 

TABLE13 

Liver Weights and Incidences of Nonneoplastic Lesions of the Liver in B6C3F, Mice 

in the 14-Week Feed Study of Oxazepam 

0 625 1,250 2,500 5,000 10,000 

Male 

Live? 10 10 10 10 10 10 

Liver Weights 

Absolute 1.634 f 0.052 1.919 f 0.044*lb 2.150 f 0.037..2.186 f 0.038.. 2.545 f 0.065*+2.966 f 0.057.' 

Relative 47.24 f 0.96 52.91 f 0.67**b 61.42 f 0.47**62.50 f 0.94.. 74.83 f 1.09** 88.09 f 1.19** 

Centrilobular Hypetrophy 0 (1.0)' (2.0) (2.0) 1011 1011 1011 101 (3.0) 1011~(2.1)

Female 

Liver 10 0 10 10 10 10 

Liver Weights 

Absolute 1.387 f 0.021 1.743 f 0.031**d1.871 f 0.046**b1.898f 0.044.. 2.391 f 0.072**2.837 f 0.061**b 

Relative 46.59 f 1.01 55.44 f 1.02**d 61.54 f 0.76**b63.20 f 1.19.. 77.35 f 1.16**93.64 f 1.03*+b 

Centrilobular Hypertrophy 0 1011(1.0)1011 (1.0) 1011 (2.0) 1011 (3.0) 1011(2.0) 

** Significantly different (PSO.01) from the control group by Williams' or Dunnett's test 

AI Significantly different (PSO.01) from the control group by Fisher's exact test 

a Number of mice with organ examined microscopically; organ weights and body weights are given in grams; organ-weight-to-bodyweight ratios are given as mg organ weight& body weight (mean f standard error) 

n=9 

Average severity of lesions in affectedmice: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked; 5 = severe 

n=10 
44 Oxazepam, NTP TR 443 

%YEAR STUDY 

Survival 

Estimates of survival probabilities formaleand 

female B6C3Fl mice in the 2-year study are shown in 

Table 14 and in the Kaplan-Meier curves in Figure 4. 

Survival of males and females receiving 125 ppm was 

similar to controls. However, there werealarge 

number of dead and moribund males and females in 

the 2,500 and 5,OOO ppm groups. Only 30% of the 

males and 4% of the females exposed to 2,500 ppm 

survived until the end of the study; most deaths in 

thesegroupsoccurredafter week 85 of the study 

TABLE14 

(Tables 15 and 16). Mortalityin 5,OOO ppm males 

was greatly increased beginning at week 65, and there 

were no survivors by week 93 of the study. Mortality 

in 5,ooO ppm females increased after week 57, and 

there were no survivors by week 89. Due to the 

increasedmortality in the 2,500 and 5,OOO ppm 

groups, the 1-week observation period at the end of 

the study was canceled except for mice being 

observed forneurobehavioral effects. The early 

deaths of exposed mice were considered due at least 

in part to hepatic neoplasia. 

Survival of B6C3F, Mice in the 2-Year Feed Study of Oxazepam 

Do= (PPW 

Male 

Animals initially in study 

15-Month interim evaluation 

Moribund 

Natural deaths 

0 

60 

10 

2 

3 

Animals surviving to study termination 45 

Percent probability of survival at end of stud9 90 

Mean survival (days)b 

Survival analyses' 

Female 

Animals initially in study 

15-Month interim evaluation 

Moribund 

Natural deaths 

Animals surviving to study termination 

668 

P<O.ool 

60 

10 

8 

3 

39 

Percent probability of survival at end of study 78 

Mean survival (days) 664 

Survival analyses P<O.ool 

125 2,500 5,000 

60 60 60 

10 10 10 

4 22 30 

2 13 20 

44 15 0 

88 30 0 

670 641 545 

P=O.987 P<O.OOl P<O.ool 

60 60 60 

10 10 10 

7 22 15 

2 26 35 

41 2 0 

82 4 0 

667 631 498 

P=0.761N P<O.ool P<O.ool 

a Kaplan-Meier determinations based on the number of animals alive on first day of terminal sacrifice 

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

(Cox, 1972) with the controls are in the dosed columns. A lower incidence in a dose group is indicated by N. 
Results 

0 15 30 bs 60 75 90 105 120 

WEEKS ON STUDY 

0 15 30 bs 60 75 90 105 120 

WEEKS ON STUDY 

FIGURE4 

Kaplan-Meier Survival Curves for B6C3F, Mice Administered Oxazepam in Feed for 2 Years 
46 Oxazepam, NTP TR 443 

TABLE15 

Mean Body Weights and Survival of Male B6C3FLMice in the 2-Year Feed Study of Oxazepam 

Week 0 vvrn 125 pprn 2.500 vvm 5,000 ppm 

on Av. Wt. No. of Av. WtWt (96of No. of Av. WtWt (96of No. of Av. WtWt (%of No. of 

Sludy (9) Survivors (9) controls) Survivors (g) controls) Survivors (9) conlrols) Survivors 

1 21.5 60 21.2 99 60 21.4 100 60 21.2 99 60 

2 22.8 60 23.7 104 60 23.8 104 60 23.4 103 60 

3 23.9 60 25.7 108 60 26.0 109 60 25.5 107 60 

4 24.7 60 26.8 109 60 27.1 110 60 26.9 109 60 

5 26.3 60 28.1 107 60 28.1 107 60 28.0 107 60 

6 27.5 60 29.3 107 60 29.2 106 60 28.7 104 60 

7 29.1 60 30.5 105 60 30.1 103 60 29.8 102 60 

8 30.0 60 31.6 105 60 30.7 102 60 30.6 102 60 

9 31.0 60 31.9 103 60 31.2 101 60 30.7 99 60 

10 32.0 60 32.9 103 60 31.7 99 60 31.5 98 60 

11 32.9 60 33.6 102 60 32.6 99 60 32.2 98 60 

12 33.8 60 34.9 103 60 33.3 99 60 32.8 97 60 

13 34.6 60 35.4 102 60 33.5 97 60 33.3 96 60 

17 38.5 60 38.2 99 60 35.9 93 60 34.9 91 60 

21 41.2 59 39.9 97 60 36.7 89 60 35.2 85 60 

25 43.5 59 41.8 96 60 38.1 88 60 36.0 83 60 

29 44.3 59 43.0 97 60 38.8 88 60 36.7 83 60 

33 46.3 59 44.4 96 60 39.4 85 60 37.1 80 60 

37 46.6 59 45.1 97 60 40.1 86 60 37.6 81 60 

41 47.1 59 46.1 98 60 40.8 87 60 38.0 81 60 

45 47.4 59 46.3 98 60 40.8 86 60 37.8 80 60 

49 47.5 59 46.9 99 60 41.0 86 60 37.7 79 60 

53 47.3 59 47.5 100 60 41.1 87 60 37.1 78 60 

57 48.0 59 47.9 100 60 41.4 86 60 36.4 76 60 

61 48.5 59 48.3 100 60 41.3 85 60 35.8 74 58 

65 48.7 59 48.3 99 60 40.5 83 60 34.7 71 57 

69 48.3 49 48.2 100 49 39.7 82 50 33.9 70 43 

73 48.8 49 48.4 99 49 38.4 79 49 33.6 69 41 

77 48.8 49 48.9 100 47 37.1 76 48 33.4 68 38 

81 48.6 49 48.6 100 47 36.1 74 47 33.0 68 33 

85 49.4 48 49.6 100 47 35.4 72 46 32.8 66 22 

89 49.4 48 48.9 99 47 34.5 70 41 32.6 66 10 

93 49.1 46 48.7 99 46 33.1 67 36 

97 48.3 46 48.2 100 46 33.0 68 29 

101 47.7 46 47.8 100 45 33.2 70 24 

104 48.3 45 47.6 99 44 33.5 69 16 

Mean for weeks 

1-13 28.5 29.7 104 29.1 102 28.8 101 

14-52 44.7 43.5 97 39.1 87 36.8 82 

53-104 48.5 48.4 100 37.0 76 34.3 71 

a Interim evaluation occurred during week 66. 
Results 47 

TABLE16 

Mean Body Weights and Survival of Female B6C3F, Mice in the 2-Year Feed Study of Oxazepam 

Weeks 0 ppm 125 ppm 2,500 ppm 5,000 ppm 

on Av. WL No. of Av. WLWL (%of No. of Av. WL WL (%of No. of Av. WL WL (%of No. of 

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

1 18.1 60 18.1 100 60 18.2 101 60 18.0 99 60 

2 19.1 60 20.7 108 60 21.0 110 60 20.5 107 57 

3 20.8 60 22.0 106 60 22.6 109 60 21.8 105 57 

4 21.4 60 22.5 105 60 22.8 107 60 23.0 108 57 

5 22.1 60 23.7 107 60 24.0 109 60 23.9 108 57 

6 23.3 60 25.1 108 60 25.1 108 60 24.7 106 57 

7 24.4 60 26.9 110 60 26.5 109 60 26.1 107 57 

8 25.5 60 27.6 108 60 27.3 107 60 26.3 103 57 

9 26.5 60 28.4 107 60 27.6 104 60 26.7 101 57 

10 27.3 60 29.2 107 60 28.2 103 60 27.2 100 57 

11 28.2 60 30.3 107 60 28.9 103 60 27.8 99 57 

12 29.4 60 31.0 105 60 29.8 101 60 28.4 97 57 

13 30.3 60 31.8 105 60 30.3 100 60 28.5 94 57 

17 35.5 60 35.9 101 60 33.4 94 60 31.5 89 57 

21 38.7 60 38.4 99 60 34.6 89 60 32.5 84 57 

25 41.7 60 40.3 97 60 36.6 88 60 33.6 81 57 

29 43.9 60 42.0 96 60 37.7 86 60 34.4 78 57 

33 45.9 60 43.2 94 60 38.4 84 60 35.2 77 57 

37 47.8 60 44.2 93 60 39.4 82 60 36.0 75 57 

41 48.7 60 45.3 93 60 40.3 83 60 36.8 76 57 

45 49.9 60 45.3 91 60 39.9 80 60 36.2 73 57 

49 50.8 60 46.2 91 60 40.3 79 60 36.9 73 57 

53 51.0 59 46.3 91 60 40.6 80 60 36.4 71 57 

57 51.9 59 46.5 90 59 40.6 78 60 35.8 69 57 

61 53.2 59 47.0 88 59 40.9 77 58 35.1 66 52 

65 53.4 59 46.6 87 59 41.0 77 58 34.2 64 52 

69 53.6 49 46.8 87 48 40.7 76 47 33.9 63 37 

73 54.4 49 47.5 87 46 39.3 72 47 33.5 62 31 

77 54.4 48 47.3 87 46 38.5 71 47 33.3 61 29 

81 55.1 48 47.3 86 46 37.8 69 47 33.1 60 25 

85 56.0 47 47.2 84 46 36.8 66 46 33.4 60 12 

89 54.6 46 47.1 86 46 36.1 66 43 

93 55.2 42 46.8 85 46 35.2 64 34 

97 54.1 39 46.5 86 44 35.6 66 28 

101 53.2 39 45.5 86 43 35.0 66 21 

Mean for weeks 

1-13 24.3 25.9 107 25.6 105 24.8 102 

14-52 44.8 42.3 94 37.8 84 34.8 78 

53-101 53.9 46.8 87 38.3 71 34.3 64 

~~ 

a Interim evaluation occurred during week 66. 
48 Oxazepam, NTP TR 443 

Body Weights, Feed Consumption, Compound

Consumption, and Clinical Findings 

Mean body weights of males receiving 125 ppm were 

similar to those of the controls throughouthe study. 

Mean body weights of males that received 2,500 and 

5,OOO ppm were more than 10% lower than those of 

the controls after week 21 (Table 15 and Figure 5). 

Mean body weights of females were more than 10% 

lower than those of controls beginning at week 61 in 

the 125 ppm group, week 21 in the 2,500 ppm group, 

and week 17 in the 5,000 ppm group (Table 16 and 

Figure 5). Feed consumption by exposed males and 

females was similar to that by controls (Tables I3 

and 14). Dietary levels of 125, 2,500, and 5,OOO ppm 

resultedinaverage daily oxazepam consumption 

levels of 12, 310, and 690 mgkg for males and 15, 

350, and 780 mgkg for females. 

In the 5,OOO ppm groups, lethargy and sedation were 

observed in a few mice during the first week of the 

study. Severalanimals exposed to 5,OOO ppm also 

exhibited decreased spontaneous exploratory behavior 

and impaired locomotor activity. As tolerance to the 

initial depressant effects of oxazepam developed, the 

incidence and severity of clinical findings in exposed 

mice decreased. Behavior and appearanceof exposed 

mice were similar tocontrols by the end of the 

second study week. 

Serum Oxazepam Concentrations 

Serum oxazepam concentrations weremeasuredat 

the 15-month interim evaluation and at the end of 

the study. Dueto excessive mortality, no blood 

samples from males or females exposed to 5,OOO ppm 

wereavailable atheend of the study. Blood 

samples were collected from 2,500 ppm females and 

males during weeks 102 and 103. Serumoxazepam 

concentrationsincreased with increasing exposure 

level in both males and females at the 15-month 

interimevaluationand at the end of 2 years 

(Tables 17and 18). Serum oxazepam concentrations 

weresimilar between males and females ineach 

exposure group. 
Results 49 

WEEKS ON STUDY 

FIGURE5 

Growth Curves €or B6C3F, Mice Administered Oxazepam in Feed €or 2 Years 
50 Oxazepam, NTP TR 443 

in the 2-Year Feed Study of Oxazepama 

Serum Oxazepam Concentrations in B6C3F, Mice at the 15-Month Interim Evaluation 

TABLE 17 

Do= (PPW 0 125 2,500 5,000 

Serum oxazepam (gg/mL) 

Male 

o+o 1.19 + 0.19 5.94 + 1.00 7.09 f 1.66 

Serum oxazepam (gg/mL) 

Female 

o+o 1.19 f 0.08 6.84 f 1.84 10.16 + 2.62 

a Mean + standard deviation for 10 animals 

Serum Oxazepam Concentrations in B6C3F, Mice in the 2-Year Feed Study of Oxazepama 

TABLE 18 

Dose (PPm) 0 125 2,500 5,000 

Serum oxazepam (gg/mL) 020 1.03 2 0.234.08 2 1.20 -b 

Female 

Serum oxazepam @g/mL) Of0 1.00 f 0.28 5.41 f 1.95 -

a Mean + standard deviation for 10 animals 

No samples collected due to100% mortality 
Results 51 

Pathology and Statistical Evaluation 

This section describes the statistically significant or 

biologically noteworthy changes in the incidences of 

nonneoplastic lesions and neoplasms in theliver and 

thyroid gland and nonneoplastic lesions in the testis 

of B6C3F1 mice. Summaries of the incidences of 

neoplasms andnonneoplastic lesions, individual 

animaltumor diagnoses, statistical analyses o f

primary neoplasms that occurred with an incidence of 

at least 5% in at least one animalgroup,and 

historical incidences for the neoplasms mentioned in 

thissection are presented inAppendix C formale 

B6C3Fl mice and Appendix D for female B6C3Fl 

mice. 

Liver: The administration of oxazepam in feed to 

B6C3Fl mice was associated with aspectrum of 

lesions similar to that observed in the Swiss-Webster 

strain. the At 15-month evaluation, interim 

centrilobular hepatocellular hypertrophy occurred in 

all males in the 125,2,500,and 5,OOO ppm groups and 

inall females in the 2,500 and 5,OOO ppm groups 

(Tables 19,20, C5, and D5). At 2 years, incidences 

of centrilobularhepatocellularhypertrophywere 

significantly increased in the 2,500 and 5,OOO ppm 

groups, and the incidence and severity increased with 

increasing dietary concentration. 

The incidences of eosinophilic foci in 2,500 ppm 

males and females and in 5,0oO ppm females and of 

hepatocellular adenoma or carcinoma (combined) in 

2,500 and 5,OOO ppm males and females were also significantly greaterthaninthecontrolsat the 

15-monthinterimevaluation.At 2 years, the incidences of hepatocellular foci (all types) in males were 

lower in the exposed groups than in the controls. 

The incidence of eosinophilic foci in 125 ppm 

females was significantly greater than controls, but 

the incidences in 2,500 and 5,OOO ppm females were 

similar to controls. The unusualdistribution of 

hepatocellular foci (a putative preneoplastic lesion) 

in the exposed groups was attributedtothe 

pronounceddose-relateddevelopment, multiplicity, 

andconfluence of hepatocellularneoplasms in the 

2,500 and 5,OOO ppm groups. 

In the 2-year study, all 2,500 and 5,OOO ppm males, all 

2,500 ppm females, and allbutthree 5,0oO ppm 

females had one or more hepatocellular neoplasms 

andthe incidences of hepatocellularneoplasms in 

these groups were significantly greater than those of 

the controls. In male and female mice, the incidences 

of both hepatocellular adenoma and hepatocellular 

carcinoma in the 2,500 and 5,OOO ppm groups were 

significantly greater than those of the controls; the 

incidence of hepatocellularadenomain 125 ppm 

females was also significantly greater than thatof the 

controls. Hepatoblastoma, a rare phenotypic variant 

of hepatocellular carcinoma, occurred in all exposed 

groups of mice but not in the control groups. Moreover, the incidences of hepatoblastoma increased with 

increasing exposure level, and metastatic foci of 

hepatoblastomaandhepatocellularcarcinomawere 

commonly observed in the lung (Tables C1 and Dl). 

The morphology of the liver neoplasms in B6C3Fl 

mice was similar to that of neoplasms in SwissWebster mice. The cellularcomponent identifying 

theseneoplasmsashepatoblastomas was characterized by sheets of cells, occasionally forming 

rosettes, with a scant vascular stroma. The cells were 

small with scant basophilic cytoplasm and round 

hyperchromatic nuclei, similar to the hepatoblasts of 

the developing fetal liver. This cell population was 

almost always a componentof a larger neoplasm with 

the morphologic characteristics of a typical carcinoma, although in a few, the predominant component 

had the characteristics of an adenoma. 

Samples of liver neoplasms fromeachcontroland 

exposuregroupwerecollectedfor analysis of the 

occurrence of hepatocellularneoplasms with an 

activated H-rus oncogene, andthe mutation spectrum 

of the H-rus geneinthoseneoplasms with an 

activated H-rus was determined(Appendix L). 

Nineteen neoplasms from the control groups,37 from 

the 125 ppm group, and 20 each from the 2,500 and 

5,0oO ppm groups of males and females were 

analyzed. The neoplasms were sampledto provide an 

approximately even distribution of hepatocellular 

adenomasandcarcinomas from each group. The 

mutation spectrum of the H-rus genes in neoplasms 

from exposed mice did not differ from the spectrum 

of mutations observed in neoplasms fromcontrols, 

but the proportion of neoplasmswith an activated 

H-rus decreased with increasingexposure level. 

While 58% of the neoplasms from control mice had 

an activated H-rus, only 1of the 40 neoplasms from 

mice receiving 2,500 or 5,OOO ppm oxazepam exhibitedasimilarmolecular lesion. In the 125 ppm 

group, 35% of the neoplasms had an activated H-rus 

oncogene, suggesting that, although the incidence of 

liver neoplasms was not statistically increased in the 

125 ppm groupcompared to that of the controls, 
52 Oxazepam, NTP TR 443 

TABLE19 

Incidences of Neoplasms and Nonneoplastic Lesions of the Liver of Male B6C3Fl Mice 

in the 2-Year Feed Study of Oxazepam 

15-Month Interim Evaluation 

Live? 

Centrilobular Hypertrophy 

Basophilic Focus 

Clear CellFocus 

Eosinophilic Focus 

Fows (any type) 

Hepatoblastoma 

Hepatocellular Adenoma 

Hepatocellular Carcinoma 

Hepatoblastoma, Hepatocellular 

Adenoma, or Carcinoma 

2-Year Study 

Liver 

Centrilobular Hypertrophy 

Basophilic Focus 

Clear Cell Focus 

Mixed Cell Focus 

Eosinophilic Focus 

Focus (any type) 

Hepatoblastoma 

0vera11 rate' 

Adjusted rated 

Terminal ratee 

First incidence (days) 

Life table testf 

Logistic regression testf 

Hepatocellular Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Hepatocellular Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

10 

0 

0 

0 

0 

0 

0 

0 

0 

0 

49 

0 

2 

13 

2 

18 

27 

0/49 (0%) 

0.0% 

0/45 (0%) 

-E 

Pc0.001 

P<O.001 

17/49 (35 %) 

37.8% 

17/45 (38%) 

729 (r)

Pc0.001 

PCO.001 

9/49 (18%) 

18.7% 

6/45 (13%) 

586 

P<0.001 

PCO.001 

10 

10.. (1.l)b 

0 

0 

1 

1 

0 

3 

1 

3 

50 

2 (2.0) 

1 

6 

1 

12 

16 

2/50 (4%) 

4.5% 

2/44 (5%) 

729 (13

P=O.234 

P=O.234 

18/50 (36%) 

37.4% 

14/44 (32%) 

453 

P=O.472 

P=O.547 

5/50 (10%) 

10.7% 

3/44 (7%) 

453 

P=0.215N 

P=0.107N 

10 

10'. (2.9) 

0 

0 

9.. 

9** 

0 

9** 

41 

9** 

50 

26" (2.4) 

0 

1 

1 

8 

8 

21/50 (42%) 

58.3% 

4/15 (27%) 

598 

PcO.001 

P<O.001 

34/50 (68%) 

87.3% 

11/15 (73%) 

486 

P<O.001 

PiO.001 

45/50 (90%) 

95.7% 

13/15 (87%) 

540 

P<O.001 

P<O.001 

10 

loL*(3.0) 

1 

1 

2 

3 

1 

9** 

9** 

10.. 

50 

43** (3.0) 

0 

0 

0 

8 

8 

13/50 (26%) 

52.9% 

O/O 

434 

Pc0.001 

P=O.O14 

32/50 (64%) 

95.1% 

o/o 

401 

PCO.001 

P=O.003 

50/50 (100%) 

100.0% 

o/o 

401 

P<O.001 

Pc0.001 
Results 53 

TABLE19 

Incidences of Neoplasms and Nonneoplastic Lesionsof the Liver of Male B6C3F, Mice 

in the 2-YearFeed Study of Oxazepam (continued) 

Dose (PPW 0 125 2,500 5,000 

2-Year Study (continued) 

Hepatoblastoma, Hepatocellular Adenoma,or Carcinomah 

Overall rate 23/49 (47%) 19/50 (38%) 50/50 (100%) SOL50 (100%)

Adjusted rate 47.9% 39.5% 100.0% 100.0% 

Terminal rate 20/45(44%) 15/44 (34%) 15/15 (100%) OD 

incidenceFirst (days) 586 453 486 401

test table Life Pc0.001 P=0.315N P<O.001 P<0.001 

PCO.001 Logistictest regression P=0.205N P<O.001 P<0.001

* Significantly different (PSO.05) from the control group by Fisher’s exact test (15-month interim evaluation)

** Significantly different (PSO.01) from the control group by Fisher’s exact test (15-month interim evaluation)or by the logistic 

regression test (2-year study) 

(T)Teminal sacrifice 

a Number of animals with liver examined microscopically 

Averageseverityoflesions in affectedmice: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked 

Number of lesion-bearing animals/number of animals necropsied or examined microscopically 

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

e Observed incidence in animals sutviving until the end of the study 

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

corresponding to pailwise comparisons between the controls and that exposed group. The life table analysis regards neoplasms in 

animals dying prior to terminal kill as being (directly or indirectly) the cause of death. The logistic regression analysis regards 

these lesions as nonfatal. A lower incidence in an exposed group is indicated by N. 

g Notapplicable;noneoplasms in animal group 

Historical incidence for 2-yearNTP feed studies with untreated control groups (mean 2 standard deviation): 485/1,366 

(35.5% -C 14.3%); range lo%&% 
54 Oxazepam, NTP TR 443 

TABLE20 

Incidences of Neoplasms and Nonneoplastic Lesionsof the Liver of Female B6C3Fl Mice 

in the 2-Year Feed Study of Oxazepam 

0 125 2,500 5,000 

15-Month Interim Evaluation 

Live? 10 10 10 10 

Basophilic Focus 

Centrilobular Hypertrophy 

0 

0 

0 

0 

0 

10"(2.7)b 

0 

lo** (3.0) 

Clear Cell Focus 0 0 0 0 

Focus (any type) 

Eosinophilic Focus 

1 

1 

0 

0 

8. * 

8.. 

7.. 

7.. 

Hepatoblastoma 0 0 0 2 

Hepatocellular Adenoma 1 1 9** lo** 

Hepatoblastoma, Hepatocellular 

Hepatocellular Carcinoma 1 0 2 10'. 

Adenoma, or Carcinoma 2 1 9** lo** 

2-Year Study 

Liver 50 50 50 50 

Basophilic Focus 

Centrilobular Hypertrophy 

4 

0 

0 

2 (1.5) 

0 

11.' (2.5) 

0 

29.. (2.9) 

Clear Cell Focus 2 3 0 0 

Mixed Cell Focus 0 0 0 1 

Eosinophilic Focus 9 19' 2 5 

Focus (any type) 14 21 2 5 

Hepatoblastoma 

Adjusted rated 

Overall rate' 

0.0% 

0/50 (0%) 

2.3% 

1/50 (2%) 

31.7% 

8/50 (16%) 

57.8% 

8/50 (16%) 

First incidence (days) 

Terminal ratee 

3

0/39 (0%) 

714 

0/41 (0%) 

614 

on (0%) 

471 

OD 

Life table test' P<O.ool P=O.519 P<0.001 P<O.001 

Logistic regression testf P<O.001 P=O.502 P=O.007 P=O.003 

Hepatocellular Adenoma 

Overall rate 25/50 (50%) 35/50 (70%) 35/50 (70%) 36/50 (72%) 

Adjusted rate 59.3% 79.5% 96.7% 100.0% 

Terminal rate 22L39 (56%) 3U41 (78%) ln (50%) OD 

First incidence (days) 598 471 393 403 

Life table test P<O.ool P=O.o62 P<O.ool P<0.001 

Logistic regression test P<O.ool P=O.O37 P=O.O14 P=O.001 

Hepatocellular Carcinoma 

Overall rate 9/50 (18%) 5/50 (10%) 49/50 (98%) 44/50 (88%) 

First incidence (days) 

Terminal rate 

Adjusted rate 

598 

7/39 (18%) 

21.6% 

714 

4/41 (10%) 

11.9% 100.0% 

393 

2n (100%) 

410

o/o 

100.0% 

Life table test P<O.001 P=0.173N P<O.ool P<0.001 

Logistic regression test P<0.001 P=0.185N P<O.ool P<O.001 
Results 55 

TABLE20 

Incidences of Neoplasms and Nonneoplastic Lesions of the Liver of Female B6C3F, Mice 

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

Do= (PPW 0 125 2,500 5,m 

&Year Study (continued) 

Hepatoblastoma, Hepatocellular Adenoma,or Carcinomah 

Overall rate 28/50 (56%) 36/50 (72%) 50/50 (1 00%) 47/50 (94%) 

Adjusted rate 66.5% 81.8% 100.0% 100.0% 

Terminal rate 25/39 (64%) 33/41 (80%) 2/2 (100%) o/o

First incidence (days) 598 471 393 403

Life table test P<0.001 P=O.126 P<O.001 P<O.001 

Logistic regression test P<O.ool P=O.o84 P<0.001 P<O.001 

* Significantly different (PSO.05) from the control group by Fisher’s exact test (15-month interim evaluation) or by the logistic 

regression test (2-year study) 

** PSO.01 

(T)Terminal sacrifice 

a Number of animals with liver examined microscopically 

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

Number of lesion-bearing animals/numberof animals necropsiedor examined microscopically 

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

e Observed incidence in animals surviving until the end of the study 

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

corresponding to paitwise comparisons between the controls and that exposed group. The life table analysis regards neoplasms in 

animals dying prior to terminal kill as being (directly or indirectly) the cause of death. The logistic regression analysis regards 

these lesions as nonfatal. A lower incidence in an exposed group is indicated by N. 

g Notapplicable;noneoplasms in animal group 

Historical incidence for 2-year NTP feed studies with untreated control groups (mean f standard deviation): 223/1,363 

(16.4% f 10.7%); range 3%-42% 
56 Oxazepam, NTP TR 443 

there was an increaseina subset similar of 

neoplasms (lacking an activated H-rus) that occurred 

with increased incidence at higher exposure levels. 

Thyroid gland: At the 15-month interim evaluation, 

the incidences of follicular cell hyperplasia in male 

and female mice receiving 2,500 and 5,000 ppm were 

significantly greater thanthose of the controls 

(Tables 21, C5, and DS). At study termination, the 

incidences of follicular cell hyperplasia in all groups 

of mice receiving oxazepam were significantly greater 

than thoseof the controls. Follicular cell hyperplasia 

occurredin nearly all mice inthe 2,500 and 

5,OOO ppm groups and in 44% of males and 68%of 

femalesin the125 ppmgroups,while only 8% of 

control males and 32%of control females were similarly affected. The lesion was focal or multifocal in 

distribution,and the extent and severity generally 

increasedwithincreasingexposure level.In female 

mice, follicular cell adenomas occurred with a doserelated positive trend, and the incidences in the 2,500 

and 5,OOO ppm groups were significantly greater than 

that of the control group. However, the incidences of 

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

only marginally greater than the range of follicular 

cell neoplasms inhistoricalcontrol females from 

recent NTP studies (0% to 9%, Table D4). Follicular cell adenomas were observed in one male in 

each exposure group, but not in the controls. 

Testis: There was a dose-related increased incidence 

of testicular atrophy (bilateral) in male mice in the 

2,500 and 5,OOO ppm groups (1/50,0/50,25/50,38/50; 

Table CS). The atrophy was primarily a decrease in 

or an absence of maturation of the spermatogenic 

cells within the tubules. The associated epididymides 

were void of maturespermatozoaandappeared 

shrunken. The shrunken epididymides of the exposed 

males often had small, focal, lymphocytic infiltrates 

(lymphocytic cellular infiltration: 2/50, 14/50, 33/50, 

21/50; Table CS). The testicularatrophyoccurred 

primarily in animals that were moribund or were 

founddead.It is not clear whether oxazepam was 

exerting adirect effect on germ-cell function or 

development, or whetherthis was related to an 

indirect effect of inanition due to oxazepam-induced 

liver neoplasms. The nature of the epididymal 

lymphocytic foci is not clear, but may relate to a 

normalphenomenonthat is made moreapparent 

when the epididymides are shrunken. 

Uterus: The incidence of cystic endometrial hyperplasia in 2,500 and 5,OOO ppm females was lower than 

that of the controls (46/50, 50/50, 19/50, 14/49; 

Table DS). 

Neurobehavioral Evaluation 

Forelimbgripstrength at12monthsin males 

receiving 125, 2,500, or 5,000 ppm and at 18 months 

in males receiving 2,500 or 5,000 ppm oxazepam was 

significantly lower than that of the controls

(Table G19). Hindlimb strength grip was not

affected (Table G20). There were no significant 

differences in forelimb and hindlimb grip strengths in 

females. Motor activity was significantly increased at 

6 and 12 months in males in the 2,500 and 5,OOO ppm 

groups, in 125, 2,500, and 5,000 ppmfemales at 

6 months, and in 2,500 and 5,000 ppmfemales at 

12 months. However, motor activity was significantly 

decreased at 18 monthsin the 5,OOO ppmgroups, 

probably indicating the debilitating effect of 

oxazepam later in the study (Table G21). Response 

to a thermal stimulus as measuredby paw lick latency 

was significantly decreased in 125 ppm males at 

18 months(Table G22). There were no significant 

differences in startle response between exposed and 

control mice. No symptoms of withdrawal were 

notedduringthe 1-week, unexposed observation 

period at the end of the study. 
Results 57 

TABLE21 

Incidences of Neoplasms and Nonneoplastic Lesionsof the Thyroid Gland of B6C3F, Mice 

in the 2-Year Feed Study of Oxazepam 

Male 

15-Month Interim Evaluation 

Thyroid Glanda 

Follicular Cell Hyperplasiab 

Follicular Cell Adenoma 

2-Year Study 

Thyroid Gland 

Follicular Cell Hyperplasia 

Follicular Cell Adenoma 

Female 

15-Month Interim Evaluation 

Thyroid Gland 

Follicular Cell Hyperplasia 

Follicular Cell Adenoma 

2-Year Study 

Thyroid Gland 

Follicular Cell Hyperplasia 

Follicular Cell Adenomad 

Overall ratee 

Adjusted ratef 

Terminal rat& 

First incidence (days) 

Logistic regression testh 

10 

0 

0 

49 

4 (1.0) 

0 

10 

0 

0 

50 

16 (1.4) 

0/50 (0%) 

0.0% 

Oyj9 (0%) -1 

P=0.007 

l Significantly different (PSO.05) from the control group 

10 10 10 

0 5. (1.6)' 7** (1.9) 

0 0 0 

50 50 50 

22.'(1.2) 49** (2.9) 47** (2.4) 

1 1 1 

10 10 10 

0 10"(1.7) lo**(2.2) 

0 0 0 

50 50 50 

34** (1.9) 49.. (3.0) 44** (3.0) 

4/50 (8%) 5/50 (10%) 6/50 (12%) 

9.8% 34.3% 46.1% 

4/41(10%) o n (0%) OD 

735 (T) 663 470 

P=O.O70 P=O.O19 P=O.O17 

by Fisher's exact test (15-month interim evaluation) 

** Significantly different (PSO.01) from the control group by Fisher's exact (15-month interim evaluation) or logistic regression tests 

(2-year study) 

QTerminal sacrifice 

a Numberofmice with thyroid gland examinedmicroscopically 

Number of lesion-bearing mice ' Averageseverityoflesions in affectedmice: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked; 5 = severe 

Historical incidence for 2-year NTP feed studies with untreated control groups (mean f standard deviation): 32/l,348 

(2.4% f 2.8%); range 0%-9% 

e Number of lesion-bearing animals/number of animals necropsied or examined microscopically 

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

Observed incidence in animals surviving until the end of the study 

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

. painvisecomparisonsbetweenthecontrols and that exposedgroup.Thelogisticregressionregardstheselesions as nonfatal. ' Notapplicable;noneoplasms in animal group 
58 Oxazepam, NTP TR 443 

GENETICTOXICITY 

Oxazepam (3 to 3,333 pgplate) did not induce 

mutations in Salmonella ryphimurium strains TA97, 

TA98, TA100, TA102, and TA1535 when tested in a 

preincubationprotocol with or withoutAroclor 

1254-induced male Sprague-Dawley rat or Syrian 

hamster liver S9 (Table El). In cytogenetic tests with 

cultured Chinese hamster ovary cells, oxazepam did 

notinducesisterchromatid exchanges or chromosomalaberrations,with or without S9(Tables E2 

and E3). Peripheral blood samplesobtainedfrom 

B6C3Fl mice in the 14-week toxicity study were 

analyzed for frequency of micronucleatednormochromatic erythrocytes; no increase in micronucleated 

normochromatic erythrocytes was observed at any of 

the exposure levels (Table E4). 

SUPPLEMENTAL STUDIES 

The marked liver neoplasm response to oxazepam 

exposureprompted the performance of supplementaryshort-term studies to further evaluate this 

effect. These studies were performed at the NIEHS 

facility under experimentalconditions described in 

Appendixes M through P. The studieswereconductedaccording to applicablehealth and safety 

requirements and NIEHS and NIH guidelinesfor the 

use of experimental animals. They were not done to 

the standardsof record keeping required under Good 

Laboratory Practice Regulations. 

Studies to Evaluate the Potential for Oxazepam to 

StimulateLiverCellReplication(Appendix M): 

Concentrations of 25, 125, 2,500, and 5,000 ppm 

oxazepam given in feed to male B6C3Fl mice for up 

to 13 weeks caused adose-relatedincrease in the 

nuclear labeling index in studies that measured the 

incorporation of bromodeoxyuridine into replicating 

liver cells. Thisincrease was statistically significant 

at all exposure levels except 25 ppm, and was limited 

to mice evaluated at 15 days. Cell replication rates 

weresimilar to that of controls in mostgroups 

evaluated at 30 days and after. 

Studies to Evaluate thePotential for Oxazepam to 

Produce Necrosis Hepatocellular (Appendir N): 

Clinical pathology and light microscopy evaluations 

wereperformed at 15-day intervals in the study in 

which liver cell replication was examined. There was 

minimal evidence suggestive of hepatocyte necrosis 

either by light microscopy or in clinical chemistry 

measures. There was, however, evidence of 

cholestasis, likely due to physical obstruction of bile 

canaliculi by swollen hepatocytes. 

Studies of Comparative Oxazepam Metabolism and 

Toxicokinetics in B6C3F, and Swiss-WebsterMice 

(Appendixes 0 and P): The metabolicfate and 

toxicokinetics of oxazepam wereevaluated and 

compared to published data from studies in humans. 

Mice andhumans both formglucuronides of 

oxazepam, and form 3- and 4-hydroxy- and methoxyderivatives of the phenyl group. Glucuronidation was 

induced in the mouse with chronic administration. 

Nonetheless, oxidative metabolism of the phenyl 

group, likely through formation of an epoxide, 

appeared to be more prevalent in the mouse than is 

reportedfor humans. A very small amount of 

oxazepam was covalently bound to liver protein 

during metabolism in the mouse. The bioavailability 

of oxazepam from feed was about 40% compared to 

a reported bioavailability of 95% in humans taking a 

therapeutic dose. Elimination half-lives of the parent 

compound did not differ in the Swiss-Webster and 

B6C3Fl mice and were similar to values reported for 

humans. Female mice generally attained slightly 

higher blood oxazepam levels for a given dose of 

oxazepam when compared to males, regardless of the 

route of administration. 
59 

DISCUSSION AND CONCLUSIONS 

The toxicity and carcinogenicity studies of oxazepam 

in Swiss-Webster and B6C3Fl mice were prompted by 

along-standingconcernover the findings of liver 

neoplasms in Swiss-Webster mice in studies in which 

relatively few animals were given oxazepam in the 

diet for less than 12 months(Fox and Lahcen, 1974). 

The Swiss-Webster mouse is not generally used in 

rodent carcinogenicity studies becauseof its short life 

spanand becausehistoricaldata to assist in the 

interpretation of findings are limited. Therefore, the 

current studies were performed with both strains of 

mice in an attempt to confirm the prior results and 

to allow for acomparison of potential neoplasm 

responses between the Swiss-Webster mouse and the 

more commonly used B6C3Fl strain. 

At the time of these studies, there were no data in 

the literature on which to base an estimated maximum exposure level for mice; however, Owen et nl. 

(1970) had reported that2 of 20 Sprague-Dawley rats 

died while receiving 5,0oO ppm oxazepam in the diet 

for 6 weeks. Therefore, in the 14-week studies, 

10,000 ppm was selected asthe highest concentration. 

One male B6C3Fl mouse in the 10,000 ppm group 

diedbecause of aurinarytractinfection,and one 

female Swiss-Webster mouse in the 10,000 ppm 

group died, presumably because of exposure to the 

chemical. The early deaths did not occur during the 

first week, when clinical findings indicating sedation 

andlethargywere attheir maximum in males and 

females of each strain. The mice were able to adapt 

tothe pharmacologicaction of oxazepam by the 

second week of the study and appeared clinically 

normal thereafter. 

In neurobehavioral assessments, oxazepam was found 

to produce two primary effects in the 14-week 

studies. The first effect was a transient reduction in 

gripstrengththat was considered to result from a 

nonspecific muscle relaxant or depressant effect. This 

was seen primarily in males of each strain and may be 

a manifestationof the sedative effects noted clinically 

duringthe early part of the study. An antianxiety 

effect, similar tothat commonly seen in animal 

studies with otherbenzodiazepine drugs, was inferred 

from findings of facilitated motor activity, enhanced 

startle response, and decreased paw lick latencies 

(Crawley and Goodwin, 1980; Freeman and

Thurmond, 1985). There were minor differences 

between the sexes and strains in some aspects of the 

neurobehavioral findings, but the general responses 

tothe drugweresimilar and followed predicted 

patterns. 

In general, exposed mice gained as much or somewhat more weight during the 14-week studies than 

did controls. Thus, body weight gain was not a factor 

in the selection of exposure levels for the chronic 

studies. The only consistent difference in organ 

weights was a dose-related increase in absolute and 

relative liver weights. The absolute and relative liver 

weights of males and females of each strain exposed 

to 10,000 ppm were nearly double those of the 

controls. Microscopically, centrilobular hepatocellular hypertrophy was observed in each strain with 

only minor evidence of focal necrosis in 

Swiss-Webster mice. Although no clinical chemistry 

studieswereperformed with theseanimals, in 

supplemental studies of a similar design with B6C3Fl 

mice, outlined in Appendix N, there was evidence for 

cholestasis, which was considered a secondary effect 

ofphysical obstruction of bile canaliculi by the 

hypertrophic hepatocytes. There was no clinical or 

microscopic evidence to suggest significant toxicity to 

hepatocytes in the B6C3Fl mice, and this was consistent with the findings in the core study. 

Increased liver weights have frequently been reported 

in many rodent studies with benzodiazepines (Owen 

et al., 1970; Kitagawa et nl., 1974; Scrollini et nl., 

1975; Irikura et ai., 1977), and the histopathologic 

appearance of the livers is typically described as 

normal,althoughhepatocyte swelling was noted by 

Irikura et nl. (1977). Diwan et al. (1986) reported 

hepatomegaly in B6C3Fl mice receiving 1,500 ppm 

oxazepam or diazepam in the dietfor 53 weeks. 

Total cytochrome-P-450 and the activity of aminopyrine N-demethylase wereincreasedin livers of 

exposed animals. Proliferation of smoothendoplasmic reticulum andenhanced sterol metabolism 
60 Oxazepam, NTP TR 443 

have been reported in studies of liver biopsies taken 

fromhumansthat received therapeutic doses of 

diazepam (Jezequel, 1974; Orlandi et af., 1975). 

A high dose of 5,OOO ppm was chosen for each strain 

of mice for the chronic studies because an increasein 

liver weight of more than about70% was thought to 

be potentially life threatening. In the chronic studies, 

the survival of both strains and mean body weight 

gains of male and female B6C3Fl mice exposed to 

2,500 and 5,OOO ppm were markedly lower than those 

of the controls. The body weight depression of all 

exposed femaleB6C3Fl mice and of 2,500 and 

5,OOO ppm B6C3Fl males could not be predicted 

based onthe body weight gains in the 14-week 

studies. Survival of 125ppmB6C3Fl mice was 

similar to that of the controls. Mice exposed to 

5,OOO ppmalsohad clinical findings indicating 

sedationand lethargy early in the study but then 

appeared clinically normal. The only clear clinical 

findings attributed to oxazepam during the later part 

of the study were abdominalswelling, presumably due 

to liver hypertrophy and neoplasia, and a spectrum of 

findings typical of moribundanimalsas the liver 

neoplasms progressed. 

Neurobehavioral evaluations were performedon both 

strains at 6-month intervals throughout the chronic 

studies. The purpose of these studies was to determine if there were any prominent changes in the 

character of the behaviors or the extent of the 

responses exhibited by the mice. Unfortunately, the 

early deaths preventedarigorousevaluation of 

behavior beyond about 12months,and effects on 

behaviorsecondary to neoplasia and reduced body 

weight also confounded interpretation of the studies. 

However, within theselimitations, there were no 

noticeable changes in behavior patternsof mice from 

those noted in the 14-week studies, and no new or 

unusualbehaviorsdevelopedduring thechronic 

studies. One notable strain difference was observed 

and involved the disinhibitory effect of oxazepam on 

motor activity. Swiss-Webster mice appeared to 

adapt to this pharmacologic effect very early in the 

study, while the effect persisted in B6C3Fl mice for 

at least 12 months. 

There were several lesions attributed to oxazepam in 

the pathologic evaluation of the tissues of mice. One 

of these effects was limited to Swiss-Webster mice 

and involved increased incidences of systemic amyloid 

depositionin tissues. Swiss-Webster mice are very 

susceptible to the formation of amyloid deposits, 

which are thought to contribute tothe relatively 

short average life span of this strain. However, the 

extent and severity of the lesions were increased in 

mice receiving oxazepam, and amyloid deposits inthe 

heart likely contributed to pulmonaryhypertension 

leading to edema and heart failure, which was considered the likely cause of death of many of these mice. 

Little is known about the similarities in etiology, if 

any, between amyloidosis in the Swiss-Webster mouse 

and the formation of amyloid plaques in the central 

nervous system (CNS) of humans with Alzheimer’s 

disease. There have been no reportsassociating 

benzodiazepine use with Alzheimer’s disease. 

Testicularatrophy was observed in B6C3Fl mice 

exposed to 2,500 and 5,000 ppm oxazepam. This 

occurred primarily in animals that were moribund or 

were found dead. It was not clear whether oxazepam 

was exerting a direct effect on germ cell function or 

development or whether this was an indirect effect of 

inanition due to oxazepam-induced liver neoplasms. 

There is support for both effects in the literature 

(i.e., treatment of Sprague-Dawley rats for 14 days 

with diazepam at 3 mgkg resulted ina significant 

reduction in serum testosterone concentrations;Calvo 

et af., 1991). Also, CD-1 mice held to 70% of control 

body weight by restricted feeding had tubular degeneration and atrophy inthe testis (Chapinet af.,1993). 

A third effect was hepatocellular hypertrophy and 

increased incidences of liver neoplasms in male and 

female mice of eachstrain.Centrilobular hypertrophy was diagnosed in the majority of exposed 

Swiss-Webster mice and the increased incidences 

weredoserelated in the B6C3Fl mice. Inmany 

instancesthisnonneoplasticlesion was difficult to 

diagnose in mice in the 2,500 and 5,OOO ppm groups, 

because there was little or no liver which was not 

part of neoplasm. this a Nonetheless,lesion 

appearedsimilar to those observed in the 14-week 

studies. 

The incidence of liver neoplasia was markedly 

increased by oxazepam exposure in male and female 

mice of each strain. In Swiss-Webster mice receiving 

2,500 or 5,000 ppm, there were increases in the 

degree of neoplasticresponse that were related to 

dose. The number of mice with carcinomas rather 

thanadenomas,and with multipleadenomas or 

carcinomas rather than single neoplasms, was higher 

in the 5,000 ppm groups than in the 2,500 ppm 
Discussion and Conclusions 61 

groups. There were also a large number of 2,500 and 

5,OOO ppmB6C3Flmicethatdevelopedmultiple 

adenomas and carcinomas, and these groupsof mice 

alsodeveloped a significantnumberofhepatoblastomas, a relatively unusual phenotypic variant of 

hepatocellularcarcinoma(Diwan et aL, 1992). The 

lower incidence of hepatoblastomas in the5,000 ppm 

groups versus the 2,500 ppm groups may have been 

due to the shorter survival of the 5,000 ppm mice. 

The overall numerical expressionof liver neoplasia in 

B6C3Flmicewasomewhathigherthanin the 

Swiss-Webstermice at comparable exposure levels, 

buthis was probablyinfluenced by the shorter 

duration of the Swiss-Webster mouse study. 

Althoughtherewasnotastatisticallysignificant 

respects, the hepaticneoplasmsandmetastases 

induced by oxazepam were considered responsible for 

the high rate of early mortality seen in B6C3Fl mice 

and may have contributed to early deaths in SwissWebster mice. 

In exposed B6C3Flmice, there was a marked increase 

in follicular cell hyperplasia of the thyroid gland at 

all exposure levels, and follicular cell adenomas were 

significantly increased inthe 2,500 and 5,OOO ppm 

groups of female mice. A relationshipbetween 

induction of hepatic microsomal enzymes and altered 

thyroidfunctionleadingtoneoplasiahasbeen 

demonstrated in the rat, and a hypothesis has been 

developed to accountfor these findings(McClain, 

1989; McClain et af., 1989). This involves induction 

increase in the overall liver neoplasm incidence ino fthetheglucuronidation activity inthe liver for 

B6C3Fl mice exposed to 125 ppm compared to the 

incidence in controls, the incidence in females (36/50, 

72%) was high considering that these mice weighed 

5 to 7 g less than the controls.As noted by Haseman 

(1992), there is a positive linear relationship between 

the maximum weekly average body weight and the 

ultimateincidenceofliverneoplasmsincontrol 

female B6C3Fl mice. The typical incidence in control 

female B6C3Fl mice that achieved a maximum body 

weight of47.5 g (similar to the maximumbody weight 

of the 125 ppm group) during 2-year studies is 25%. 

Also, two malesandonefemalein the 125ppm 

grouphadahepatoblastoma. No hepatoblastomas 

wereobservedinthecontrolsinthisstudy,and 

historically, none were found in 1,366 control male 

mice and only onewas found in 1,363female control 

mice, again indicatingthat this is an unusualneoplasm type and that its occurrence is likely chemical 

related. 

Diwan et al. (1989) noted that formation of hepatoblastomasin hybrid D2B6Flmiceinitiatedwith 

N-nitrosodiethylamineandgivenO.O5%phenobarbital 

indrinkingwaterwasincreasedoverthatin mice 

receivingonlytheinitiator. They alsoshowedthat 

this response was different in different strains,or in 

hybrids of the same strains but from matings of the 

opposite sex. This finding is somewhat similarto the 

present studies with oxazepam. Historically, phenobarbital alone had been thought to causeincreased 

incidencesofhepatocellularadenomasandcarcinomas,whichhadalowmetastaticpotentialand 

wouldnotresultinincreasedmortality(McClain, 

1990). Although the responses of the mouse liver to 

phenobarbital and oxazepam appear similar in many 

thyroxine, causing enhanced biliary excretion. This 

leads to a persistent increase in thyroid-stimulating 

hormone levels, which fosters increasedthyroid 

follicularcell and hyperplasia neoplasia. 

Comparative studies, in metabolism outlined 

Appendix P, indicated that glucuronidation is a major 

metabolic pathway for oxazepam,andinduction of 

this activity occurswithrepeateddosing.Naive 

Swiss-Webster mice rely onglucuronidationfor 

elimination of oxazepam to a somewhat lesser extent 

than do B6C3Fl mice, but clearly, induction of 

glucuronidation activity is seenwitheachstrain. 

Thus, it appears that further study of the effects of 

repeatedxposure to oxazepamonthyroxine 

metabolism in these two strains of mice is needed to 

evaluate whether this hypothesis can account for the 

sex and strain specificity noted forfollicular cell 

adenomas in these studies. 

Prior studies of the potentialcarcinogenicity of 

benzodiazepines (reviewed in the Introduction) have 

provided somewhat mixed results, with no suggestion 

of a positive response comparablein magnitude to 

that seen in the liver in the current studies. Assays 

of the potentialforbenzodiazepines to causegene 

mutation or otherforms of geneticdamageare 

typically negative (Carlo et af., 1989), and this 

includes the findingsinthisreportwithoxazepam 

(Appendix E). However, there have been numerous 

reports thatthe benzodiazepines can act as neoplasm 

promoters in various tissues (see Introduction), and 

oxazepam was clearly shown to enhance the liver 

neoplasm response of B6C3Fl mice given an initiating dose ofN-nitrosodiethylamine(Diwan et af., 

1986). Therefore, a number of studies supplemental 
62 Oxazepam, NTP TR 443 

to the current studies were performed in an attempt 

to more fully describe the effects of oxazepam on the 

liver in these strains of mice and to characterize some 

of the molecular changes in the neoplasms from these 

animals. These are described in moredetailin 

Appendixes L through P. 

Dietary administration of oxazepam to male B6C3Fl 

mice was found to induce a transient increase in liver 

cell replication (Appendix M). The increase was 

clearly evident only at the 15-day time point in the 

125,2,500 and5,000ppm groups, although there was 

asuggestion of an increase at 25 ppm, a lower 

exposure level than any used in the chronic studies. 

This response is similar to that observed in phenobarbital studies. Smith et ul. (1991) noted a liver cell 

labeling index of 30% in male CD-1 mice receiving 

0.1% phenobarbital in the diet for 1week versus 2% 

incontrols.However, by 5 weeks of exposure the 

labeling indices were not different from those of the 

controls.Similar findings of a lack of evidence for 

sustained increased liver cell replication were noted 

by Ward et ul. (1988) in their study of male B6C3Fl 

mice exposed for 40 weeks to 500 ppm phenobarbital 

in drinking water. 

The neoplasms observed in the livers of the BGC3F, 

mice in the 2-year study of oxazepam were sampled 

and analyzed for the frequency and mutation spectrum of activatedH-rusoncogenes(AppendixL). 

Oncogenes are a large groupof genes whose products 

play a roleinthecontrol of cell replication and 

differentiation, and when alteredthroughpoint 

mutations or rearrangement, or when overexpressed, 

are thought tocontribute to neoplasmformation 

(Travali et al., 1990). The H-ras oncogene is 

frequently found activated in mouse liver neoplasms, 

and the frequency of the occurrence andthe positions 

of point mutations found in thesegenes, which result 

in activation of the oncogene, have been 

characterized and used to distinguish between 

B6C3Fl mice given phenobarbital. The absence of 

mutated H-rus genes supports the notionthat 

oxazepam and phenobarbital are not potentmutagens 

and suggests that these agents foster an environment 

that favors the developmentof, or "promotes," 

neoplasmsthat do not express thisgenetic lesion. 

Approximately 20% to 30% of the liver neoplasms 

that develop in the control male BBBF, mouse do 

not have an activated H-rus oncogene (Fox et al., 

1990). Nearly all of the neoplasmsfoundin the 

livers of mice receiving 2,500 or 5,000 ppm lacked an 

activated H-rus, and 65% of the neoplasms analyzed 

in the 125 ppm group also lacked a mutated form of 

this gene. The implication of this is that although 

the liver neoplasm incidence was not statistically 

differentbetween the controls and the 125ppm 

group, oxazepam was able to increase the incidence 

of neoplasms formed that lacked an activated H-rus 

oncogene.Withincreasedexposure, there is an 

apparent increased suppression of neoplasms that 

contain an activated oncogene. 

As previously indicated, the intention of the 125 ppm 

exposure level was to produceaserumoxazepam 

concentrationclose to the therapeuticrangefor 

humans, which is about 0.3 to 1 pg/mL (Greenblatt 

et af., 1980; Salzman et al., 1983). Exposure to 

125 ppm resulted in serum or plasma oxazepam concentrations of about 1 pg/mL in the 2-year B6C3Fl 

studyandin the toxicokineticstudiesoutlined in 

Appendix 0. However, using different analysis 

methods,serum levels were determined to be from 

2 to about 6 pg/mL in mice exposed to 125 ppm in 

the cell replication studies(Appendix M). The 

reason for this discrepancy is not clear. Nonetheless, 

the results of the cell replication and neoplasm 

oncogene studies indicate that oxazepamis capable of 

exerting an influence on neoplasm development in 

the liver at exposure levels which result inblood 

oxazepam concentrations that are not very different 

from those typically achieved in humans taking the 

chemical-induced neoplasms and neoplasms that arisedrug. 

"spontaneously" in controls (Goodman et ul., 1991). 

While the mutation spectrum of the H-ras gene in 

neoplasmsfrom exposed mice (primarily from the 

125 ppm group) did not differ from the spectrum in 

neoplasmsfromcontrol mice, the incidence of 

neoplasms with an activatedH-rasgene declined 

dramatically with increasing exposure level. These 

findings arequite similar to thosereported. by 

Fox et ul. (1990) in their study of the frequency of 

liver neoplasms with activated H-ras oncogenes in 

This apparent similarity in exposure levels does not 

apply when considering the totalexposure level 

required to achieve plasma levels. these The

estimated average exposure level for the BGC3F, mice 

exposed to oxazepam in feed at 125 ppm was between 

10 and29 mg/kg per day over the course of the 

2-year study. A typical adult human therapeutic dose 

is 15 to 30 mg(0.2 to 0.4 mg/kg) taken asneeded 

(Goodnzan and Gifnlan's, 1990). While the half-life 
Discussion and Conclusions 63 

for elimination from the blood is similar in humans 

and in mice, the total amount of drug handledby the 

liver is proportionatelylargerinmiceconsuming 

125 ppminthe feed thaninhumanstakinga 

therapeutic dose, even considering when the

differencesinbioavailability(Appendix 0). The 

disproportionality between the amount of oxazepam 

consumed and the resulting blood concentrations is 

evenmorepronouncedatthehigheroxazepam 

concentrations used in these feed studies. 

A comparisonofthemetabolic pathways for 

oxazepamfortheSwiss-Websterand the B6C3Fl 

mouse with that reported for humans is outlined in 

Figures P2 and P3. As discussed in Appendix P, the 

primaryhumanin pathway theinvolves 

glucuronidation of the parent molecule and excretion 

in the urine. Glucuronidationof oxazepam or of oxyor methoxy-derivativesis also a major pathway inthe 

Swiss-Websterand mouse, B6C3F, and 

glucuronidation is enhanced with repeated exposure. 

There is evidenceforoxidativemetabolism of the 

phenyl group, particularly at the3 and 4 positions, in 

the human as well as in the mouse, and this activity 

accounts for alargerfraction of the administered 

material in the mouse under the conditions studied. 

Formation of the 3- and 4-oxy derivativessuggests 

thatapotentiallyreactive 3,4-epoxide may be an 

intermediate, and may account for thefact that some 

of the radiolabeled material cannotbe extracted from 

liver proteins. Whetheroxidativemetabolism is in 

any way involved in the liver neoplasms seen inthese 

studies has not been determined. 

Many aspects of the neoplasms and other responses 

of mice to oxazepam resemble those reported with 

phenobarbital.Benzodiazepinesandphenobarbital 

are known to modulate nervous impulsesin the CNS 

through interactions with independent receptors on 

GABAergicneurons (see Introduction).Thereare 

benzodiazepinereceptorsin the peripheral tissues 

includingthe liver (Olsen et al., 1986). Whether 

activation of the peripheral benzodiazepine receptor 

influences the neoplastic process, orif phenobarbital 

may also activate this receptoror influence the same 

biological processes, may be fruitful areasof research. 

Phenobarbital is currently considered an agent that is 

"possibly carcinogenic to humans" by the International Agency for Research on Cancer(IARC), 

basedprimarily on evidence of increasedhepatocellular neoplasms in experimental studies with rats 

andmice(IARC,1987).There has beennoclear 

evidencelinking liver neoplasms inhumanswith 

phenobarbital exposure, although some authors have 

attributed an increase in CNS neoplasms in selected 

patient populations to the drug, while other studies 

haverejectedthislink(ClemmesenandHjalgrimJensen, 1978; Gold et al., 1978; Olsen et al., 1989). 

The IARC has reported that there is inadequate evidence to determine whether diazepamis carcinogenic 

to humans orto experimental animals (IARC, 1987). 

CONCLUSIONS 

Under the conditions of these feed studies, there was 

clear evidence of carcinogenic activity*of oxazepam in 

maleandfemaleSwiss-Webstermicebasedon 

increased incidences of hepatocellular adenoma and 

carcinoma. There was clear evidence of carcinogenic 

activizy of oxazepam in male and female B6C3Fl mice 

based on increased incidencesof hepatoblastoma and 

hepatocellularadenomaandcarcinoma.Increased 

incidences of hyperplasia of thyroid gland follicular 

cellsin maleandfemaleB6C3Flmiceand of 

follicular cell adenomas in female B6C3F, mice were 

also related to oxazepam exposure. 

Administration of oxazepam to Swiss-Webster mice 

resulted in centrilobular hepatocellular hypertrophy 

andincreasedincidencesandseverity of systemic 

amyloidosis. Administration of oxazepam to B6C3Fl 

micealsoresultedincentrilobularhepatocellular 

hypertrophy. 

* Explanation of Levels of Evidence of CarcinogenicActivity is onpage 10. A summary of theTechnical Reports Review 

Subcommittee comments and the public discussion on this report appear on page 12. 
64 Oxazepam, NTP TR 443 
65 

REFERENCES 

AmericanConference of CovernmentalIndustrial 

Hygienists (ACCIH) (1987). Threshold Limit Values 

and Biological Exposure Indices for 1987-88. 

Cincinnati, OH. 

~ O ~ ~ (1986). O S U Top 200 Drugs of 1985: A 1.4%

increasein refills nudges 1985 Rxs 1.1%aheadof 

1984 volume. Bham. Times 52, 25-33. 

Armitage, P. (1971). Statistical Methods in Medical 

Research, pp. 362-365. John Wiley and Sons, 

New York. 

Ahby, J., andTennant, R.W. (1991). Definitive 

relationshipsamongchemicalstructure,carcinogenicity, and mutagenicity for301 chemicals tested by 

the US. NTP. Mutat. Res. 257, 229-306. 

Balbi, A, Muscettola, G., Staiano, N., Martire, C., 

and De hrenzo, F. (1980). Psychotropic drugs: 

Evaluationofmutagenic effect. Phamzacol. Res. 

C~mmun.12,423-431. 

Barnard, EA, Stephenson, EA., Sigel, E., 

Mamalaki, C., and Bilbe, G. (1984). Structureand 

properties ofthe brain CAl3Abenzodiazepine

receptor complex. Adv. fip. Med. Biol. 175,235254. 

Batzinger, W.P., Ou, S.-Y.L.,and Bueding, E. (1978). 

Antimutageniceffects of 2(3)-tert-butyl-4-

hydroxyanisoleand of antimicrobialagents. Cancer 

Res. 38, 4478-4485. 

Baum, C. (1986). Memo to A. Wilk,FDA. 

Bignami, M.,Morpurgo, G.,Pagliani, R., Carere, A., 

Conti, C., andDiGiuseppe, G. (1974). Nondisjunctionandcrossing-overinduced by pharmaceutical drugs inAspergillus nidulans. Mutnt. Res. 26, 

159-170. 

Boorman, G.A., Montgomery, C.A.;Jr., Eustis, S.L., 

Wolfe, M.J., McConnell, E.E., andHardisty, J.F. 

(1985). Qualityassuranceinpathologyforrodent 

carcinogenicity studies. In Handbook of Carcinogen 

Testing (H.A. Milman and E.K. Weisburger, Eds.), 

pp. 345-357. Noyes Publications, Park Ridge, NJ. 

Bregman, A.A. (1970). Invirro effectsoftranquilizers on human chromosomes. EMS Mewsl. 3, 41. 

Calvo, D.J., Campos, M.B., Calandra, R.S., 

Medina, J.H., and Ritta, M.N. (1991). Effect of long 

termdiazepamadministration on testicularbenzodiazepine receptors and steroidogenesis.Life Sci 49, 

519-525. 

Carlo, P., Finollo, R., Ledda, A., and Brambilla, C. 

(1989). Absence of liver DNA fragmentation in rats 

treatedwithhighoral doses of 32 benzodiazepine 

drugs. Fundam. Appl. Toxicol. 12,34-41. 

Chapin, R.E., Gulati, D.K., Fail, P.A., Hope, E., 

R u s s e l l ,S . R . ,H e i n d e l , J.J., G e o r g e ,J . D . , 

Grizzle, T.B., and Teague, J.T. (1993). The effects of 

feed restriction on reproductivefunctionin Swiss 

CD-1 mice. Fundam. Appl. Toxicol. 20, 15-22. 

Clemmesen, J., andHjalgrim-Jensen, S. (1978). Is 

phenobarbital carcinogenic? A follow-up of

8078 epileptics. Ecotoxicol. Environ. Safe9 1, 

457-470. 

Code of Federal Regulations (CFR) 21, Part 58. 

Committeeon the Review of Medicines (CRM) 

(1980). Systematic review of the benzodiazepines. 

Guidelinesfordata sheets on diazepam,chlordiazepoxide,medazepam,clorazepate,lorazepam, 

oxazepam,temazepam,triazolam,nitrazepam,and 

flurazepam. Br. Med. J. 280, 910-912. 
66 Oxazepam, NTP TR 443 

Cooper, J.R., Bloom,F.E.,and Roth, R.H., Eds. 

(1978). The Biochemical Basisof Neuropharntacology, 

3rd ed. Oxford University Press, New York. 

Cox, D.R. (1972). Regression models and life tables. 

J. R Stat. Soc. B34, 187-220. 

Crawford, B.D. (1985).Perspectives on the somatic 

mutation model of carcinogenesis. In Advancesin 

ModernEnvironmentalToxicology:Mechanisntsand 

Toxicity of ChemicalCarcinogens and Mutagens 

(MA Mehlman, W.G. Flamm andR.J. Lorentzen, 

Eds.), pp. 13-59, Princeton Scientific Publishing Co., 

Inc., Princeton, NJ. 

Crawley,J.,andGoodwin,F.K.(1980).Preliminary 

report of a simpleanimalbehaviormodelfor the 

anxiolyticeffects of benzodiazepines. Pharnzacol. 

Biochent.Behav. 13, 167-170. 

Crebelli,R., Conti, G., Conti, L., and Carere, A. 

(1989). The E.E.C. genomicmutationprogram: 

Results with Aspergillusnidulans.Environ. Mol. 

Mutagen. 14 (Suppl. 15), 41-42. 

Crebelli,R., Conti, G., Conti. L., and Carere, A. 

(1991). In vitro studieswith nine known or suspected 

spindle poisons: Results in testsforchromosome 

malsegregationinAspergillus nidulans. Mutagenesis6, 

131-136. 

Degraeve, N., Chollet, C., Moutschen, J., 

Moutschen-Dahmen, M., and Gilet-Delhalle, J. 

(1985).Investigation of the potential mutagenic 

activity of benzodiazepines in mice. Mum. Res. 147, 

290. 

De Iglesia,Barsoum,Gough, la A., N., A.,

Mitchell, L., Martin, R.A., di Fonzo, C., and 

McGuire,E.J.(1981).Carcinogenesisbioassay of 

prazepam (Verstran) in rats and mice. Toxicol. Appl. 

Pharntacol. 57, 39-54. 

Dinse,G.E., and Lagakos, S.W. (1983). Regression 

analysis of tumour prevalence data. Ayyl. Sratisf. 32, 

236-248. 

Dinse,G.E.,andHaseman,J.K.(19S6).Logistic 

regressionanalysis of incidental-tumor data from 

animalcarcinogenicityexperiments. Fundanl. Appl. 

Toxicol. 6, 44-52. 

Diwan,B.A.,Rice,J.M., and Ward,J.M.(1986). 

Tumor-promoting activity of benzodiazepine tranquilizers,diazepamandoxazepam, in mouseliver. 

Carcinogenesis 7, 789-794. 

Diwan,B.A.,Ward,J.M.,andRice,J.M.(1989). 

Promotion of malignant "embryonal" liver tumors by 

phenobarbital:Increasedincidenceand shortened 

latency of hepatoblastomas in (DBAD X C57BL/6)Fl 

mice initiated with N-nitrosodiethylamine. 

Carcinogenesis 10, 1345-1348. 

Diwan,B.A.,Ward,J.M.,andRice,J.M.(1992). 

Origin and pathology of hepatoblastoma in mice. In 

TheRole of CellTypesinHepatocarcinogenesis 

(A.E.Sirica,Ed.), pp. 71-87. CRC Press, Boa 

Raton, FL. 

Dunnett, C.W. (1955). A multiple comparison procedure for comparing severaltreatments with a control. 

J. Ant. Stat. AS.SOC. 50, 1095-1121. 

Eadie, M.J. (1984).Currentlyavailablebenzodiazepines. Med. J. Aust. 8, 827-832. 

Finkle, B.S., McCloskey,K.L.,and Goodman, L.S. 

(1979). Diazepam and drug-associated deaths. JAW 

242, 429-434. 

Florey, K. (Ed.) (1974). AnalyticalProfiles of Drug 

Substances. Vol. 3, pp. 442-464.AcademicPress, 

New York. 

Fox,K.A., and Lahcen, R.B. (1974).Liver-cell 

adenomas and peliosis hepatis in mice associatedwith 

oxazepam. Res.Contntun.Chent. Parhol. Pharmacol. 

8, 481-488. 

Fox, T.R., Schumann, Watanabe, A.M., P.G.,

Yano, B.L., Maher, V.M., andMcCormick,J.J. 

(1990). Mutational analysis of the H-ras oncogene in 

spontaneous C57BL/6 x C3H/He mouse liver tumors 

and tumors inducedwith genotoxic and nongenotoxic 

hepatocarcinogens. Cancer Res. 50, 4014-4019. 

Freeman,G.B., and Thurmond, J.B. (1985).Brain 

aminesandeffects of chlordiazepoxide on motor 

activity in response to stress. Pharnzacol.Biochenz. 

Behav. 22, 665-670. 
Galloway, S.M., Armstrong, M.J., Reuben, C., 

Qlman, S., Brown, B., Cannon, C., Bloom, AD., 

Nakamura, F., Abed, M., Duk, S., Wimpo, J., 

Margolin, B.H., Wesnick, MA, Anderson, B., and 

Zeiger, E. (198’7). Chromosomeaberrationsand 

sister chromatid exchanges in Chinese hamster ovary 

cells: Evaluationsof 108 chemicals. Environ. Mol. 

Mulagen. B@ (Suppl. IO), 1-175. 

Gat, J.J., Chu, KC., andTarone, R.E. (19’79). 

Statistical issues in interpretationof chronic bioassay 

tests for carcinogenicity. J. Nad. Cancer Inst. 62, 

997-9743. 

Gold, E., Gordis, L.,Tonascia, J., andSzklo, M. 

(1978). Increased risk of braintumorsinchildren 

exposed to barbiturates. J. Marl. Cancer Znsr. 61, 

1031-1034. 

Goodman and Gilntan ’s The Pharnlacological Basis of 

Therapeutics (1990). 8th ed. (A.6. Gilman,

T.W. Ball, A.S. Nies, and P. Taylor, Eds.). Pergamon 

Press, New York. 

Goodman, J.I., Ward, J.M., Pogp, J.A., Klaunig, J.E., 

and Fox, T.R. (1951). Mouse liver carcinogenesis: 

Mechanismsand relevance. Fundam. Appl. Toxicol. 

17, 651-665. 

Qreenblatt, D.J., Shader, and (1978). R.I. 

Pharmacokineticunderstanding of antianxietydrug 

therapy. South. Med. J. 71, 2-9. 

Qreenblatt, D.J., Divoll, M., Harmatz, J.S., and 

Shader, R.I. (1980). Qxazepamkinetics,effects of 

age and sex. J. Pharnlacol. Bp. Ther. 215, 86-91. 

Hartz, S.C., Heinonen, Q.P., Shapiro, S., Siskind, V., 

andSlone, ID. (1975). Antenatal exposure to 

meprobamateandchlordiazepoxideinrelation to 

malformations, mental development, and childhood 

mortality. N. En@. J. Med. 292, ’726-728. 

Haseman, J.K. (1984). Statistical issues in the design, 

analysis and interpretation of animal carcinogenicity 

studies. Environ. Health Perspect. 58, 385-392. 

Haseman, J.K. (1992). Value of historical controls in 

the interpretation of rodent tumor data. Drug In$ J. 

26, 191-20. 

Haseman, J.K., Huff, J., and Boorman, G.A. (1984). 

Use of historicalcontroldataincarcinogenicity 

studiesinrodents. Toxicol. Pathol. 12, 126-135. 

Haseman, J.K., Huff, J.E., Rao, G.N., Arnold, J.E., 

Boorman, G.A., and McConnell, E.E. (1985).

Neoplasms observed in untreated and corn oil gavage 

controlgroups of F344/N rats and (C57BL/6N x 

C3H/HeN)F, (B6C3Fl) mice. JNCZ 75, 975-984. 

Hollander, M., Wolfe, and D.A. (1973).

Nonparantetric Statistical Methods, pp. 120-123. 

John Wiley and Sons, New York. 

Horrobin, D.F., Ghayur, T., andKarmali, R.A. 

(1979). Mindandcancer. Lancet 1, 978. 

Wsu, T.C., Liang, J.C., and Shirley, L.R. (1983). 

Aneuploidyinduction by mitoticarrestants.Effects 

of diazepam on diploid Chinese hamstercells. Mutat. 

Res. 822, 201-209. 

Husum, B., Wulf, H.C., Wiebuhr, and E.,

Rasmussen, J.A. (1985). SCE in lymphocytes of 

patients treated with single, largedoses of diazepam. 

Mutat. Res. 155, 71-73. 

IntegratedLaboratory Systems(ILS)(1990). P.O. 

Box 13501, Research Triangle Park, NC 27707. 

International Agency for Research on Cancer (IARC) 

(1987). IARCMonographson the Evaluation of 

CarcinogenicRisks to Humans,Supplement 7. 

World Health Organization, United Kingdom. 

Irikura, T., Sugimoto, T., Shoji, S., andAijima, H. 

(1977). Acute, subacute, and chronic toxicity studies 

on flurazepam free base. Oyo Yakuri 4, 637-657. 

Ishidate, M., Jr., Hayashi, M., Sawada, M., 

Matsuoka, K., Yoshikawa, K., Qno, M., and 

Nakadate, M. (1978).Cytotoxicity test on medical 

drugs - Chromosome aberration tests with Chinese 

hamster cells in vitro. Eisei Shikenjo Hokoku 96, 

55-61. 

Ishimura, K., Sawai, M., Yamamoto, K., Neda, K., 

and Sato, H. (1975). Studies onthe chromosomes of 

ratbonemarrow cells treated withbenzodiazepine 

derivative in vivo. Teratology 12, 199. 
68 Oxazepam, NTP TR 443 

Jablonska, JK, Knobloch, IC, Majka, J., and 

Wisniewska-Knypl, J.M. (1975). Stimulatory effects 

of chlordiazepoxide, diazepam and oxazepam on the 

drug-metabolizingin enzymes microsomes. 

Toxicology 5, 103-111. 

Jezequel, A.M., Koch, M., and Orlandi, F. (1974). A 

morphometric studyof the endoplasmic reticulum in 

human Correlation hepatocytes: between 

morphological and biochemical data in subjects under 

treatment with certain drugs. Gut 15, 737-747. 

Jonckheere, AR. (1954). A distribution-free 

k-sample test against ordered alternatives. Biontetrika 

41, 133-145. 

Kanto, J.H. (1982). Use of benzodiazepinesduring 

pregnancy, labourand lactation, with particular 

reference to pharmacokinetic considerations. Drugs 

23,354-380. 

Kaplan, E.L., and Meier, P. (1958). Nonparametric 

estimationfromincomplete observations. J. Ant. 

Stat. Assoc. 53,457-481. 

Kataoka, Y., Shibata, K., Gomita, Y., and Ueki, S. 

(1982). The mammillary body is a potential site of 

antianxiety actionof benzodiazepines. Brain Res. 241, 

374-377. 

Kawachi, T., Yahagi, T., Kada, T., Tazima, Y., 

Ishidate, M., Sasaki, M., and Sugiyama, T. (1980). 

Cooperativeprogramme on short-term assays for 

carcinogenicity in Japan. LARC Sci. Publ. 27, 

323-330. 

Kitagawa, H., Yano, H., Tsukakoshi, R., Umeda, M., 

and Yoshiro, F. (1974). The chronic toxicity study of 

7-chloro-2,3-dihydro-2-oxo-5-phenyl-lH1,4-benzodiazepine-3-carboxylicacid,monopotassium 

salt, hydroxide monopotassium (clorazepate 

dipotassium)in rats. Oyo Yakuri 2,89-106. 

Kleinerman, R.k, Brinton, L.A., Hoover, R.,and 

Fraumeni, J.F., Jr. (1984). Diazepam use and progression of breast cancer. Cancer Res. 44,1223-1225. 

Krueger, ICE., and Papadopoulos, V. (1992). 

Mitochondrialbenzodiazepinereceptorsand the 

regulation of steroid biosynthesis. Annu. Rev.

Phamacol. Toxicol. 32,211-237. 

Lafi, A., and Parry, J.M. (1988). A study of the 

induction of aneuploidy and chromosome aberrations 

after medazepam, diazepam, midazolam and

bromazepamtreatment. Mutagenesis 1, 78. 

Lafi, A., Parry, E.M., and Parry, J.M. (1987). The 

effects of benzodiazepines uponthe fidelity of mitotic 

cell division in culturedChinesehamster cells. 

Mutat. Res. 189, 319-332. 

Levitan, E.S., Schofield, P.R., Burt, D.R., Rhee, L.M., 

Wisden, W., Kohler, M., Fujita, N., Rodriguez, H.F., 

Stephenson, A, Darlison, M.G., Barnard, E.A., and 

Seeburg, P.H. (1988). Structural and functional basis 

for GABA, receptorheterogeneity. Nature 335, 

76-79. 

Livezey,G.T., Marczynski, T.J., McGrew, E.A., and 

Beluhan, F.Z. (1986a). Prenatal exposure to

diazepam: Late teratogenic postnatal effect. 

Neurobehav. Toxicol. Teratol. 8, 433-440. 

Livezey, G.T., Marczynski, T.J., and Isaac, L. (1986b). 

Prenataldiazepam:Chronic anxiety and deficits in 

brain receptors in mature rat progeny. Neurobehav. 

Toxicol. Teratol. 8, 425-432. 

MacGregor, J.T., Wehr, C.M., and Langlois,R.G. 

(1983). A simple fluorescent staining procedure for 

micronuclei and RNA in erythrocytes using Hoechst 

33258 and pyronin Y. Mutat. Res. 120, 269-275. 

MacGregor, J., Wehr, C.M., Henika, P.R., and 

Shelby, M.D. (1990). The in vivo erythrocyte 

micronucleustest:Measurement at steady state 

increases assay efficiency and permits integrationwith 

toxicity studies. Fundant. Appl. Toxicol. 14, 513-522. 

Marcucci, F., Guaitani, A., Kvetina,J., Mussini, E., 

andGarattini, S. (1968). Speciesdifference in 

diazepam metabolism and anticonvulsant effect. Eur. 

J. Pharntacol. 4, 467470. 

Maronpot,R.R.,and Boorman, G.A. (1982). 

Interpretation of rodent hepatocellular proliferative 

alterationsandhepatocellulartumorsin chemical 

safety assessment. Toxicol. Pathol. 10, 71-80. 

Martin, W.R., McNicholas, L.F., and Cherian, S. 

(1982). Diazepamandpentobarbitaldependence in 

the rat. Life Sciences 31, 721-730. 
References 69 

Matsuoka, A., Hayashi, M., and Ishidate, M., Jr. 

(1979). Chromosomal aberration tests on 29

chemicals combined withS9 mix in vitro. Mutat. Res. 

66, 277-290. 

Matula, T.I., and Downie, R. (1983). Evaluation of 

diazepam and oxazepam in in vino microbial mutagenicity tests andinan in vivo promoter assay. 

Environ. Mutagen. 5, 478. 

McClain, R.M. (1989). The significanceofhepatic 

microsomalenzymeinductionandalteredthyroid 

functionin rats: Implicationsforthyroidgland 

neoplasia. Toxicol. Pathol. 17, 294-306. 

McClain,R.M. (1990). Mouse liver tumors and 

Miller, B.M., and Adler, I.-D. (1989). Suspect spindle 

poisons: Analysis of c-mitotic effects in mouse bone 

marrow cells. Mutagenesis 4, 208-215. 

Miller, J.A., andMiller, E.C. (1977). Ultimate 

chemical asmutagenic carcinogens reactive 

electrophiles.In Origins of Human Cancer 

(H.H.Hiatt, J.D. Watson, and J.A. Winsten, Eds.), 

pp. 605-628. ColdSpringHarborLaboratory, Cold 

Spring Harbor, NY. 

Miller, R.P., and Becker, B.A. (1975). Teratogenicity 

of oraldiazepamanddiphenylhydantoin in mice. 

Toxicol. Appl. Pharntacol. 32, 53-61. 

microsomalenzyme-inducing drugs: ExperimentalNationalCancerInstitute(NCI) (1976). Guidelines 

andclinicalperspectiveswithphenobarbital.InforCarcinogen 

MouseLiver Carcinogenesis: Mechanisms and Species 

Conlparisons (D.E. Stevenson, J.A. Popp, J.M. Ward, 

R.M. McClain, T.J. Slaga,and H.C. Pitot, Eds.) 

pp. 345-365. Alan R. Liss, Inc., New York. MD. Bethesda, 

McC1ain9 R.’*, Levin, A*A., Posch9 R*C*, and

Downing, J.C. (1989). The effect of phenobarbitalon 

the metabolismandexcretion of thyroxine in rats. 

Toxicol. Appl. Pharnrncol. 99, 216-228. 

McConnell, E.E., Solleveld,H.A., Swenberg, J.A., and 

Boorman, G.A. (1986). Guidelinesforcombining 

neoplasmsforevaluation of rodentcarcinogenesis 

studies. JNCI 76, 283-289. 

McKnight, B., and Crowley, J. (1984). Tests for 

differencesintumorincidence based onanimal 

carcinogenesisexperiments. J. Am. Stat. Assoc. 79, 

639-648. 

The Merck Index (1983). 10th ed. (M. Windholz, 

Ed.), p. 994. Merck and Company, Rahway, NJ. 

Bioassay in SmallRodents.Technical 

ReportSeriesNo. 1. NIHPublication No. 76-801. 

U.S. Department of Health, Education, and Welfare, 

PublicHealth Service, National Institutes of Health, 

NationalInstitutes of Health(NIH) (1978). Open Formula Rat and Mouse Ration (NIH-07). 

Specification NIH-ll-1335. u.s. Department of 

Health,Education,andWelfare,PublicHealth 

Service, National Institutesof Health, Bethesda, MD. 

Olsen, R.W., Wamsley, J.K., Lee, R.J., and Lomax, P. 

(1986). Benzodiazepine/barbiturate/GABA receptorchloride ionophone complex in a genetic model for 

generalized epilepsy. Adv. Neurol. 44, 365-378. 

Olsen, J.H., Boice, J.D., Jensen, J.P.A., and 

Fraumeni, J.F., Jr. (1989). Canceramongepileptic 

patientsexposed to anticonvulsant drugs. J. Natl, 

Cancer Inst. 81, 803-808. 

Milkovich, L., and van den Berg, B.J. (1974). Effects Owen, G., Smith, T.H.F., and Agersborg, H.P.K., Jr. 

of prenatalmeprobamateandchlordiazepoxide (1970).Toxicity of somebenzodiazepinecompounds 

hydrochloride on embryonicfetal human and with CNS activity, Toxicol. Appl. Pharnzacol. 16,

development. N.Engl. J. Med. 291, 1268-1271. 556-570. 
70 Oxazepam, NTP TR 443 

Parry, J.M., Parry, E.M., Calvert, R., Lafi, A., and 

Somers, A. (1986). The mechanisms of induction of 

chromosome aneuploidy as revealed by the study of 

aberrations of mitotic cell division. Frog. Clin. Biol. 

Res. 209A,325-332. 

Parry, J.M., Parry, E.M., Warr, T., Lynch, A., and 

James, S. (1989). The detection of aneugensusing 

yeasts and cultured mammalian cells. Environ. Mol. 

Mutagen. 14 (Suppl.15), 151. 

Pedersen, W., and Lavik, N.J. (1991). Adolescents 

and benzodiazepines: Prescribed use, self-medication 

and intoxication. Acta Psychiatr. Scand. 84, 94-98. 

Petersen, K.W., Sherwood, H.L., and Petersen, H.D. 

(1978). The cytogeneticeffects of diazepamand 

chlordiazepoxide. Anat. Rec. 190, 620. 

Petrescu, M., Cuparencu, B., Horak, J., Petrescu, L., 

Florescu, P., Kory, M., Tomus, C., andRusu, V. 

(1981). Certainaspects of the toxicity of some 

benzodiazepines. Agressologie 22, 209-217. 

Physicians’ Desk Reference (PDR) (1991). 45th ed. 

Medical Economics Data, Montvale, NJ. 

Preat, V., de Gerlache, J., Lans, M., and 

Roberfroid,M. (1987). Promoting effect of 

oxazepam in rat hepatocarcinogenesis.Carcinogenesis 

8, 97-100. 

Preiss, A.M., Scheutwinkel-Reich, M., Fiille, I., 

Grohmann, H.G., Stan, and H.-J. (1982).

Investigation, withthe Salntonellalmicrosometest, of 

psychopharmaceuticalsusedinmeatproduction. 

Mutat. Res. 104, 333-337. 

Rajaonarison, J.F., Lacarelle, B., DeSousa, G.,

Catalin, J., andRahmani,R. (1991). In vitro 

glucuronidation of 3’-azido-3’-deoxythymidine by 

human liver: Role of UDP-glucuronosyltransferase 

2 form. Drug Metab. Dispos. 4, 809. 

Randall, L.O., Schallek, W., Scheckel, C.L., 

Stefko, P.L., Banziger, R.F., Pool, W., and Moe, R.A. 

(1969). Pharmacological on studiesflurazepam 

hydrochloride (’) (RO 5-6901), a newpsychotropic 

agentofbenzodiazepine the class. Arch. Int. 

Phamacodyn. Ther. 178, 216-241. 

Randall, L.O., Schenckel, C.L., and Pool, W. (1970). 

Pharmacology of medazepam (’) andmetabolites. 

Arch. Int. Pharntacodyn. Ther. 185, 135-148. 

Remandet, B., Gouy, D., Berthe, J., Mazue, G., and 

Williams, G.M. (1984). Lack of initiating or promoting activity of six benzodiazepine tranquilizers in 

rat liver limited bioassays monitored by histopathologyand assay of liverandplasma enzymes. 

Fundant. Appl. Toxicol. 4, 152-163. 

Rentington’s Pharntaceutical Sciences (1980). 16th ed. 

(A. Osol, Ed.). Mack Company, Publishing 

Easton, PA. 

Richards,Schoch, J.G., P., Mohler, H., and

Haefely, W. (1986). Benzodiazepine receptors

resolved. Experientia 42, 121-126. 

Robinson, R.L., Van Ryzin, R.J., Stoll, R.E., 

Jensen, R.D., and Bagdon,R.E.(1984).Chronic 

toxicity/carcinogenesis study of temazepaminmice 

andrats. Fundam. Appl. Toxicol. 4, 394-405. 

Ryan, C.L., and Pappas, B.A. (1986). Intrauterine 

diazepamexposure:Effects on physical and neurobehavioraldevelopmentin the rat. Neurobehav. 

Toxicol. Teratol. 8, 279-286. 

Saano, V. (1988). Central-type andperipheral-type 

benzodiazepinereceptors. Ann. Clin. Res. 20, 

348-355. 

Saito,H.,Kobayashi, H., Takeno, S., andSakai, T. 

(1984). Fetal toxicity of benzodiazepinesinrats. 

Res. Contntun. Chent. Pathol. Pharmacol. 46, 437. 

(Abst.) 

Salzman, C., Shader, R.I., Greenblatt, D.J., and 

Harmatz, J.S. (1983). Long vs. short half-life benzodiazepinesin the elderly. Arch. Gen. Psychiaq 40, 

293-297. 

Sasaki, M., Sugimura, K., Yoshida, M.A., and Abe, S. 

(1980). Cytogenic effectsof 60 chemicals on cultured 

humanandChinesehamster cells. Senshokutai 

(fiontosonza) 20, 574-584. 

Schmid, W., and Staiger, G.R. (1969). Chromosome 

studiesonbonemarrowfromChinesehamsters 

treatedbenzodiazepine and with tranquillizers 

cyclophosphamide. Mutat. Res. 7, 99-108. 
References 71 

Schmidt, W. (1976). The micronucleus test for cytogenetic analysis. In Chentical Mutagens, Principles 

and Methods for their Detection (A. Hollaender, Ed.), 

Vol. 4,pp. 31-53, Plenum Press, New York. 

Scrollini, F., Caliari, S., Romano, A, and Torchio, P. 

(1975). Toxicologicalpharmacological and 

investigations of pinazepam (7-chloro-l-propargy1-5-

phenyl-3H-1,4-benzodiazepin-2-one):A new psychotherapeutic agent. Drug Res. 25, 934-940. 

Shirley, E.(1977). A non-parametricequivalent of 

Williams’ test for contrasting increasingdose levels of 

atreatment. Biontetrics 33, 386-389. 

§isenwine, S.F., andTio, C.O. (1986). The metabolic 

disposition of oxazepamin rats. Drug Metab. Dispos. 

n4,41-45. 

Sisenwine, S.F., Tio, C.O., Liu, A.L., and 

Politowski, J.F. (1987). The metabolicfate of 

oxazepam in mice. Drug Metab. Dispos. 15, 579-580. 

Smith, P.F.,Q’Brian, K.A., and Keenan, K.P. (1991). 

Evaluation of bromodeoxyuridine labeling in hepatomegaly produced by peroxisomal proliferationor 

P-450inductioninrodents. In Chemically Induced 

Cell Proliferation: Implications for Risk Assessment 

(BE. Butteworth, T.J. Slaga, W. Farland,and 

M. “lain, Eds.), pp. 285-289. Wiley-Lis, 

New York. 

Snyder, S.W., Verma, A., and Trifiletti, R.R. (1987). 

The peripheral-typebenzodiazepinereceptor: A 

protein of mitochondrial outer membranes utilizing 

porphyrinsasendogenous ligands. FASEB J 1, 

282-288. 

Staiger, G.R. (1969). Chlordiazepoxide and 

deazepam: Absence of effects on the chromosomes 

of diploidhumanfibroblast cells. Mutat. Res. 7, 

109-115. 

Stoll, B.A. (1976). Psychosomatic factors and tumour 

growth. In Risk Factors in Breast Cancer (B.A. Stoll, 

Ed.), pp. 193-203.William HeinemannMedical 

Books, London. 

Straus, D.S. (1981). Somatic mutation, cellular 

differentiation, and cancer causation. JNcI 6,, 

233-241. 

Tarone, R.E. (1975). Tests fortrendin life table 

analysis. Bionzetrika 62, 679-682. 

Tennant, R.W., Margolin, B.H., Shelby, M.D., 

Zeiger, E., Haseman, J.K., Spalding, J., Caspary, W., 

Resnick, M., Stasiewicz, S., Anderson, B., and 

Minor, R. (1987). Prediction of chemicalcarcinogenicityin rodentsfrom in vitro genetic toxicity 

assays. Science 236, 933-941. 

Tocus, E.C., Nelson, W.C., and Vocci, F.J., Jr. (1983). 

Regulatoryperspectivesand the benzodiazepines. 

J. Psychoactive Drugs 15, 151-157. 
72 Oxazepam, NTP TR 443 

Torigoe, K. (1979). Sisterchromatid exchange in 

children treated with anticonvulsantdrugs. Acta Med 

Biol. 27, 65-72. 

Travali, S., Koniecki, J., Petralia, S., and Baserga, R. 

(1990). Oncogenesingrowthandevelopment. 

FASEB J. 4,3209-3214. 

Trosko, J.E., and Horrobin, D.P. (1980). The activity 

of diazepam in a Chinese hamsterV79 lung cell assay 

for tumour promoters. ZRCS J. Med Sci 8,887. 

Tucker, J.C. (1985). Benzodiazepines and the

developing rat: A critical review. Neurosci. Biobehav. 

Rev. 9, 101-111. 

Vega, P., Carrasco, M., Sanchez, E., and del Villar, E. 

(1984). Structure-activity relationship in the effect of 

1,4-benzodiazepines on morphine, aminopyrine and 

oestrone metabolism. Res. Contmun. Chenz. Pathof. 

Pharmacol. 44, 179-198. 

Verma, A., and Snyder, S.H. (1989). Peripheral type 

benzodiazepinereceptors. Annu.Rev. Pharntacol. 

Toxicol. 29, 307-322. 

Vorne, M., andIdanpaan-Heikkila, J. (1975). 

Inhibition of drug metabolizing enzymes by diazepam 

in rat liver. Erperientia 31, 962-963. 

Walkenstein, S.S., Wiser, R., Gudmundsen, C.H., 

Kimmel, H.B., and Corradino, R.A. (1964). Absorption, metabolism, and excretion of oxazepam and its 

succinate half-ester. J. Phamz. Sci 53,1181-1186. 

Ward, J.M., Hagiwara, A., Anderson, L.M., 

Lindsey, K., andDiwan, B.A. (1988). The chronic 

hepatic or renal toxicity of di(2-ethylhexyl)phthalate, 

acetaminophen, sodium barbital, and phenobarbital 

inmale B6C3Fl mice: Autoradiographic,immunohistochemical, and biochemical evidence for levels of 

DNA synthesis not associated with carcinogenesis or 

tumorpromotion. Toxicol. Appl. Pharntacol. 96, 

494-506. 

Waskell, L. (1978). A study of the mutagenicity of 

anestheticsandtheirmetabolites. Mutat. Res. 57, 

141-153. 

Wee, E.L., and Zimmerman, E.F. (1983). Involvement of GABA in palate morphogenesis and its 

relation to diazepamteratogenesis in two mouse 

strains. Teratology 28, 15-22. 

White, B.J., Driscoll, E.J., Tjio, J.-H. and 

Smilack, Z.H. (1974). Chromosomal aberration rates 

and intravenously given diazepam. JAM4 230, 

414-417. 

Whittaker, S.G., Zimmermann, F.K, Dicus, B., 

Piegorsch, W.W., Resnick, M.A., and Fogel, S. 

(1990). Detection of inducedmitoticchromosome 

loss in Saccharomyces cerevisiae -An interlaboratory 

assessment of 12 chemicals. Mutat. Res. 241,225-242. 

Williams, D.A. (1971). A test for differences between 

treatmentmeans when several dose levels are 

compared with zero control. a dose Biometrics

27, 103-117. 

Williams, D.A. (1972). The comparison of several 

dose levels with azero dose control. Biomemks 

28, 519-531. 

Williams, G.M., Mori, H., and McQueen, C.A. 

(1989). Structure-activityrelationships in the rat 

hepatocyteDNA-repairtestfor 300 chemicals. 

Mutat. Res. 221, 263-286. 

Xu, W., and Adler, I.-D. (1990). Clastogenic effects 

of known and suspectspindlepoisonsstudied by 

chromosome analysis in mouse bone marrow cells. 

Mutagenesis 5, 371-374. 

Zeiger, E., Anderson, B., Haworth, S., Lawlor, T., and 

Mortelmans, K. (1992). Salnzonella mutagenicity 

tests. V. Results from the testing of 311 chemicals. 

Environ. Mol. Mutagen. 19 (Suppl. 21), 2-141. 

Zhurkov, V.S. (1975). Investigation of the mutagenic 

activity of drug preparations and food additives in a 

culture of human lymphocytes. Sov.Genet. 11, 

528-530, 
73 

APPENDIX A 

SUMMARY OF LESIONS 

IN MALE SWISS-WEBSTER MICE 

IN THE 57-WEEK FEED STUDY 

OF OXAZEPAM 

TABLEA1 Summary of the Incidence of Neoplasms in Male Swiss-Webster Mice 

in the 57-Week Feed Study of Oxazepam .................................. 74 

TABLEA2 Individual Animal Tumor Pathology of Male Swiss-Webster Mice 

in the 57-Week Feed Study of Oxazepam .................................. 77 

TABLEA3 Statistical Analysis of Primary Neoplasms in Male Swiss-Webster Mice 

in the 57-Week Feed Study of Oxazepam .................................. 98 

TABLEA4 Summary of the Incidence of Nonneoplastic Lesions in Male Swiss-Webster Mice 

in the 57-Week Feed Study of Oxazepam .................................. 101 
74 Oxazepam, NTP TR 443 

TABLEA1 

Summary of the Incidenceof Neoplasms in Male Swiss-Webster Mice in the 57-Week Feed Studyof Oxazepama 

Disposition Summary 

Animals initially in study 

Early deaths 

Moribund 

Natural deaths 

SuMvors 

Terminal sacrifice 

Animals examined microscopically 

Alimentary System 

Intestine small, duodenum 

Adenoma 

Intestine small, jejunum 

Liver 

Hepatoblastoma 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic Sarcoma 

Sarcoma, metastatic, skeletal muscle 

Pancreas 

Sarcoma, metastatic, skeletal muscle 

Salivary glands 

Carcinoma 

Histiocytic Sarcoma 

Stomach, glandular 

Cardiovascular System 

Hart 

Sarcoma, metastatic, skeletal muscle 

Endocrine System 

Adrenal cortex 

Adenoma 

Capsule, adenoma 

Adrenal medulla 

Thyroid gland 

Cardiovascular System 

None 

Genital System 

Epididymis 

Histiocytic sarcoma 

Testes 

0 PPm 

60 

10 

5 

45 

60 

1 (2%) 

1 (2%) 

1 (2%) 

(60)

1 (2%) 

(60)

1 (2%) 

1 (2%) 

(60) 

(60)

1 (2%) 

2,500 ppm 

60 

13 

28 

19 

60 

(57)

1 (2%) 

(59) 

(60)

1 (2%) 

4 (7%) 

1 (2%)

14 (23%) 

21(35%) 

(59) 

5,000 ppm 

60 

24 

26 

10 

60 

(58) 

(56) 

(60) 

13 (22%) 

6 (10%) 

8 (13%) 

42 (70%) 
Lesions in Male Swiss-Webster Mice 75 

TASLEA1 

Summary of the Incidence.of Neoplasms in Male Swiss-Webster Micein the 57-Week Feed Studyof Oxazepam 

(continued) 

Hematopoietic System 

Bone marrow 

Lymph node 

Inguinal, Sarcoma, metastatic, skeletal 

muscle 

Lumbar, histiocytic Sarcoma 

Mediastinal, histiocytic Sarcoma 

Pancreatic, sarcoma, metastatic, skeletal 

muscle 

Renal, histiocytic Sarcoma 

Lymph node, mandibular 

Histiocytic Sarcoma 

Lymph node, mesenteric 

Histiocytic Sarcoma 

Sarcoma, metastatic, skeletal muscle 

Spleen 

Histiocytic sarcoma 

Sarcoma, metastatic, skeletal muscle 

Thymus 

Integumentary System 

Skin 

Hemangiosarcoma 

Neurofibrosarcoma 

Musculoskeletal System 

Skeletal muscle 

Sarcoma 

Nervous System 

Brain 

Respiratory System 

Lung 

Alveolaribronchiolar adenoma 

Alveolar/bronchiolar adenoma, multiple 

Alveolaribronchiolar carcinoma 

Alveolar/bronchiolar carcinoma, multiple 

Histiocytic sarcoma 

Sarcoma, metastatic, skeletal muscle 

Nose 

Special Senses System 

None 

(60) (60) (59)

1 (2%) 1 (2%) 

1 (2%) 

(60) (60) (60)

10 (17%) 5 (8%) 7 (12%) 

2 (3%) 1 (2%) 

5 (8%) 3 (5%) 

1 (2%) 

1 (2%) 

1 (2%) 

(60) (60) (60) 
76 Oxazepam, NTP TR 443 

TABLEA1 

Summary of the Incidence of Neoplasms in Male Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam 

(continued) 

Urinary System 

Kidney 

Histiocytic sarcoma 

Sarcoma, metastatic, skeletal muscle 

Urinary bladder 

Histiocytic sarcoma 

Systemic Lesions 

Multiple organsb 

Histiocytic sarcoma 

Lymphoma malignant 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed 

Lymphoma malignant undifferentiated cell 

Neoplasm Summary 

Total animals with primary neoplasms' 

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 neoplasm 

(60) 

1 (2%)

1 (2%) 

(60)

1 (2%) 

(60)

1 (2%) 

1(2%) 

2 (3%) 

3 (5%) 

4 (7%) 

26 

33 

14 

14 

17 

19 

1 

9 

(60) (60) 

(59) (59) 

(60) (60) 

2 (3%) 2 (3%) 

3 (5%) 

5 (8%) 1 (2%) 

42 54 

61 83 

36 51 

43 57 

17 25 

18 26 

a Number of animals examined microscopically at site and number of animals with lesion 

Number of animals with any tissue examined microscopically ' Primaly neoplasms: allneoplasmsexcept metastatic neoplasms 
Lesions in Male Swiss-Webster Mice 77 

TABLEA2 

Individual Animal Tumor Pathology of Male Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam: 0 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular adenoma 

Histiocytic Sarcoma 

Sarcoma, metastatic, skeletal muscle 

Pancreas 

Sarcoma, metastatic, skeletal muscle 

Salivary glands 

Carcinoma 

Histiocytic Sarcoma 

Stomach, forestomach 

Stomach, glandular 

Tongue 

Tooth 

Cardiovascular System 

Blood vessel 

Heart 

Sarcoma, metastatic, skeletal muscle 

Endocrine System 

Adrenal cortex 

Adenoma 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

General Body System 

Tissue NOS 

Genital System 

Epididymis 

Histiocytic Sarcoma 

Preputial gland 

Prostate 

Seminal vesicle 

Testes 

+: Tissueexaminedmicroscopically 

A: Autolysis precludes examination 

1222222233333333333333333 

6457889922334579999999999 

1479113577184817777777777 

2212111212221111111111111 

2093998181309898888889999 

8828084322661731356894567 

......................... 

........................ 

......................... 

......................... 

......................... 

......................... 

+++M+++++++++++++++++++++ 

+++++++++++++++++M+++++++ 

......................... X 

X 

X ......................... X 

......................... 

X 

......................... 

......................... + 

+ 

......................... 

......................... X 

......................... X 

......................... 

......................... 

+M+MM++M+M+++++++++++++++ 

......................... 

......................... 

......................... X ......................... 

......................... 

......................... 

......................... 

M: Missing tissue X Lesionpresent 

I: Insufficienttissue Blank Notexamined 
78 Oxazepam, NTP TR 443 

TABLEA2 

Individual Animal Tumor Pathology of Male Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam: 0 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular adenoma 

Histiocytic sarcoma 

Sarcoma, metastatic, skeletal muscle 

Pancreas 

Sarcoma, metastatic, skeletal muscle 

Salivary glands 

Carcinoma 

Histiocytic sarcoma 

Stomach, forestomach 

Stomach, glandular 

Tongue 

Tooth 

Cardiovascular System 

Blood vessel 

Heart 

Sarcoma, metastatic, skeletal muscle 

Endocrine System 

Adrenal cortex 

Adenoma 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

General Body System 

Tissue NOS 

Genital System 

Epididymis 

Histiocytic sarcoma 

Preputial gland 

Prostate 

Seminal vesicle 

Testes 

. , 

3333333333333333333333333 

9999999999999999999999999 

1111111111111111111111111 

1222222222222222222222222 

9000000001111111122222222 

9012345190145678901234561 

......................... 

......................... 

......................... 

+ ....................... + ....................... ......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

+++++++++++++++++M+++++++ 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 
Lesions in Male Swiss-Webster Mice 79 

TABLEA2 

Individual Animal Tumor Pathology of Male Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam: 

(continued) 

Number of Days OD Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular adenoma 

Histiocytic Sarcoma 

Sarcoma, metastatic, skeletal muscle 

Pancreas 

Sarcoma, metastatic, skeletal muscle 

Salivary glands 

Carcinoma 

Histiocytic Sarcoma 

Stomach, forestomach 

Stomach, glandular 

Tongue 

Tooth 

Cardiovascular System 

Blood vase1 

Heart 

Sarcoma, metastatic, skeletal muscle 

Endocrine System 

Adrenal cortex 

Adenoma 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

General Body System 

Tissue NOS 

Genital System 

Epididymis 

Histiocytic Sarcoma 

Preputial gland 

Prostate 

Seminal vesicle 

Testes 

3333333333 

9999999999 

7777777777 

2222222222 

2333333334 

9012345790 

++++++++++ 

+++++++M++ 

+++++M++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

X 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++MM 

++++++++++ 

+ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

0 ppm 

Total 

Tissues/ 

Tumors 

60 

57 

59 

59 

59 

60 

59 

59 

60 

1 

1 

1 

60 

1 

60 

1 

1 

60 

60 

1 

1 

60 

60 

1 

60 

1 

60 

60 

54 

58 

60 

1 

60 

1 

60 

60 

60 

60 
80 Oxazepam, NTP TR 443 

TABLEA2 

Individual Animal Tumor Pathology of Male Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam: 0 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Inguinal, sarcoma, metastatic, 

skeletal muscle 

Lumbar, histiocytic sarcoma 

Mediastinal, histiocytic Sarcoma 

Pancreatic, sarcoma, metastatic, 

skeletal muscle 

Renal, histiocytic sarcoma 

Lymph node, mandibular 

Histiocytic sarcoma 

Lymph node, mesenteric 

Histiocytic sarcoma 

Sarcoma, metastatic, skeletal muscle 

Spleen 

Histiocytic sarcoma 

Sarcoma, metastatic, skeletal muscle 

Thymus 

Integumentary System 

Mammary gland 

Skin 

Hemangiosarcoma ~~~ ~ ~ ~ 

Musculoskeletal System 

Bone 

Skeletal muscle 

Sarcoma 

Nervous System 

Brain 

Peripheral nene 

Spinal cord 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Alveolarbronchiolar adenoma, 

multiple 

Alveolarbronchiolar carcinoma 

Histiocytic Sarcoma 

Sarcoma, metastatic, skeletal muscle 

Nose 

Trachea 

1222222233333333333333333 

6457889922334579999999999 

1479113577184817777777777 

2212111212221111111111111 

2093998181309898888889999 

8828084322661731356894567 

......................... + + + + + 

X 

X 

X 

X 

X 

++++++++++++++++++++++M++ 

X 

M++++++++++++++++++++++++ 

X 

X 

......................... X 

X 

++++++M++++++++++++++++++ 

MMMMMM+MMMMM++M+MMMMMMM+M 

......................... 

......................... 

......................... X 

......................... + ++++ + + 

+ + ++++ + 

......................... 

X 

X 

X 

......................... 

......................... 
Lesions in Male Swiss-Webster Mice 81 

TABLEA2 

Individual Animal Tumor Pathology of Male Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam: 0 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Inguinal, Sarcoma, metastatic, 

skeletal muscle 

Lumbar, histiocytic sarcoma 

Mediastinal, histiocytic sarcoma 

Pancreatic, Sarcoma, metastatic, 

skeletal muscle 

Renal, histiocytic Sarcoma 

Lymph node, mandibular 

Histiocytic Sarcoma 

Lymph node, mesenteric 

Histiocytic Sarcoma 

Sarcoma, metastatic, skeletal muscle 

Spleen 

Histiocytic Sarcoma 

Sarcoma, metastatic, skeletal muscle 

Thymus 

Integumentary System 

Mammary gland 

Skin 

Hemangiosarcoma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Sarcoma 

Nervous System 

Brain 

Peripheral newe 

Spinal cord 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Alveolarbronchiolar adenoma, 

multiple 

Alveolarbronchiolar carcinoma 

Histiocytic Sarcoma 

Sarcoma, metastatic, skeletal muscle 

Nose 

Trachea 

3333333333333333333333333 

9999999999999999999999999 

7777777777777777777777777 

1222222222222222222222222 

9000000001111111122222222 

9012345790145678901234567 

......................... + 

......................... 

......................... 

......................... 

......................... 

MMMMMMMMMMMMMM+MMMMM+MMMM 

......................... X 

......................... 

......................... 

......................... 

+ 

......................... x x x x xxx 

X 

X x xx 

......................... 

......................... 
82 Oxazepam, NTP TR 443 

TABLEA2 

Individual Animal Tumor Pathology of Male Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam: 0 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Inguinal, Sarcoma, metastatic, 

skeletal muscle 

Lumbar, histiocytic Sarcoma 

Mediastinal, histiocytic Sarcoma 

Pancreatic, Sarcoma, metastatic, 

skeletal muscle 

Renal, histiocytic Sarcoma 

Lymph node, mandibular 

Histiocytic Sarcoma 

Lymph node, mesenteric 

Histiocytic Sarcoma 

Sarcoma, metastatic, skeletal muscle 

Spleen 

Histiocytic Sarcoma 

Sarcoma, metastatic, skeletal muscle 

Thymus 

Integumentary System 

Mammary gland 

Skin 

Hemangiosarcoma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Sarcoma ~~ ~ 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Alveolarbronchiolar adenoma, 

multiple 

Alveolarbronchiolar carcinoma 

Histiocytic Sarcoma 

Sarcoma, metastatic, skeletal muscle 

Nose 

Trachea 

3333333333 

9999999999 

7111177111 

2222222222 

2333333334 

9012345190 

++++++++++ 

+ 

+++M++++M+ 

++++++++++ 

++++++++++ 

++++++++++ 

MM+MMM+MMM 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

X X X 

X 

++++++++++ 

++++++++++ 

Total 

Tissues/ 

Tumors 

60 

7 

1 

1 

1 

1 

1 

57 

1 

59 

1 

1 

60 

1 

1 

59 

9 

60 

1 

60 

60 

1 

60 

7 

8 

60 

10 

2 

5 

1 

1 

60 

60 
Lesions in Male Swiss-Webster Mice 

TABLEA2 

Individual Animal Tumor Pathology of Male Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam: 0 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

~ ~~ 

Special Senses System 

Ear 

Eye _______~~~~ 

Urinary System 

Kidney 

Hisiiocytic sarcoma 

Sarcoma, metastatic, skeletal muscle 

Urethra 

Urinary bladder 

Histiocytic sarcoma 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed 

Lymphoma malignant undifferentiated 

cell type 

1222222233333333333333333 

6457889922334579999999999 

1479113577184817777777777 

2212111212221111111111111 

2093998181309898888889999 

8828084322661731356894561 

+ + + 

......................... X 

X 

+ 

......................... X 

......................... X 

X 

X X 

X xx 

X x xx 
84 Oxazepam, NTP TR 443 

TABLEA2 

Individual Animal Tumor Pathology of Male Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam: 0 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Special Senses System 

Ear 

Eye 

Urinary System 

Kidney 

Histiocytic sarcoma 

Sarcoma, metastatic, skeletal muscle 

Urethra 

Urinary bladder 

Histiocytic sarcoma 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed 

Lymphoma malignant undifferentiated 

cell type 

3333333333333333333333333 

9999999999999999999999999 

7777717777777777777717777 

1222222222222222222222222 

9000000001111111122222222 

9012345790145678901234567 

+ + 

......................... 

......................... 

~~~~~ ~~~ ~ ~~~~~ 

......................... 
Lesions in Male Swiss-Webster Mice 

TABLEA2 

Individual Animal Tumor Pathology of Male Swiss-Webster Mice in the 57-Week Feed Studyof Oxazepam: 0 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Special Senses System 

Ear 

Eye 

Urinary System 

Kidney 

Histiocytic Sarcoma 

Sarcoma, metastatic, skeletal muscle 

Urethra 

Urinary bladder 

Histiocytic Sarcoma 

Systemic Lesions 

Multiple organs 

Histiocytic Sarcoma 

Lymphoma malignant 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed 

Lymphoma malignant undifferentiated 

cell type 

3333333333 

9999999999 

7777777777 

2222222222 

2333333334 

9012345790 

+ 

++++++++++ 

++++++++++ 

++++++++++ 

Total 

Tissues1 

Tumors 

4 

2 

60 

1 

1 

1 

60 

1 

60 

1 

1 

2 

3 

4 
86 Oxazepam, NTP TR 443 

TABLEA2 

Individual Animal Tumor Pathology of Male Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam: 2,500 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Adenoma 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatoblastoma 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tongue 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Capsule, adenoma 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

General Body System 

None 

Genital System 

Epididymis 

Penis 

Preputial gland 

Prostate 

Seminal vesicle 

Testes 

0111122222223333333333333 

1045802699990112223334445 

4759365856675277891690110 

2222222222222222222222222 

9796454754495787899558466 

5004843311746896798736698 

......................... 

+++++++++++++++++++++++M+ 

......................... 

++++A+++ ++++++++++++++++ 

++++A++++++++++++++++++++ 

......................... 

++++A++++++++++++++++++++ 

++++A++++++++++++++A+++++ 

......................... 

X X x xx x xx 

X X ......................... 

......................... 

......................... 

++++A++++++++++++++++++++ 

+ 

++M++++++++++++++++++++++ 

......................... 

.......................... 

++++++++++++++M++++++++++ 

......................... 

++++++++++M++++++M+++++++ 

M++M++++++++++M+++++++M++ 

......................... 

++++++ ++ +++++++++++++++ 

+ + + + +++ ++++ ......................... 

++++++ ++ +++++++++++++++ 

M++++++++++++++++++++++++ 

......................... 
Lesions in Male Swiss-Webster Mice 87 

TABLEA2 

Individual Animal Tumor Pathology of Male Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam: 2,500 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Adenoma 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatoblastoma 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tongue 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Capsule, adenoma 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

General Body System 

None 

Genital System 

Epididymis 

Penis 

Preputial gland 

Prostate 

Seminal vesicle 

Testes 

3333333333333333333333333 

5556666677788889999999999 

1690125956923573777777777 

2222222222222222222222222 

6948955865868479445566667 

21536291105602534958035’71 

......................... 

+++++++M+++++++++++++++++ 

......................... 

......................... 

......................... 

+++++++A+++++++++++++++++ 

......................... 

......................... 

......................... X 

X X X A

., xx X X 

x xx x x x x x xxxx ......................... 

......................... 

......................... 

......................... + 

......................... 

......................... 

......................... 

+++++++++++++++++++++M+++ 

......................... 

+++M+++++++M+++M+++M+M++M 

++++M+++++M++M+++++++++++ 

......................... 

......................... 

+ + +++ ++++ +++ 

+++M+++++++++++++++++++++ 

......................... 

......................... 

......................... 
88 Oxazepam, NTP TR 443 

TABLEA2 

Individual Animal Tumor Pathologyof Male Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam: 2,500 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Adenoma 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatoblastoma 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tongue 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Capsule, adenoma 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

General Body System 

None 

Genital System 

Epididymis 

Penis 

Preputial gland 

Prostate 

Seminal vesicle 

Testes 

3333333333 

9999999999 

1111777177 

2222222223 

7777888990 

2419248270 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++M+++++++ 

X 

++++++++++ 

++++++++++ 

++++++++++ 

X 

X X 

xx xxx xx 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

X 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

+.+++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

Total 

Tissues/ 

Tumors 

60 

58 

60 

58 

59 

57 

1 

59 

58 

60 

1 

4 

1 

14 

21 

60 

60 

60 

59 

2 

59 

60 

60 

1 

58 

60 

52 

53 

60 

58 

23 

59 

58 

59 

60 
Lesions in Male Swiss-Webster Mice 89 

TABLEA2 

Individual Animal Tumor Pathology of Male Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam: 2,500 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Integumentary System 

Mammary gland 

Skin 

Hemangiosarcoma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Nervous System 

Brain 

Peripheral newe 

Spinal cord 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Alveolarbronchiolar adenoma, 

multiple 

Alveolarbronchiolar carcinoma 

Alveolarbronchiolar carcinoma, 

multiple 

Nose 

Trachea 

Special Senses System 

Ear 

Eye

Harderian gland 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Lymphoma malignant lymphocytic 

Lymphoma malignant undifferentiated 

cell type 

0111122222223333333333333 

1045802699990112223334445 

4759365856675277891690110 

2222222222222222222222222 

9796454754495787899558466 

5004843311746896798736698 

......................... + ++ + 

+++++M+++++++++++++++ +++ 

++++MM++++++++++M+M+++M++ 

......................... 

+++++++++++++M++++++M++++ 

+MMMMMMMM+MMMMMMMMMMMMMMM 

......................... X 

......................... 

......................... 

......................... 

+ + ++ + + 

+ ++ + + 

......................... 

X 

......................... 

......................... 

+ 

+ 

......................... 

++++A++++++++++++++++++++ 

......................... xx 

X X xx 
90 Oxazepam, NTP TR 443 

TABLEA2 

Individual Animal Tumor Pathology of Male Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam: 2,500 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Integumentary System 

Mammary gland 

Skin 

Hemangiosarcoma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Alveolar/bronchiolar adenoma, 

multiple 

Alveolarbronchiolar carcinoma 

Alveolarbronchiolar carcinoma, 

multiple 

NOS2 

Trachea 

Special Senses System 

Ear 

Eye

Harderian gland 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Lymphoma malignant lymphocytic 

Lymphoma malignant undifferentiated 

cell type 

3333333333333333333333333 

5556666677788889999999999 

1690125956923573777777777 

2222222222222222222222222 

6948955865868479445566667 

2153629110560253495803571 

......................... 

+ + + 

+++++++++++++++++M+++++++ 

......................... 

......................... 

+++++++++++++++++M+++++++ 

MMMMMMMMMMM+MMMMM+MMMMMM+ 

......................... 

......................... 

......................... 

......................... + 

+ 

......................... X X xx 

X 

X ......................... 

......................... 

+ 

......................... 

......................... 

......................... 

X 
Lesions in Male Swiss-Webster Mice 91 

TABLEA2 

Individual Animal Tumor Pathology of Male Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam: 2,500 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Integumentary System 

Mammary gland 

Skin 

Hemangiosarcoma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Alveolarbronchiolar adenoma, 

multiple 

Aveolarbronchiolar carcinoma 

Aveolarbronchiolar carcinoma, 

multiple 

Nose 

Trachea 

Special Senses System 

Ear 

Eye

Harderian gland 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Lymphoma malignant lymphocytic 

Lymphoma malignant undifferentiated 

cell type 

3333333333 

9999999999 

1171771177 

2222222223 

7177888990 

2479248270 

++++++++++ 

+++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

+MMMMMMMMM 

++++++++++ 

++++++++++ 

+'+++++++++ 

++++++++++ 

++++++++++ 

X 

X 

X 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

Total 

Tissues1 

Tumors 

60 

7 

56 

55 

60 

57 

6 

60 

1 

60 

60 

60 

7 

6 

60 

5 

1 

3 

1 

60 

60 

1 

1 

1 

60 

59 

60 

2 

5 
92 Oxazepam, NTP TR 443 

TABLEA2 

Individual Animal Tumor Pathology of Male Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam: 5,000 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

General Body System 

None 

Genital System 

Epididymis 

Penis 

Preputial gland 

Prostate 

Seminal vesicle 

Testes 

0122222222222233333333333 

5623377778889900001112234 

7551711231562622560148841 

3333333333333333333333333 

1525433501332622532324530 

9107858680963019328473001 

......................... 

......................... 

++++++++++++A++++++++++++ 

......................... 

+++++A++++++A++++++++++++ 

++++++++++++A++++++++++++ 

+++++++++ ++A++++++++++++ 

+++++A++++++A++++++++A+A+ 

......................... X X 

X 

X X X 

x x xx x x x x x xx x x x x ......................... 

......................... 

......................... 

......................... 

++++++++M++++++++++++++++ 

......................... 

......................... 

++++++++++++++++M+ ++++++ ......................... 

++M+++++++M++++++++++++++ 

+++++++M++++++++++M++++++ 

......................... 

......................... ++ +++ + ......................... 

......................... 

......................... 

......................... 
Lesions in Male Swiss-Webster Mice 93 

TABLEA2 

Individual Animal Tumor Pathology of Male Swiss-Webster Micein the 57-Week Feed Study of Oxazepam: 5,000 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellularcarcinoma 

Hepatocellularcarcinoma, multiple 

Hepatocellularadenoma 

Hepatocellularadenoma, multiple 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Hart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

General Body System 

None 

Genital System 

Epididymis 

Penis 

Preputial gland 

Prostate 

Seminal vesicle 

Testes 

3333333333333333333333333 

4445556666666777777778889 

1451581234589001134593555 

3333333333333333333333333 

1210340405150140112042445 

4562767238896279374542192 

......................... 

+++++++++++M++++++M++M++M 

......................... 

......................... 

+M+++AM++++++++++++++++++ 

++++++M++++++++++++++++++ 

++++++M++++++++++++++++++ 

++++A++++++++++++++++++++ 

......................... xx x xx xx X 

X X X 

X X X 

x xx xx xxxxxxxxxxxxxxx ......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

+++++++++++++++M++++M++++ 

......................... 

+M++M+M++++++M+++++++M+++ 

+M+++++++++++++++++++++++ 

......................... 

......................... + ++ +++ ++ +++ +++++ ......................... 

......................... 

......................... 

......................... 
94 Oxazepam, NTP TR 443 

TABLEA2 

Individual Animal Tumor Pathology of Male Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam: 5,000 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

General Body System 

None 

Genital System 

Epididymis 

Penis 

Preputial gland 

Prostate 

Seminal vesicle 

Testes 

3333333333 

9999999999 

7777777777 

3333333333 

0112334455 

4156130545 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++M+++ 

M+++++++++ 

++++++++++ 

X X X 

X X 

xx 

xxxx xxx 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++-I-+++++++ 

++++++++++ 

++++++++++ 

M+++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

+ + 

++M++++M++ 

+M++++++++ 

++++++++++ 

++++++++++ 

Total 

Tissues/ 

Tumors 

60 

56 

59 

59 

55 

58 

56 

54 

60 

13 

6 

8 

42 

60 

60 

60 

60 

59 

60 

60 

56 

60 

52 

57 

60 

60 

24 

58 

59 

60 

60 
Lesions in Male Swiss-Webster Mice 95 

TABLEA2 

Individual Animal Tumor Pathology of Male Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam: 5,OOO ppm 

(continued) 

Number of Days on Study 

CarcassIDNumber 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Integumentary System 

Mammary gland 

Skin 

Neurofibrosarcoma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Nose 

Trachea 

Special Senses System 

Ear 

Eye 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed 

Lymphoma malignant undifferentiated 

cell type 

0122222222222233333333333 

5623377778889900001112234 

7551711231562622560148841 

3333333333333333333333333 

1.525433501332622532324530 

9107858680963019328473001 

......................... + + + 

++++++++++++++++M++++++++ 

++++++++++++ ++++++++++++ ......................... 

+++++M+++MMM++M+++M+M++++ 

MMMMMM+MMMMMMMMMMMMMMMMMM 

++++++++++++A++++++++++++ 

X 

......................... 

......................... 

......................... 

+ + + ++ 

+ ++ + + 

......................... X X X X ......................... 

......................... 

+ + ++ + 

+ 

......................... 

......................... 

......................... X X 

X 
96 Oxazepam, NTP TR 443 

TABLEA2 

Individual Animal Tumor Pathology of Male Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam: 5,000 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Integumentary System 

Mammary gland 

Skin 

Neurofibrosarcoma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Nose 

Trachea 

Special Senses System 

Ear 

Eye 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed 

Lymphoma malignant undifferentiated 

cell type 

3333333333333333333333333 

4445556666666777777778889 

1451581234589001134593555 

3333333333333333333333333 

1210340405150140112042445 

4562767238896279374542192 

......................... 

+++++++M+M++++++M++++++++ 

++++++++++M++++++++++M+++ 

......................... 

......................... 

MMMMMM+MMMMMM+MMMMMMMMMMM 

......................... 

......................... 

......................... 

......................... + + + + 

+ + + + 

......................... X xx ......................... 

......................... 

+ 

......................... 

++++++M++++++++++++++++++ 

......................... 

X 
Lesions in Male Swiss-Webster Mice 97 

TABLE A2 

Individual Animal Tumor Pathology of Male Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam: 5,000 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Integumentary System 

Mammary gland 

Skin 

Neurofibrosarcoma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Nose 

Trachea 

Special Senses System 

Ear 

Eye 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed 

Lymphoma malignant undifferentiated 

cell type 

3333333333 

9999999999 

7777777777 

3333333333 

0112334455 

4156130545 

++++++++++ 

+ 

+++M++++++ 

++++++++++ 

++++++++++ 

+M+++++++M 

+M+MMMMMMM 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

X 

X 

Total 

Tissues/ 

Tumors 

60 

4 

55 

57 

60 

51 

5 

59 

1 

60 

60 

60 

9 

9 

60 

7 

60 

60 

6 

1 

60 

59 

60 

2 

3 

1 
98 Oxazepam, NTP TR 443 

TABLEA3 

Statistical Analysisof Primary Neoplasms in Male Swiss-Webster Mice in the 57-Week Feed Studyof Oxazepam 

Liver: Hepatocellular Adenoma 

Overall ratea 

Adjusted rateb 

Terminal rate' 

First incidence (days) 

Life table testd 

Logistic regression testd 

Cochran-Armita e testd I Fisher exact test 

Liver: Hepatocellular Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Liver: Hepatocellular Adenoma or Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Lung: Alveolar/bronchiolar Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Lung: Alveolarhonchiolar Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

1/60 (2%) 

2.2% 

1/45 (2%) 

397 (T)

P<O.001 

P<O.001 

P<0.001 

0/60(0%) 

0.0% 

0/45 (0%) e -

P<0.001 

P<O.001 

P<O.001 

1/60(2%) 

2.2% 

1/45 (2%) 

397 (T)

P<O.001 

P<0.001 

P<0.001 

12/60(20%) 

26.7% 

12/45 (27%) 

397 (T) P=O.123 

P=0.353N 

P=O.l17N 

5/60 (8%) 

10.5% 

4/45 (9%) 

281 

P =0.266N 

P=0.043N 

P=0.030N 

35/60 (58%) 

88.7% 

15/19 (79%) 

268 

P<0.001 

P<0.001 

P<O.001 

5/60 (8%) 

21.7% 

3/19 (16%) 

356 

P=O.003 

P=O.OlO 

P=O.O29 

35/60 (58%) 

88.7% 

15/19 (79%) 

268 

P<0.001 

P<0.001 

Pc0.001 

6/60 (10%) 

28.9% 

5/19 (26%) 

369 

P=O.482 

P=O.570 

P=0.100N 

4/60 (7%) 

14.2% 

1/19 (5%) 

268 

P=O.407 

P=0.551N 

P=0.500N 

50/60 (83%) 

98.0% 

9/10 (90%) 

231 

P<0.001 

P<O.001 

P<0.001 

19/60 (32%) 

72.0% 

5/10 (50%) 

302 

P<O.001 

P<0.001 

P<0.001 

52/60 (87%) 

98.1% 

9/10 (90%) 

231 

P<O.001 

P<O.001 

P<O.001 

7/60 (12%) 

26.0% 

0/10 (0%) 

272 

P=O.164 

P =0.329N 

P=0.159N 

0/60(0%) 

0.0% 

OD0 (0%) -

P=0.217N 

P=O.O51N 

P=0.029N 
Lesions in Male Swiss-Webster Mice 

TABLEA3 

Statistical Analysisof Primary Neoplasms in Male Swiss-Webster Mice in the 57-WeekFeed Study of Oxazepam 

(continued) 

Lung: Alveolar/bronchiolar Adenoma or Carcinoma 

Overall rate 14/60 (23%) 9/60 (15%) 7/60 (12%) 

Adjusted rate 30.2% 35.7% 26.0% 

Terminal rate 13/45 (29%) 5/19 (26%) on0 (0%) 

First incidence (days) 281 268 272 

Life table test P=O.rn P=O.291 P=O.286 

Logistic regression test P=0.158N P=0.415N P=0.155N 

Cochran-Annitage test P=0.056N 

Fisher exact test P=0.177N P=0.074N 

All Organs: Malignant Lymphoma or Histiocytic Sarcoma 

Overall rate 10/60 (17%) 7/60 (12%) 6/60 (10%) 

Adjusted rate 17.6% 13.9% 28.3% 

Terminal rate 0/45 (0%) 0/19 (0%) 2/10 (20%) 

First incidence (days) 244 145 272 

Life table test P=0.415N P=0.435N P=0.519N 

Logistic regression test P=0.005N P=0.036N P=0.007N 

Cochran-Armitage test P=0.169N 

Fisher exact test P=0.301N P=0.211N 

All Organs: Malignant Lymphoma (Lymphocytic,Mixed, or Undifferentiated Cell Type) 

Overall rate 9/60 (15%) 7/60 (12%) 6/60 (10%) 

Adjusted rate 16.0% 13.9% 28.3% 

Terminal rate 0/45 (0%) 0/19 (0%) 2/10 (20%) 

First incidence (days) 244 145 272 

Life table test P=0.508N P=0.526N P=O.603 

Logistic regression test P=0.014N P=O.WN P=0.021N 

Cochran-Armitage test P=0.243N 

Fisher exact test P=0.395N P=0.291N 

All Organs:Benign Neoplasms 

Overall rate 14/60 (23%) 36/60 (60%) 51/60 (85%) 

Adjusted rate 30.4% 91.5% 98.0% 

Terminal rate 13/45 (29%) 16/19 (84%) 9/10 (90%) 

First incidence (days) 344 268 231 

Life table test P<O.001 P<0.001 P<O.001 

Logistic regression test P<O.001 P<O.001 P<O.001 

Cochran-Armitage test P<O.ool 

Fisher exact test P<O.001 P<O.001 

All Organs: Malignant Neoplasms 

Overall rate 17/60 (28%) 17/60 (28%) 25/60 (42%) 

Adjusted rate 29.6% 44.1% 80.3% 

Terminal rate 5/45 (11%) 4/19 (21%) 6/10 (60%) 

First incidence (days) 244 145 271 

Life table test P<0.001 P=O.170 P<O.001 

Logistic regression test P=O.321 P =0.203N P=0.555N 

Cochran-Armitage test P=O.O72 

Fisher exact test P=0.580N P=O.o90 
100 Oxazepam, NTP TR 443 

TABLEA3 

Statistical Analysisof Primary Neoplasms in Male Swiss-Webster Mice in the 57-WeekFeed Study of Oxazepam 

(continued) 

All Organs:Benign 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Annitage test 

Fisher exact test 

(T)Terminal sacrifice 

or Malignant Neoplasms 

0 PPm 2,500 PPm 5,Ooo PPm 

26/60 (43%) 42/60 (70%) 54/60 (90%) 

45.4% 92.6% 100.0% 

14/45 (31%) 16/19 (84%) lono (100%) 

244 145 231 

P<O.001 P<O.001 P<O.001 

P<O.001 P=O.005 P<0.001 

P <0.001 

P=O.003 P<O.001 

a Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for adrenal gland, 

bone marrow, brain, epididymis, gallbladder, heart, kidney, larynx, liver, lung, nose, pancreas, parathyroid gland, pituitary gland, preputial gland, 

prostate gland, salivary gland, spleen, testes, thyroid gland, and urinaly bladder; for other tissues, denominator is number of animals necropsied. 

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

Observed incidence at terminal kill 

Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding 

to painvise comparisons between the controls and that exposed group. The life table analysis 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-Annitage 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 Notapplicable;noneoplasms in animal group 
Lesions in Male Swiss-Webster Mice 101 

TABLEA4 

Summary of the Incidence of Nonneoplastic Lesions in Male Swiss-Webster Mice in the 57-WeekFeed Study 

of Oxazepama 

Disposition Summary 

Animals initiallyin study 

Early deaths 

Moribund 

Natural deaths 

Sulvivors 

Terminal sacrifice 

Animals examined microscopically 

Alimentary System 

Intestine large, colon 

Amyloid deposition 

Lumen, hemorrhage 

Intestine large, rectum 

Amyloid deposition 

Intestine large, cecum 

Amyloid deposition 

Ulcer 

Lumen, hemorrhage 

Intestine small, duodenum 

Amyloid deposition 

Ulcer 

Intestine small, jejunum 

Amyloid deposition 

Ulcer 

Intestine small, ileum 

Amyloid deposition 

Inflammation, granulomatous 

Liver 

Amyloid deposition 

Basophilic focus 

Eosinophilic focus 

Fatty change 

Hematopoietic cell proliferation 

Infarct 

Infiltration cellular, lymphocyte 

Inflammation, chronic 

Inflammation, subacute 

Necrosis 

Centrilobular, hypertrophy 

Oval cell, hyperplasia 

Pancreas 

Amyloid deposition 

Duct, cyst 

Salivary glands 

Amyloid deposition 

Stomach, forestomach 

Acanthosis 

Amyloid deposition 

Hyperkeratosis 

60 

10 

5 

45 

60 

(59)

6 (10%) 

(60)

39 (65%) 

1(2%) 

(59)

31(53%) 

1(2%) 

(59)

46 (78%) 

1(2%) 

(60)

22 (37%) 

1(2%) 

1 (2%) 

1 (2%) 

10 (17%) 

12 (20%) 

(60) 

60 

13 

28 

19 

60 

(60)

29 (48%) 

(58) 

5 (9%) 

(59)

24 (41%) 

1(2%) 

(57)

49 (86%) 

(59)

49 (83%) 

(58)

48 (83%) 

(60)

31 (52%) 

22 (37%) 

1(2%) 

1(2%) 

2 (3%) 

13 (22%) 

46 (77%) 

1(2%) 

(60)

1(2%) 

(60)

44 (73%) 

(60)

1(2%) 

3 (5%) 

60 

24 

26 

10 

60 

(59)

29 (49%) 

1 (2%) 

(59)

9 (15%) 

(55)

29 (53%) 

1(2%) 

1 (2%) 

(58) 

50 (86%) 

(56)

48 (86%) 

1(2%) 

(54)

43 (80%) 

(60)

28 (47%) 

1 (2%) 

22 (37%) 

1(2%) 

2 (3%) 

1(2%) 

1 (2%) 

8 (13%) 

47 (78%) 

(60) 

1(2%) 

(60)

44 (73%) 

(60) 

4 (7%) 

1(2%) 
102 Oxazepam, NTP TR 443 

TABLEA4 

Summary of the Incidence of Nonneoplastic Lesions in Male Swiss-Webster Mice in the 57-WeekFeed Study 

of OXaZepam (continued) 

Alimentary System (continued) 

Stomach, glandular 

Amyloid deposition 

Mineralization 

Epithelium, hyperplasia 

Cardiovascular System 

Heart 

Amyloid deposition 

Degeneration 

Inflammation, subacute 

Atrium, thrombosis 

Valve, thrombosis 

Ventricle, thrombosis 

Endocrine System 

Adrenal cortex 

Amyloid deposition 

Hyperplasia 

Capsule, hyperplasia 

Adrenal medulla 

Hyperplasia 

Islets, pancreatic 

Hyperplasia 

Parathyroid gland 

Amyloid deposition 

Thyroid gland 

Amyloid deposition 

Inflammation. chronic 

General Body System 

None 

Genital System 

Preputial gland 

Cyst

Inflammation, suppurative 

Seminal vesicle 

Fibrosis 

Testes 

Edema 

Germinal epithelium, atrophy 

(60)

8 (13%) 

(60)

43 (72%) 

2 (3%) 

1(2%) 

1(2%) 

1(2%) 

(60)

17 (28%) 

4 (7%) 

3 (5%) 

(60)

2 (3%) 

(60)

4 (7%) 

(54)

10 (19%) 

(60)

19 (32%) 

(59)

29 (49%) 

3 (5%) 

(60)

52 (87%) 

34 (57%) 

2 (3%) 

1(2%) 

(60)

37 (62%) 

(60)

22 (37%) 

1(2%) 

1(2%) 

(60)

52 (87%) 

35 (58%) 

(60)

41 (68%) 

(52)

42 (81%) 

(60)

48 (80%) 

1(2%) 
Lesions in Male Swiss-Webster Mice 103 

TABLEA4 

Summary of the Incidence of Nonneoplastic Lesions in Male Swiss-Webster Mice in the 57-Week Feed Study 

of Oxazepam (continued) 

Hematopoietic System 

Lymph node 

Inguinal, hyperplasia, lymphoid 

Mediastinal, hyperplasia, lymphoid 

Pancreatic, inflammation, granulomatous 

Renal, hyperplasia, lymphoid 

Lymph node, mandibular 

Amyloid deposition 

Hyperplasia, lymphoid 

Lymph node, mesenteric 

Amyloid deposition 

cyst

Hyperplasia, lymphoid 

Infiltration cellular, plasma cell 

Necrosis 

Spleen 

Amyloid deposition 

Hematopoietic cell proliferation 

Hyperplasia, lymphoid 

Necrosis 

Capsule, fibrosis 

Thymus 

Amyloid deposition 

Vein, thrombosis 

Integumentary System 

Skin 

Edema 

Hyperkeratosis 

Ulcer 

Musculoskeletal System 

None 

Nervous System 

Peripheral nerve 

Axon, degeneration 

2 (29%) 

(57) 

8 (14%) 

2 (4%) 

(59)

25 (42%) 

2 (3%) 

1 (2%) 

(60)

16 (27%) 

3 (5%) 

7 (12%) 

(59) 

(60)

1 (2%) 

2 (3%) 

3 (5%) 

(7)

2 (29%) 

(7)

1 (14%) 

1 (14%) 

1 (14%) 

(56)

29 (52%) 

1 (2%) 

(55)

36 (65%) 

2 (4%) 

1 (2%) 

(60)

32 (53%) 

2 (3%) 

4 (7%) 

1 (2%) 

(57) 

(60) 

1 (2%) 

(7)

1 (14%) 

(55)

29 (53%) 

(57)

35 (61%) 

1 (2%) 

2 (4%) 

1 (2%) 

(60)

30 (50%) 

3 (5%) 

1 (2%) 

(51)

1 (2%) 

1 (2%) 

(59) 

(9) 
104 Oxazepam, NTP TR 443 

TABLEA4 

Summary of the Incidence of Nonneoplastic Lesions in Male Swiss-Webster Mice in the57-WeekFeed Study 

of OXaZepam (continued) 

0 PPm 2,500 PPm 5,Ooo PPm 

Respiratory System 

Lung (60) (60) (60)

Fibrosis 27 (45%) 25 (42%) 

Inflammation, chronic 6 (10%) 3 (5%) 2 (3%) 

Inflammation, granulomatous 1 (2%) 

Inflammation, subacute 1 (2%) 2 (3%) 11 (18%) 

Thrombosis 1 (2%) 1 (2%) 1 (2%) 

Alveolar epithelium, hyperplasia 1 (2%) 4 (7%) 3 (5%) 

Alveolus, infiltration cellular, mononuclear 

cell 1 (2%) 31 (52%) 37 (62%) 

Alveolus, metaplasia,osseous 1 (2%) 

Alveolus, metaplasia, squamous 1 (2%) 

Special Senses System 

Ear 

External ear, ulcer 

Eye

Degeneration 

Urinary System 

Kidney (60) (60)

Cyst 1 (2%)

Hydronephrosis 1 (2%) 

Infarct 2 (3%) 1 (2%) 1 (2%) 

Inflammation, granulomatous 2 (3%) 

Inflammation, suppurative 1 (2%) 1 (2%) 

Artery, thrombosis 1 (2%) 

Glomerulus, amyloid deposition 42 (70%) 35 (58%) 42 (70%) 

Interstitium, amyloid deposition 3 (5%) 14 (23%) 10 (17%) 

Renal tubule, necrosis 1 (2%) 

Urethra (1)

Transitional epithelium, hyperplasia 1 (100%) 

Urinary bladder (60) (59) 

Calculus gross obselvation 1 (2%) 

Calculus micro obselvation only 1 (2%) 

Hemorrhage 1 (2%) 

Inflammation, suppurative 2 (3%) 

Transitional epithelium, hyperplasia 1 (2%) 2 (3%) 

a Number of animals examined microscopically at site and number of animals with lesion 
105 

APPENDIX B 

SUMMARY OF LESIONS 

IN FEMALE SWISS-WEBSTER MICE 

IN THE 57-WEEK FEED STUDY 

OF OXAZEPAM 

TABLEB1 Summary of the Incidence of Neoplasms in Female Swiss-WebsterMice 

in the 2-Year Feed Studyof Oxazepam .................................... 106 

TABLEB2 IndividualAnimalTumorPathology of FemaleSwiss-WebsterMice 

in the 2-Year Feed Study of Oxazepam .................................... 109 

TABLEB3 Statistical Analysis of Primary Neoplasms inFemaleSwiss-WebsterMice 

in the 2-Year Feed Studyof Oxazepam .................................... 127 

TABLEB4 Summary of the Incidence of NonneoplasticLesions 

in Female Swiss-Webster Mice in the 2-Year Feed Study of Oxazepam ............ 130 
106 Oxazepam, NTP TR 443 

TABLEB1 

Summary of the Incidence of Neoplasms in Female Swiss-Webster Mice in the 57-Week Feed Study of Oxazepama 

Disposition Summary 

Animals initially in study 60 

Early deaths 

Moribund 9 

Natural deaths 4 

Sulvivors 

Died last week of study 

Terminal sacrifice 47 

Missing 

Animals examined microscopically 60 

Alimentary System 

Gallbladder 

Intestine large, cecum 

Intestine small, duodenum 

Adenoma 

Intestine small, jejunum 

Carcinoma 

Liver 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma 

Pancreas 

Salivary glands 

Stomach, glandular 

Tooth 

Adamantinoma NOS 

Cardiovascular System 

Heart 

Endocrine System 

Adrenal cortex 

Adenoma 

Adrenal medulla 

Pituitary gland 

Thyroid gland 

Follicular cell. adenoma 

General Body System 

None 

60 

13 

18 

1 

27 

1 

59 

(58) 

(56) 

(56)

1 (2%) 

(59) 

(59)

1 (2%) 

9 (15%) 

13 (22%) 

60 

11 

31 

17 

1 

59 

(56)

1 (2%) 

(59)

6 (10%) 

5 (8%) 

8 (14%) 

39 (66%) 

1 (2%) 

(59) 

(59) 

(57) 

(2) 
Lesions in Female Swiss-Webster Mice 107 

TABLEB1 

Summary of the Incidence of Neoplasms in Female Swiss-Webster Mice in the 57-Week Feed Studyof Oxazepam 

(continued) 

Genital System 

ovary

Hemangioma 

Uterus 

Polyp stromal 

Sarcoma stromal 

Hematopoietic System 

Bone marrow 

Lymph node 

Pancreatic, histiocytic sarcoma 

Lymph node, mandibular 

Histiocytic sarcoma 

Lymph node, mesenteric 

Histiocytic sarcoma 

Spleen 

Histiocytic sarcoma 

Thymus 

Histiocytic sarcoma 

Integumentary System 

None 

Musculoskeletal System 

Skeletal muscle 

Sarcoma stromal, metastatic, uterus 

Nervous System 

Brain 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Alveolar/bronchiolar carcinoma 

Nose 

Special Senses System 

None 

Urinary System 

Kidney 

Urinary bladder 

(59) (58)

1 (2%) 

(60) (59)

1 (2%) 

1 (2%) 

(60) (59)

6 (10%) 4 (7%) 

5 (8%) 3 (5%) 

(60) (59) 

(55)

1 (2%) 

(57)

1 (2%) 

(59)

6 (10%) 

1 (2%) 

(59) 
10s Oxazepam, NTP TR 443 

TABLEB1 

Summary of the Incidence of Neoplasms in Female Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam 

(continued) 

Systemic Lesions 

Multiple organsb 

Histiocytic sarcoma 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed 

Lymphoma malignant undifferentiated cell 

Neoplasm Summary 

Total animals with primary neoplasms' 

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 neoplasm 

Total animals with neoplasmsof uncertain 

benign or malignant 

Total uncertain neoplasms 

origin 

(60) 

6 (10%) 

3 (5%) 

24 

26 

9 

9 

16 

16 

1 

1 

1 

1 

(59) (59)

1 (2%) 

1 (2%) 

8 (14%) 1 (2%) 

4 (7%) 5 (8%) 

37 49 

46 75 

26 47 

29 55 

16 17 

17 20 

a Number of animals examined microscopically at site and number of animals with lesion 

Number of animals with any tissue examined microscopically ' Primaryneoplasms:allneoplasmsexcept metastatic neoplasms 
Lesions in Female Swiss-Webster Mice 109 

TABLEB2 

Individual Animal Tumor Pathology of Female Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam: 0 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tooth 

Adamantinoma NOS 

Cardiovascular System 

Blood vessel 

Hart ~ ~~~ ~ 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

None 

Genital System 

Clitoral gland 

ovary

Hemangioma 

Uterus 

Polyp stromal 

Sarcoma stromal 

+: Tissueexaminedmicroscopically 

A: Autolysisprecludesexamination 

1123333333333333333333333 

8970256677799999999999999 

4326281222526777777777777 

0000000000000000000000000 

0346132101451000000011111 

9870023935268124567812346 

......................... 

......................... 

+A+++++++++++++++++++++++ 

+A+++++++++++++++++++++++ 

+A++++++++M++++++++++++++ 

......................... 

+A+++++++++++++++++++++++ 

+A+++++A+++++++++++++++++ 

......................... 

+ + ......................... 

......................... 

+A+++++++++++++++++++++++ 

+A+++++++++++++++++++++++ 

+ + 

X 

......................... 

......................... 

......................... 

......................... 

......................... 

++M+M++++++++++++M+++++++ 

......................... 

......................... X 

......................... 

+++++++++++++++++M+++++++ 

X ......................... X 

X 

M Missing tissue X Lesion present 

I: Insufficienttissue Blank:Notexamined 
110 Oxazepam, NTP TR 443 

TABLEB2 

Individual Animal Tumor Pathology of Female Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam: 0 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tooth 

Adamantinoma NOS 

Cardiovascular System 

Blood vessel 

Heart ~~-~~ ~~ ~ ~ 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

None 

Genital System 

Clitoral gland 

ovary

Hemangioma 

Uterus 

Polyp stromal 

Sarcoma stromal 

3333333333333333333333333 

9999999999999999999999999 

1111111111111111111111111 

0000000000000000000000000 

1222222222333333334444444 

1012456789013456190134568 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

+ ......................... 

......................... 

......................... 

......................... 

......................... 

.......................... 

......................... 

+++++++M+++++++++++++++++ 

......................... 

++++++++++++M++++M+++++++ 

++M++++++++++++++M+++++++ 

......................... 

......................... 

......................... 

......................... 
Lesions in Female Swiss-Webster Mice 111 

TABLEB2 

Individual Animal Tumor Pathology of 

(continued) 

Number of Days on Study 

Carcass ID Number 

~ ~ 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tooth 

Adamantinoma NOS 

Cardiovascular System 

Blood vessel 

Hal? 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

None 

Genital System 

Clitoral gland 

ovary

Hemangioma 

Uterus 

Polyp stromal 

Sarcoma stromal 

Female Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam: 0 ppm 

7771777777 

9999999999 

3333333333 

9012345789 

4555555555 

0000000000 

Tumors 

Tissues/ 

Total 

++++++++++ 60 

++++++++++ 60 

++++++++++ 59 

++++++++++ 59 

++++++++++ 58 

++++++++++ 59 

++++++++++ 59 

++++++++++ 58 

++++++++++ 60 

X 1 

3 

++++++++++ 60 

++++++++++ 60 

++++++++++ 59 

++++++++++ 59 

2 

1 

++++++++++ 60 

++++++++++ 60 

++++++++++ 60 

++++++++++ 59 

++++++++++ 60 

++++++++++ 55 

+++++++++M 57 

++++++++++ 60 

1 

++++++++++ 60 

++++++++++ 59 

1 

++++++++++ 60 

1 

1 
112 Oxazepam, NTP TR 443 

TABLEB2 

Individual Animal Tumor Pathology of Female Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam: 0 ppm 

(continued) 

1123333333333333333333333 

Number of Days on Study 8970256677199999999999999 

4326281222526777777177717 

0000000000000000000000000 

Carcass ID Number 0346132101451000000011111 

9810023935268124567812346 

Hematopoietic System 

Bone marrow ......................... Lymph node ++ +++ + + + 

Lymph node, mandibular +++++++++++++++++++M+++++ 

Lymph node, mesenteric ......................... Spleen ......................... Thymus +M++M++++++++++++++++++++ 

Integumentary System 

Mammary gland ++++M+M++++++++++++++++++ 

Skin ......................... 

Musculoskeletal System 

Bone ......................... Skeletal muscle ......................... Sarcoma stromal, metastatic, uterus X 

Nervous System 

Brain ......................... Peripheral nerve ++ + + 

Spinal cord + + 

Respiratory System 

Lung ......................... Alveolarlbronchiolar adenoma X 

Alveolarbronchiolar carcinoma X 

Nose ......................... Trachea ......................... 

Special Senses System 

Ear + 

Urinary System 

Kidney ......................... Urinary bladder ......................... 

Systemic Lesions 

Multiple organs ......................... Lymphoma malignant mixed x x x xx 

Lymphoma malignant undifferentiated 

cell type X xx 
Lesions in Female Swiss-Webster Mice 113 

TABLEB2 

Individual Animal Tumor Pathology of Female Swiss-Webster Mice in the 57-Week Feed Studyof Oxazepam: 0 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Integumentary System 

Mammary gland 

Skin 

Musculoskeletal System 

Bone 

Skeletal muscle 

Sarcoma stromal, metastatic, uterus 

Nervous System 

Brain 

Peripheral newe 

Spinal cord 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Alveolarbronchiolar carcinoma 

Nose 

Trachea 

Special Senses System 

Ear 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Lymphoma malignant mixed 

Lymphoma malignant undifferentiated 

cell type 

3333333333333333333333333 

9999999999999999999999999 

7777711777777777777771777 

0000000000000000000000000 

1222222222333333334444444 

IO12456789013456790134568 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

X X X 

xx X X ......................... 

......................... 

......................... 

++++++++.M++++++++++++++++ 

......................... 
114 Oxazepam, NTP TR 443 

TABLEB2 

Individual Animal Tumor Pathology of Female Swiss-Webster Mice in the 57-Week Feed Studyof Oxazepam: 0 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Integumentary System 

Mammary gland 

Skin 

Musculoskeletal System 

Bone 

Skeletal muscle 

Sarcoma stromal, metastatic, uterus 

Nervous System 

Brain 

Peripheral newe 

Spinal cord 

Respiratory System 

Lung

Alveolarfironchiolar adenoma 

Alveolarfironchiolar carcinoma 

NO% 

Trachea 

Special Senses System 

Ear 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Lymphoma malignant mixed 

Lymphoma malignant undifferentiated 

cell type 

3333333333 

9999999999 

7777777777 

0000000000 

4555555555 

9012345789 

++++++++++ 

++M+++++++ 

++++++++++ 

++++++++++ 

++++++M++M 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

X X 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

Total 

TissUesl 

Tumors 

60 

8 

58 

60 

60 

56 

58 

60 

60 

60 

1 

60 

4 

2 

60 

6 

5 

60 

60 

1 

60 

59 

60 

6 

3 
Lesions in Female Swiss-Webster Mice 115 

TABLEB2 

Individual Animal Tumor Pathology of Female Swiss-Webster Micein the 57-Week Feed Study of Oxazepam: 

2,500 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Adenoma 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Hart 

Endocrine System 

Adrenal cortex 

Adenoma 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

None 

Genital System 

Clitoral gland 

ovary

Uterus 

0112222223333333333333333 

lo63589990122222223344566 

7609611449500126680649037 

1101001000101100101010100 

0161771688170096170818068 

0276258863072406113957724 

........................ 

++++++++++++++M+++++++++ 

+++++A++++++++++++++++++ 

+++++A++++++++++++++++++ 

+++++A+++++++++++++++M+A 

+++++A+++++++A++++M+++++ 

X ........................ 

........................ 

........................ 

xx X X 

X X X x x 

........................ 

........................ 

........................ 

........................ 

........................ 

........................ 

........................ 

........................ 

........................ 

++++++++MM+MM+M++M++M+++ 

+++++M++++++++++++++++++ 

........................ 

........................ 

+++++++++++++++++++M++++ 

........................ 
116 Oxazepam, NTP TR 443 

TABLEB2 

Individual Animal Tumor Pathology of Female Swiss-Webster Mice in the 57-Week Feed Studyof Oxazepam: 

2,500 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Adenoma 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adenoma 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

None 

Genital System 

Clitoral gland 

Ovary

Uterus 

3333333333333333333333333 

Ill8999999999999999999999 

2469234111111111111111111 

0000010000000000000000000 

6999119666611111888889999 

9143916134503468012582518 

......................... 

......................... 

......................... 

++++++++++++++++M++++ +++ ......................... 

......................... 

......................... 

++++++++++++M++++++++++++ 

......................... X 

X X X 

xx xxx X 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... X ..................... +++ ......................... 

++M++++++++++++++++++++++ 

......................... 

......................... 

++++++++++++++++++++++M++ 

......................... 

......................... 
Lesions in Female Swiss-Webster Mice 117 

TABLEB2 

Individual Animal Tumor Pathologyof Female Swiss-Webster Mice in the 57-Week Feed Studyof Oxazepam: 

2,500 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Adenoma 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adenoma 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

None 

Genital System 

Clitoral gland 

Ovary

Uterus 

3333333333 

9999999999 

7777777777 

0111111111 

9000001112 

9156893490 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

X X 

X X 

+ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++M+++++ 

++++++++++ 

++++++++++ 

X 

++++++++++ 

++++++++++ 

++++++++++ 

Total 

Tissues1 

Tumors 

59 

58 

58 

56 

56 

56 

1 

59 

56 

59 

1 

9 

13 

1 

59 

59 

59 

59 

59 

59 

59 

1 

58 

59 

50 

58 

59 

1 

58 

58 

59 
118 Oxazepam, NTP TR 443 

TABLEB2 

Individual Animal Tumor Pathology of Female Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam: 

2,500 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Integumentary System 

Mammary gland 

Skin 

Musculoskeletal System 

Bone 

Skeletal muscle 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Alveolarbronchiolar carcinoma 

Nose 

Trachea 

Special Senses System 

Ear 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed 

Lymphoma malignant undifferentiated 

cell type 

0112222223333333333333333 

lo63589990122222223344566 

7609611449500126680649037 

1101001000101100101010100 

0161771688170096170818068 

0276258863072406113957724 

........................ + ++ ++ ++ + + ........................ 

++ ..................... ........................ 

+M++++++++++++++++++++++ 

++M++++++++++++MM+++++MM 

........................ 

........................ 

........................ 

........................ + + + + 

+ ++ + + 

........................ X 

X ........................ 

........................ 

+ 

........................ 

........................ 

........................ X 

xx X xxx x 

X X X 
Lesions in Female Swiss-Webster Mice 119 

TABLEB2 

Individual Animal Tumor Pathology of Female Swiss-Webster Mice in the 57-Week Feed Studyof Oxazepam: 

2,500 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

~ ~~~ ~____ 

Integumentary System 

Mammary gland 

Skin ~ ~~~ ~~~ 

Musculoskeletal System 

Bone 

Skeletal muscle 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Alveolarhronchiolar adenoma 

Alveolarbronchiolar carcinoma 

Nose 

Trachea 

Special Senses System 

Ear 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed 

Lymphoma malignant undifferentiated 

cell type 

3333333333333333333333333 

7778999999999999999999999 

2469234777777777777777777 

0000010000000000000000000 

6999719666677777888889999 

9143976134503468012582578 

......................... + 

++++++M++M+++++++++++++++ 

......................... 

......................... 

+++++++++++++M+++++++++++ 

+++M+++++++++M++++++++M++ 

......................... 

......................... 

......................... 

......................... + 

+ 

......................... X X 

X X 

......................... 

......................... 

......................... 

......................... 

......................... 

X 
120 Oxazepam, NTP TR 443 

TABLEB2 

Individual Animal Tumor Pathology of Female Swiss-Webster Micein the 57-Week Feed Study of Oxazepam: 

2,500 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

~~ ~ ~ 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Integumentary System 

Mammary gland 

Skin ~~ 

Musculoskeletal System 

Bone 

Skeletal muscle 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

Respiratory System 

~ ~~ 

Lung 

Alveolar/bronchiolar adenoma 

Alveolarbronchiolar carcinoma 

Nose 

Trachea 

Special Senses System 

Ear 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed 

Lymphoma malignant undifferentiated 

cell type 

3333333333 

9999999999 

7717777777 

0111111111 

9000001112 

9156893490 

++++++++++ 

++++++++++ 

+++++++++M 

++++++++++ 

++++++++++ 

++M+++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

X 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

X 

Total 

Tissues/ 

Tumors 

59 

10 

57 

57 

59 

51 

50 

59 

59 

59 

59 

5 

6 

59 

4 

3 

59 

59 

1 

59 

59 

59 

1 

8 

4 
Lesions in Female Swiss-Webster Mice 121 

TABLEB2 

Individual Animal Tumor Pathology of Female Swiss-Webster Micein the 57-Week Feed Study of Oxazepam: 

5,000 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Carcinoma 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic Sarcoma 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tooth 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

PituitaIy gland 

Thyroid gland 

General Body System 

None 

Genital System 

Clitoral gland 

ovary

Hemangioma 

Uterus 

Polyp stromal 

1122233333333333333333333 

1989900111222222233334445 

1744556468011557867790290 

1111111111111111111111111 

5536142544734246562637762 

6829251346972311208501527 

........................ 

+++++++++++++M++++++++++ 

+A+++A+++++A++++++++++++ 

+A+++A+++++A++++++++++++ 

+A++MA+++++A+++++++++A++ 

+A+++M+++++A++++++++++++ 

........................ 

+A++++++A++A+++++++A++++ 

........................ X X 

X X X x x 

X x xxx x xxxx x 

X 

........................ 

........................ 

....................... 

....................... 

+ + 

++++++++++++++++++M+++++ 

........................ 

........................ 

++++++++++M+++++++++++++ 

........................ 

+++M+MM+++++M++MMM+MMM+M 

........................ 

........................ 

++++++++++++++++++++M+++ 

++++++++++++++++++M+MM++ 

++++++++++++++++++M+M+++ 
122 Oxazepam, NTP TR 443 

TABLEB2 

Individual Animal Tumor Pathology of Female Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam: 

5,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Carcinoma 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tooth 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

General Body System 

None 

Genital System 

Clitoral gland 

ovary

Hemangioma 

Uterus 

Polyp stromal 

3333333333333333333333333 

5556666677777888999999999 

0570345656669115057777777 

1111111111111111111111111 

3452576424552366352233334 

4872934750149176554636893 

......................... 

........................ 

......................... 

......................... 

++++++A++++++M+++++++++++ 

......................... 

++++++++++++++A++++++++++ 

X 

++++++++++++++++++++++++M 

......................... x x 

X x x 

X X X 

xx x xxxxxxxx xxx xxxxxx 

+ + ......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

+++++++++++++M+++++++++++ 

......................... 

M+++++++++++++++++++++M++ 

+++++++++++++++++M+++++++ 

......................... 

++++++++++++++M++++++++++ 

+++++++++++++++M+++++++++ 

X ......................... X 
Lesions in Female Swiss-Webster Mice 123 

TABLEB2 

Individual Animal Tumor Pathology of Female Swiss-Webster Mice in the 57-Week Feed Studyof Oxazepam: 

5,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Carcinoma 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic Sarcoma 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tooth 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

~~~ 

General ~~ay system 

None 

Genital System 

Clitoral gland 

Ovary

Hemangioma 

Uterus 

Polyp stromal 

3333333333 

9999999999 

7777777777 

1111111111 Total 

4566777778 Tissues/ 

9038046780 Tumors 

++++++++++ 59 

++++++++++ 56 

++++++M+++ 55 

++++++++++ 56 

+++M++++++ 51 

++++++++++ 56 

++++++++++ 56 

1 

++++++++++ 54 

++++++++++ 59 

xx 6 

X X 5 

8 

xxxxxx xx 39 

1 

+ 3 

++++++++++ 59 

++++++++++ 59 

++++++++++ 57 

++++++++++ 57 

2 

++++++++++ 58 

++++++++++ 59 

++++++++++ 59 

++++++++++ 57 

++++++++++ 59 

+++++++++M 45 

++++++++++ 58 

++++++++++ 59 

++++++++++ 57 

++++++++++ 55 

1 

++++++++++ 57 

1 
124 Oxazepam, NTP TR 443 

TABLEB2 

Individual Animal Tumor Pathology of Female Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam. 

5,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Pancreatic, histiocytic sarcoma 

Lymph node, mandibular 

Histiocytic sarcoma 

Lymph node, mesenteric 

Histiocytic sarcoma 

Spleen 

Histiocytic sarcoma 

Thymus

Histiocytic sarcoma 

Integumentary System 

Mammary gland 

Skin 

Musculoskeletal System 

Bone 

Skeletal muscle 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Aveolarbronchiolar adenoma 

Aveolarbronchiolar carcinoma 

NO% 

Trachea 

Special Senses System 

Ear 

Eye 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Histiocytic Sarcoma 

Lymphoma malignant mixed 

Lymphoma malignant undifferentiated 

cell type 

1122233333333333333333333 

1989900111222222233334445 

1744556468011557867790290 

1111111111111111111111111 

5536742544734246562637762 

6829251346972311208501527 

........................ 

+ + ++ 

X 

++++++++M+++++++++++++++ 

X 

........................ X 

........................ X 

++++M+M++++++++M++++++++ 

X 

++++++M+++++M++M++MM+++M 

........................ 

........................ 

........................ 

........................ + + 

+ + + 

........................ xx 

X 

........................ 

........................ 

+ + 

........................ 

........................ 

........................ X 

x x X X 
Lesions in Female Swiss-Webster Mice 125 

TABLEB2 

Individual Animal Tumor Pathology of Female Swiss-Webster Micein the 57-Week Feed Study of Oxazepam: 

5,000 ppm (continued) 

3333333333333333333333333 

Number of Days on Study 5556666677777888999999999 

0570345656669115057777777 

1111111111111111111111111 

Carcass ID Number 3452576424552366352233334 

4872934750149176554636893 

Hematopoietic System 

Bone marrow ......................... Lymph node + + + 

Pancreatic, histiocytic sarcoma 

Lymph node, mandibular ......................... Histiocytic sarcoma 

Lymph node, mesenteric ......................... Histiocytic sarcoma 

Spleen ......................... Histiocytic sarcoma 

Thymus +++++++++++++++++++++M+++ 

Histiocytic sarcoma 

Mammary gland ++M++++MM+MM+++M+++++M+++ 

Skin ......................... 

Musculoskeletal System 

Bone ......................... Skeletal muscle ......................... 

Nervous System 

Brain ......................... Peripheral nerve + + + 

Spinal cord + + + 

Respiratory System 

Lung ......................... Alveolarbronchiolar adenoma xx 

Alveolarbronchiolar carcinoma 

NO% ......................... Trachea ......................... 

Special Senses System 

Ear 

Eye + 

Urinary System 

Kidney ......................... Urinary bladder ......................... ~ ~~ ~~ ~ ~ ~ ~ ~~~ ~~ ~~ ~~~~ 

Systemic Lesions 

Multiple organs ......................... Histiocytic sarcoma 

Lymphoma malignantmixed X 

Lymphoma malignant undifferentiated 

cell type X 
126 Oxazepam, NTP TR 443 

TABLEB2 

Individual Animal Tumor Pathology of Female Swiss-Webster Mice in the 57-Week Feed Studyof Oxazepam: 

5,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Pancreatic, histiocytic sarcoma 

Lymph node, mandibular 

Histiocytic sarcoma 

Lymph node, mesenteric 

Histiocytic sarcoma 

Spleen 

Histiocytic sarcoma 

Thymus 

Histiocytic sarcoma 

Integumentary System 

Mammary gland 

Skin 

Musculoskeletal System 

Bone 

Skeletal muscle 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Alveolarbronchiolar carcinoma 

Nose 

Trachea 

Special Senses System 

Ear 

Eye 

Urinary System 

Kidney 

Urinaly bladder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant mixed 

Lymphoma malignant undifferentiated 

dl type 

3333333333 

9999999999 

7777777777 

1111111111 

4566777778 

9038046780 

++++++++++ 

++++++++++ 

++++M+++MM 

++++++++++ 

++++++++++ 

M+++M+++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

X X 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

Total 

Tissues1 

Tumors 

59 

7 

1 

58 

1 

56 

1 

59 

1 

55 

1 

44 

59 

59 

59 

59 

5 

6 

59 

6 

1 

59 

59 

2 

1 

59 

59 

59 

1 

1 

5 
Lesions in Female Swiss-Webster Mice 127 

TABLEB3 

Statistical Analysis of Primary Neoplasms in Female Swiss-Webster Micein the 57-Week Feed Study of Oxazepam 

Liver: Hepatocellular Adenoma 

Overall ratea 

Adjusted rateb 

Terminal rate' 

First incidence (days) 

Life table testd 

Logistic regression testd 

Cochran-Armita e testd ! Fisher exact test 

Liver: Hepatocellular Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Liver: Hepatocellular Adenoma or Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Lung: Alveolar/bronchiolar Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Lung: Alveolarbronchiolar Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

0/60(0%) 

0.0% 

0/47 (0%) -e

P<0.001 

P<O.001 

P<O.001 

1/60 (2%) 

2.1% 

1/47 (2%) 

397 (T)

P<O.001 

P<O.OOl 

P<O.001 

1/60 (2%) 

2.1% 

1/47 (2%) 

397 (-9

P<0.001 

P<0.001 

P<O.001 

6/60 (10%) 

12.8% 

6/47 (13%) 

397 (r)

P=O.103 

P =0.385 

P=O.552 

5/60 (8%) 

10.6% 

5/47 (11%) 

397 (-9

P=0.317N 

P=O.lllN 

P=0.074N 

22/59 (37%) 

52.6% 

lorn (36%) 

291 

P<0.001 

P<O.001 

P<0.001 

1/59 (2%) 

3.6% 

1/28 (4%) 

397 (-9

P=O.642 

P=O.642 

P=O.748 

23/59 (39%) 

55.2% 

11/28 (39%) 

291 

P<O.001 

P<0.001 

P<O.001 

4/59 (7%) 

12.4% 

3/28 (11%) 

294 

P=O.593 

P=0.515N 

P=0.382N 

3/59 (5%) 

8.9% 

2/28 (7%) 

294 

P=0.620N 

P=0.458N 

P=0.368N 

47/59 (80%) 

95.7% 

15/17 (88%) 

284 

P<O.001 

P<O.001 

P<O.001 

11/59 (19%) 

51.6% 

8/17 (47%) 

337 

P<O.001 

P<O.001 

P=O.002 

47/59 (80%) 

95.7% 

15/17 (88%) 

284 

P<0.001 

P<0.001 

P<O.001 

6/59 (10%) 

24.2% 

2/17 (12%) 

325 

P=O.105 

P=O.393 

P=0.607 

1/59 (2%) 

2.6% 

0/17 (0%) 

342 

P=0.413N 

P=0.213N 

P=0.107N 
128 Oxazepam, NTP TR 443 

TABLEB3 

Statistical Analysis of Primary Neoplasms in Female Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam 

(continued) 

Lung: Alveolar/bronchiolar Adenoma or Carcinoma 

Overall rate 11/60 (18%) 6/59 (10%) 7/59 (12%) 

Adjusted rate 23.4% 19.4% 26.2% 

Terminal rate 11/47 (23%) 5/28 (18%) 2/17 (12%) 

First incidence (days) 397 0 294 325 

Life table test P=O.266 P=0.510N P=O.259 

Logistic regression test P=0.407N P=0.346N P=0.506N 

Cochran-Armitage test P=0.183N 

Fisher exact test P=0.156N P=0.234N 

All Organs: Malignant Lymphoma (Lymphocytic,Mixed, or Undifferentiated Cell Type) 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

All Organs: Malignant Lymphoma or Histiocytic Sarcoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

All Organs: Benign Neoplasms 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

All Organs: Malignant Neoplasms 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

9/60 (15%) 13/59 (22%) 6/59 (10%) 

15.4% 26.2% 16.1% 

0/47 (0%) 2128 (7%) 1/17 (6%) 

184 106 197 

P=O.543 P=O.117 P=0.581N 

P=O.OlON P=0.260N P =0.036N 

P=0.278N 

P=O.226 P=033N 

9/60 (15%) 13/59 (22%) 7/59 (12%) 

15.4% 26.2% 17.9% 

0/47 (0%) 2128 (7%) 1/17 (6%) 

184 106 197 

P=O.447 P=O.117 P=O.516 

P=0.017N P=0.260N P =0.056N 

P=0.370N 

P=O.226 P=0.409N 

9/60 (15%) 26/59 (44%) 47/59 (80%) 

18.5% 61.2% 95.7% 

8/47 (17%) 13/28 (46%) 15/17 (88%) 

322 291 284 

P<O.001 P<O.001 P<O.001 

P<O.001 P<O.001 P <0.001 

P<O.001 

P<0.001 P <0.001 

16/60 (27%) 16/59 (27%) 17/59 (29%) 

27.7% 34.7% 61.0% 

6/47 (13%) 5/28 (18%) 9/17 (53%) 

184 106 197 

P=O.O37 P=0.230 P=0.025 

P=0.390N P =0.236N P=0.427N 

P=0.437 

P=O.560 P=O.477 
Lesions in Female Swiss-Webster Mice 129 

TABLEB3 

Statistical Analysis of Primary Neoplasms in Female Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam 

(continued) 

All Organs:Benign or Malignant Neoplasms 

Overall rate 24/60 (40%) 37/59 (63%) 49/59 (83%) 

Adjusted rate 41.8% 73.8% 95.8% 

Terminal rate 14/47 (30%) 16/28 (57%) 15/17 (88%) 

First incidence (days) 184 106 197 

Life table test P <0.001 P<O.ool P<0.001 

Logistic regression test P<O.001 P=O.O37 P<O.001 

Cochran-Armitage test P <0.001 

Fisher exact test P=O.O11 P<O.001 

(TTerminal sacrifice 

a Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for adrenal gland, 

bone marrow, brain, clitoral gland, gallbladder, heart, kidney, larynx, liver, lung, nose, ovary, pancreas, parathyroid gland, pituitary gland, salivary 

gland, spleen, thyroid gland, and urinary bladder; for other tissues, denominator is number of animals necropsied. 

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

Observed incidence at terminal kill 

Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding 

to pairwise comparisons between the controls and that exposed group. The life table analysis 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 Notapplicable;noneoplasms in animal group 
130 Oxazepam, NTP TR 443 

TABLEB4 

Summary of the Incidence of Nonneoplastic Lesions in Female Swiss-Webster Mice in the 57-Week Feed Study 

of Oxazepam" 

Disposition Summary 

Animals initially in study 

Early deaths 

Moribund 

Natural deaths 

Survivors 

Died last week of study 

Terminal sacrifice 

Missing 

Animals examined microscopically 

Alimentary System 

Intestine large, colon 

Amyloid deposition 

Hemorrhage 

Intestine large, rectum 

Amyloid deposition 

Intestine large, cecum 

Amyloid deposition 

Hemorrhage 

Ulcer 

Intestine small, duodenum 

Amyloid deposition 

Ulcer 

Intestine small, jejunum 

Amyloid deposition 

Ulcer 

Intestine small, ileum 

Amyloid deposition 

Liver 

Amyloid deposition 

Eosinophilic focus 

Hematopoietic cell proliferation 

Hepatodiaphragmatic nodule 

Infarct 

Inflammation, chronic 

Inflammation, granulomatous 

Inflammation, subacute 

Necrosis 

Centrilobular, hypertrophy 

Mesentely 

Inflammation, granulomatous 

Inflammation, subacute 

Inflammation, suppurative 

Fat, necrosis 

Pancreas 

Amyloid deposition 

Ectopic tissue 

Salivary glands 

Amyloid deposition 

60 

9 

4 

47 

60 

(59)

6 (10%) 

(59)

1(2%) 

(58)

6 (10%) 

(59)

45 (76%) 

1 (2%) 

(59)

28 (47%) 

3 (5%) 

(58)

52 (90%) 

(60)

28 (47%) 

5 (8%) 

1 (2%) 

4 (7%) 

10 (17%) 

3 (5%) 

(3)

1 (33%) 

1(33%) 

1(33%) 

(60)

1 (2%) 

(60)

35 (58%) 

60 

13 

18 

1 

27 

1 

59 

(58)

24 (41%) 

1 (2%) 

(56)

4 (7%) 

(56)

23 (41%) 

1 (2%) 

2 (4%) 

(56)

40 (71%) 

2 (4%) 

(59)

42 (71%) 

2 (3%) 

(56)

43 (77%) 

(59)

31 (53%) 

20 (34%) 

4 (7%) 

1(2%) 

1(2%) 

4 (7%) 

8 (14%) 

51 (86%) 

(1)

1(100%) 

(59)

3 (5%) 

1 (2%) 

(59)

36 (61%) 

60 

11 

31 

17 

1 

59 

(55)

30 (55%) 

(56)

12 (21%) 

(51)

34 (67%) 

(56)

47 (84%) 

(56)

49 (88%) 

(54)

46 (85%) 

(59)

35 (59%) 

14 (24%) 

2 (3%) 

1 (2%) 

1(2%) 

7 (12%) 

53 (90%) 

(3)

1 (33%) 

1(33%) 

1 (33%) 

(59)

1(2%) 

(59)

40 (68%) 
Lesions in Female Swiss-Webster Mice 131 

TABLEB4 

Summary of the Incidence of Nonneoplastic Lesions in Female Swiss-Webster Mice in the 57-Week Feed Study 

of Oxazepam (continued) 

Alimentary System (continued) 

Stomach, forestomach 

Amyloid deposition 

Hemorrhage 

Hyperkeratosis 

Stomach, glandular 

Amyloid deposition 

Mineralization 

Cardiovascular System 

Blood vessel 

Mineralization 

Heart 

Amyloid deposition 

Degeneration 

Atrium, thrombosis 

Coronary artery, inflammation, chronic 

Valve, inflammation, chronic 

Valve, thrombosis 

Endocrine System 

Adrenal cortex 

Amyloid deposition 

Hematopoietic cell proliferation 

Infiltration cellular, mononuclearcell 

Capsule, hyperplasia 

Adrenal medulla 

Hyperplasia 

Parathyroid gland 

Amyloid deposition 

Thyroid gland 

Amyloid deposition 

Follicular cell, hyperplasia 

General Body System 

None 

Genital System 

Clitoral gland 

Inflammation, suppurative 

ovary

Amyloid deposition 

Angiectasis 

Cyst

Arteriole, inflammation, chronic 

0 PPm 

(59) 

(59)

2 (3%) 

(60) 

(60) 

50 (83%) 

1(2%) 

2 (3%) 

1 (2%) 

(60)

24 (40%) 

2 (3%) 

(59)

1(2%) 

(55)

24 (44%) 

(60)

22 (37%) 

1(2%) 

(60)

1 (2%) 

(59)

24 (41%) 

1 (2%) 

7 (12%) 

3 (5%) 

2,500 ppm 

(59)

1(2%) 

1(2%) 

1(2%) 

(59)

17 (29%) 

2 (3%) 

(59)

1 (2%) 

(59)

44 (75%) 

23 (39%) 

1 (2%) 

1(2%) 

(59)

32 (54%) 

2 (3%) 

1 (2%) 

(58)

1(2%) 

(50)

32 (64%) 

(59)

38 (64%) 

(58) 

(58)

13(22%) 

5 (9%) 

4 (7%) 

5,000 ppm 

(57)

3 (5%) 

5 (9%) 

(57)

19 (33%) 

3 (5%) 

(58) 

(59)

53 (90%) 

31 (53%) 

(59)

41 (69%) 

3 (5%) 

1(2%) 

(57) 

(45)

33 (73%) 

(59)

46 (78%) 

(57)

2 (4%) 

(55)

15 (27%) 

1(2%) 

1 (2%) 

4 (7%) 
132 Oxazepam, NTP TR 443 

TABLEB4 

Summary of the Incidence of Nonneoplastic Lesions in Female Swiss-Webster Mice in the 57-Week Feed Study 

of Oxazepam (continued) 

Genital System (continued) 

Uterus 

Amyloid deposition 

Dilatation 

Infiltration cellular, polymorphonuclear 

Inflammation, suppurative 

Thrombosis 

Endometrium, hyperplasia, cystic 

Hematopoietic System 

Lymph node 

Axillary, hematopoietic cell proliferation 

Inguinal, hyperplasia, lymphoid 

Mediastinal, hematopoietic cell proliferation 

Renal, hematopoietic cell proliferation 

Lymph node, mandibular 

Amyloid deposition 

Hematopoieticcell proliferation 

Infiltration cellular, plasmacell 

Lymph node, mesenteric 

Amyloid deposition 

Crst 

Hematopoietic cell proliferation 

Hyperplasia, lymphoid 

Inflammation, suppurative 

Spleen 

Amyloid deposition 

Fibrosis 

Hematopoieticcell proliferation 

Hyperplasia, lymphoid 

Thymus 

Amyloid deposition 

Hyperplasia, lymphoid 

Integumentary System 

Skin 

Hyperkeratosis 

Ulcer 

Musculoskeletal System 

Skeletal muscle 

Hemorrhage 

Inflammation, chronic 

Nervous System 

Peripheral nelve 

Axon, degeneration 

(60)

26 (43%) 

1(2%) 

1(2%) 

1(2%) 

13 (22%) 

1(13%) 

(58)

3 (5%) 

1 (2%) 

(60)

16 (27%) 

1(2%) 

5 (8%) 

1(2%) 

(60)

12 (20%) 

5 (8%) 

14 (23%) 

(56) 

1(2%) 

(60)

1(2%) 

2 (3%) 

(4)

2 (50%) 

(59)

28 (47%) 

2 (3%) 

(10) 

1(10%) 

1(10%) 

1(10%) 

(57)

23 (40%) 

1 (2%) 

(57)

30 (53%) 

1(2%) 

1(2%) 

6 (11%) 

(59)

20 (34%) 

4 (7%) 

5 (8%) 

(57) 

1(2%) 

(59) 

1(2%) 

(5)

1(20%) 

(57)

40 (70%) 

1(2%) 

(7) 

1(14%) 

(56)

35 (63%) 

1(2%) 

(59)

39 (66%) 

1(2%) 

2 (3%) 

1(2%) 

(55) 

(59) 

1(2%) 

(5) 
Lesions in Female Swiss-Webster Mice 133 

TABLEB4 

Summary of the Incidence of Nonneoplastic Lesions in Female Swiss-Webster Mice in the 57-Week Feed Study 

of Oxazepam (continued) 

Respiratory System 

Lung (60) (59) (59)

Fibrosis 1 (2%) 14 (24%) 8 (14%) 

Infiltration cellular, lymphocyte 1 (2%) 1 (2%) 

Inflammation, chronic 3 (5%) 2 (3%) 6 (10%) 

Inflammation, granulomatous 1 (2%) 

Inflammation, subacute 4 (7%) 4 (7%) 3 (5%) 

Thrombosis 1 (2%) 

Alveolar epithelium, hyperplasia 6 (10%) 3 (5%) 2 (3%) 

Alveolus, infiltration cellular, mononuclear 

Cell 2 (3%) 16 (27%) 26 (44%) 

NO%? (60) (59) (59)

Amyloid deposition 1 (2%) 

Inflammation, suppurative 1 (2%) 

Special Senses System 

None 

Urinary System 

Kidney (60) (59) (59) 

Cyst 1 (2%) 

Infarct 1 (2%) 

Necrosis 1 (2%) 

Glomerulus, amyloid deposition 48 (80%) 39 (66%) 39 (66%) 

Interstitium, amyloid deposition 5 (8%) 5 (8%) 7 (12%) 

a Number of animals examined microscopically at site and number of animals with lesion 
134 Oxazepam, NTP TR 443 
135 

APPENDIX C 

SUMMARY OF LESIONS 

IN MALE B6C3Fl MICE 

IN THE 2-YEAR FEED STUDY 

OF OXAZEPAM 

TABLECl Summary of the Incidence of Neoplasms in Male B6C3F, Mice 

in the 2-Year Feed Study of Oxazepam .................................... 137 

TABLE C2 Individual Animal Tumor Pathologyof Male B6C3Fl Mice 

in the 2-Year Feed Study of Oxazepam .................................... 142 

TABLE C3 Statistical Analysis of Primary Neoplasms in Male B6C3Fl Mice 

in the 2-Year Feed Study of Oxazepam .................................... 164 

TABLEC4 Historical Incidenceof Liver Neoplasms in Untreated Male B6C3F, Mice.......... 167 

TABLEC5 Summary of the Incidence of NonneoplasticLesionsinMale B6C3F,Mice 

in the 2-Year Feed Study of Oxazepam .................................... 168 
136 Oxazepam, NTP TR 443 
Lesions in Male B6C3Fl Mice 137 

TABLEC1 

Summary of the Incidence of Neoplasms in Male B6C3F, Mice in the 2-YearFeed Study of Oxazepama 

0 PPm 125 ppm 2,500 ppm 5,000 ppm 

Disposition Summary 

Animals initially in study 60 60 60 60 

ISM& intsim Nnlkrrtian 10 10 10 10 

Early deaths 

Moribund 2 4 22 30 

Natural deaths 3 2 13 20 

survivors 

Terminal sacrifice 45 44 1.5 

Animals examined microscopically 60 60 60 60 

I5-Month Interim Evaluation 

Alimentary System 

Liver (10) (10) (10)

Hepatoblastoma 1 (10%) 

Hepatocellular carcinoma 1 (10%) 4 (40%) 1 (10%) 

Hepatocellular carcinoma, multiple 8 (80%) 

Hepatocellular adenoma 3 (30%) 1 (10%) 

Hepatocellular adenoma, multiple 8 (80%) 9 (90%) 

Cardiovascular System 

None 

Endocrine System 

None 

General Body System 

None 

Genital System 

Testes 

Interstitial cell, adenoma 

Hematopoietic System 

None 

Integumentary System 

None 

Musculoskeletal System 

None 
138 Oxazepam, NTP TR 443 

TABLEC1 

Summary of the Incidence of Neoplasms in Male B6C3F, Mice in the 2-Year Feed Study of Oxazepam (continued) 

15-Month Interim Evaluation (continued) 

Nervous System 

None 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Hepatocellular carcinoma, metastatic, liver 

Special Senses System 

None 

Urinary System 

None 

2-Year Study 

Alimentary System 

Gallbladder 

Histiocytic sarcoma 

Intestine large, colon 

Intestine small, duodenum 

Polyp adenomatous 

Intestine small, jejunum 

Carcinoma 

Liver 

Hemangiosarcoma, multiple 

Hemangiosarcoma, metastatic, skin 

Hepatoblastoma 

Hepatoblastoma, multiple 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma 

Sarcoma, metastatic, kidney 

Mesentery 

Histiocytic sarcoma 

Fat, hepatocellular carcinoma, metastatic, 

liver 

Pancreas 

Hepatoblastoma, metastatic, liver 

Histiocytic sarcoma 

Salivary glands 

Stomach, forestomach 

Squamous cell carcinoma 

Squamous cell papilloma 

0 PPm 

(10)

2 (20%) 

(47) 

(49) 

(49) 

1(2%) 

(49)

1 (2%) 

(49)

1 (2%) 

8 (16%) 

1 (2%) 

10 (20%) 

7 (14%) 

1 (2%) 

(2)

1 (50%) 

(49) 

1 (2%) 

(50) 

(50)

1 (2%) 

1 (2%) 

125 ppm 

(10)

1 (10%) 

(50) 

1 (2%) 

(50) 

(50) 

(50)

1 (2%) 

(50) 

1 (2%) 

2 (4%) 

3 (6%) 

2 (4%) 

9 (18%) 

9 (18%) 

1 (2%) 

(2)

1(50%) 

2,500 

(10) 

1 (2%) 

20 (40%) 

1 (2%) 

3 (6%) 

42 (84%) 

6 (12%) 

28 (56%) 

1 (2%) 

(2) 

5,000 ppm 

12 (24%) 

1 (2%) 

5 (10%) 

45 (90%) 

5 (10%) 

27 (54%) 
Lesions in Male B6C3F, Mice 139 

TABLEC1 

Summary of the Incidence of Neoplasms in Male B6C3Fl Mice in the 2-Year Feed Study of Oxazepam (continued) 

0 PPm 125 ppm 2,500 5,000 PPm 

2-Year Study (continued) 

Cardiovascular System 

None 

Endocrine System 

Adrenal cortex 

Adenoma 

Capsule, adenoma 

Capsule, hepatoblastoma, metastatic, liver 

Capsule, histiocytic Sarcoma 

Adrenal medulla 

Pheochromocytomabenign 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

None 

Genital System 

Prostate 

Hepatoblastoma, metastatic, liver 

Histiocytic sarcoma 

Hematopoietic System 

Bone marrow 

Hemangiosarcoma 

Lymph node 

Lumbar, histiocytic sarcoma 

Popliteal, histiocytic sarcoma 

Renal, hepatoblastoma, metastatic, liver 

Lymph node, mandibular 

Carcinoma, metastatic, harderiangland 

Histiocytic sarcoma 

Lymph node, mesenteric 

Histiocytic sarcoma 

Sarcoma, metastatic, kidney 

Lymph node, mediastinal 

Histiocytic sarcoma 

Spleen 

Hemangiosarcoma 

Hemangiosarcoma, metastatic, skin 

Histiocytic Sarcoma 

Thymus 

Histiocytic sarcoma 

(49) 

6 (12%) 

1 (2%) 

(49) 

(48)

1 (2%) 

(49) 

(44) 

(48)

1 (2%) 

(49) 

1 (2%) 

(43)

1 (2%) 

(50)

1 (2%) 

(1) 

1(100%) 

(45)

1 (2%) 

1 (2%) 

(49)

1 (2%) 

(1)

1 (100%) 

(50)

1 (2%) 

1 (2%) 

(43)

1 (2%) 
140 Oxazepam, NTP TR 443 

TABLEC1 

Summary of the Incidence of Neoplasms in Male B6C3F, Mice in the 2-YearFeed Study of Oxazepam (continued) 

0 PPm 125 ppm 2,500 5,000 ppm 

2-Year Stud’ (continued) 

Integumentary System 

Skin 

Subcutaneous tissue, hemangiosarcoma 

Subcutaneous tissue, melanoma NOS 

Musculoskeletal System 

Skeletal muscle 

Hemangiosarcoma 

Nervous System 

None 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Alveolarlbronchiolar adenoma, multiple 

Alveolarbronchiolar carcinoma 

Alveolarbronchiolar carcinoma, multiple 

Carcinoma, metastatic, harderian gland 

Hemangiosarcoma 

Hepatoblastoma, metastatic, liver 

Hepatocellular carcinoma, metastatic, liver 

Histiocytic sarcoma 

Sarcoma, metastatic, kidney 

Special Senses System 

Ear 

External ear, fibrosarcoma 

Eye

Carcinoma, metastatic, harderian gland 

Ciliary body, adenoma 

Harderian gland 

Adenoma 

Carcinoma 

Bilateral, adenoma 

Urinary System 

Kidney 

Histiocytic sarcoma 

Sarcoma 

Urinary bladder 

Histiocytic sarcoma 

(50) 

(50)

9 (18%) 

2 (4%) 

2 (4%) 

1 (2%) 

(2)

1 (50%) 

(3)

1 (33%) 

1 (33%) 

1 (33%) 

(49)

1 (2%) 

(50)

1 (2%) 

(50)

11 (22%) 

6 (12%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

3 (6%) 

1 (2%) 

(1)

1 (100%) 

(2)

1 (50%) 

1 (50%) 

(2)

1 (50%) 

1 (50%) 

(50)

1 (2%) 

1 (2%) 

(50)

5 (10%) 

I (14%) 

10 (20%) 

1 (2%) 

(50) 

(50)

1 (2%) 

5 (10%) 

16 (32%) 

(1)

1 (100%) 
Lesions in Male B6C3Fl Mice 141 

TABLEC1 

Summary of the Incidence of Neoplasms in Male B6C3Fl Mice in the 2-Year Feed Study of Oxazepam (continued) 

125 ppm 2,500 5,000 ppm 

2-Year Study (continued) 

Systemic Lesions 

Multiple organsb 

Histiocytic sarcoma (50)

1(2%) 

(50)

1 (2%) 

(50) (50) 

Lymphoma malignant lymphocytic 1 (2%) 

Lymphoma malignant mixed 2 (4%) 1(2%) 1(2%) 

Neoplasm Summary 

Total animals with primary neoplasmsc 

15-Month interim evaluation 2 5 9 10 

2-Year study 37 35 50 50 

Total primary neoplasms 

15-Month interim evaluation 2 6 13 19 

2-Year study 57 63 112 99 

Total animals with benign neoplasms 

15-Month interim evaluation 2 5 9 9 

2-Year study 30 30 37 33 

Total benign neoplasms 

15-Month interim evaluation 2 5 9 9 

2-Year study 39 43 42 35 

Total animals with malignant neoplasms 

15-Month interim evaluation 1 4 10 

2-Year study 17 18 47 50 

Total malignant neoplasms 

15-Month interim evaluation 1 4 10 

2-Year study 18 20 69 64 

Total animals with metastatic neoplasms 

15-Month interim evaluation 1 

2-Year study 2 4 21 21 

Total metastatic neoplasms 

15-Month interim evaluation 1 

2-Year study 2 7 27 21 

Total animals with uncertain neoplasms -

benign or malignant 

2-Year study 1 

Total uncertain neoplasms 

2-Year study 1 

a Number of animals examined microscopically at site and number of animals with lesion 

Number of animals with any tissue examined microscopically 

Primary neoplasms: a11 neoplasms except metastatic neoplasms 
142 Oxazepam, NTP TR 443 

TABLE C2 

Individual Animal Tumor Pathology of Male B6C3F, Mice in the 2-Year Feed Study of Oxazepam: 0 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Polyp adenomatous 

Intestine small, jejunum 

Carcinoma 

Intestine small, ileum 

Liver 

Hemangiosarcoma, multiple 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma 

Mesentery 

Histiocytic sarcoma 

Pancreas 

Histiocytic sarcoma 

Salivary glands 

Stomach, forestomach 

Squamous cell carcinoma 

Squamous cell papilloma 

Stomach, glandular 

Tooth 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Capsule, adenoma 

Capsule, histiocytic sarcoma 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

General Body System 

None 

+: Tissueexaminedmicroscopically 

A: Autolysis precludes examination 

1566111111711111111111171 

1822022222222222222222223 

1641199999999999999999990 

0000000000000000000000000 

2205400001112233334455560 

1033812561296802455925804 

......................... 

+M++M+++M++++++++++++++++ 

A++++++++++++++++++++++++ 

A++++++++++++++++++++++++ 

A++++++++++++++++++++++++ 

A++++++++++++++++++++++++ 

......................... 

X 

A++++++++++++++++++++++++ 

A++++++++++++++++++++++++ 

X 

xx X x x 

X 

xx X X 

xx X xx 

X 

+ 

X 

A++++++++++++++++++++++++ 

X 

......................... 

......................... 

X 

......................... + 

......................... 

......................... 

M++++++++++++++++++++++++ 

xx X 

X 

M++++++++++++++++++++++++ 

A++++++++++++++++++++++++ 

M++M+++++++++++++++++++++ 

M++++++++++++++++++++++++ 

A++++++++++++++++++++++++ 

M. Missing tissue X Lesion present 

I: Insufficienttissue Blank:Notexamined 
Lesions in Male B6C3F, Mice 143 

TABLEC2 

Individual Animal Tumor Pathology of Male B6C3Fl Mice in the 2-Year Feed Study of Oxazepam: 0 ppm (continued) 

~~~~ 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Polyp adenomatous 

Intestine small, jejunum 

Carcinoma 

Intestine small, ileum 

Liver 

Hemangiosarcoma, multiple 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic Sarcoma 

Mesentery 

Histiocytic Sarcoma 

Pancreas 

Histiocytic Sarcoma 

Salivary glands 

Stomach, forestomach 

Squamous cell carcinoma 

Squamous cell papilloma 

Stomach, glandular 

Tooth 

Cardiovascular System 

Blood vessel 

Hart 

Endocrine System 

Adrenal cortex 

Capsule, adenoma 

Capsule, histiocytic Sarcoma 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

General Body System 

None 

7777777777777777777777777 

3333333333333333333333333 

0000000000000000000000000 

0000000000000000000000000 Total 

0011112222333444444555555 Tissues/ 

7804672347379012367014679 Tumors 

......................... 50 

......................... 47 

......................... 49 

......................... 49 

......................... 49 

......................... 49 

X 1 

......................... 49 

1 

......................... 49 

......................... 49 

1 

X X X 8 

1 

X X xx X X 10 

X X 7 

1 

+ 2 

1 

......................... 49 

1 

......................... 50 

......................... 50 

X 1 

1 

......................... 50 

1 

......................... 50 

......................... 50 

......................... 49 

X X X 6 

1 

......................... 49 

......................... 49 

......................... 48 

......................... 48 

X 1 

......................... 49 
144 Oxazepam, NTP TR 443 

TABLEC2 

Individual Animal Tumor Pathology of Male B6C3F, Mice in the 2-Year Feed Study of Oxazepam: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Genital System 

Epididymis 

Preputial gland 

Prostate 

Histiocytic Sarcoma 

Seminal vesicle 

Testes 

Hematopoietic System 

Bone marrow 

Lymph node 

Lumbar, histiocytic sarcoma 

Lymph node, mandibular 

Lymph node, mesenteric 

Histiocytic sarcoma 

Spleen 

Histiocytic sarcoma 

Thymus 

Histiocytic Sarcoma 

Integumentary System 

Mammary gland 

Skin 

Musculoskeletal System 

Bone 

Skeletal muscle 

Nervous System 

Brain 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Alveolarbronchiolar adenoma, 

multiple 

Alveolarlbronchiolar carcinoma 

Hepatocellular carcinoma, metastatic, 

liver 

NO.% 

Trachea 

Special Senses System 

Eye

Carcinoma, metastatic, harderian 

gland 

Harderian gland 

Adenoma 

Carcinoma 

Bilateral, adenoma 

1566111111111711111111171 ~~ 

1822022222222222222222223 

I641199999999999999999990 

0000000000000000000000000 

2205400001112233334455560 

1033812561296802455925804 

......................... 

......................... 

M++++++++++++++++++++++++ 

X 

M++++++++++++++++++++++++ 

......................... 

......................... + 

X 

MM++++++++M++++++++++++++ 

M+++M++++++++++++++++++++ 

X 

A++++++++++++++++++++++++ 

X 

+++++++++++++++++M+M+++++ 

X 

MMMMMMMMMMMMMMMMMMMMMMMMM 

......................... 

......................... 

......................... 

......................... 

......................... X xx X X 

X 

X 

X 

......................... 

......................... 

+ + 

X 

+ + + 

X 

X 

X 
Lesions in Male B6C3Fl Mice 145 

TABLEC2 

Individual Animal Tumor Pathology of Male B6C3Fl Mice in the 2-Year Feed Study of Oxazepam: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Genital System 

Epididymis 

Preputial gland 

Prostate 

Histiocytic sarcoma 

Seminal vesicle 

Testes 

Hematopoietic System 

Bone marrow 

Lymph node 

Lumbar, histiocytic sarcoma 

Lymph node, mandibular 

Lymph node, mesenteric 

Histiocytic sarcoma 

Spleen 

Histiocytic sarcoma 

Thymus 

Histiocytic sarcoma 

Integumentary System 

Mammary gland 

Skin 

Musculoskeletal System 

Bone 

Skeletal muscle 

Nervous System 

Brain 

Respiratory System 

Lung 

Alveolaribronchiolar adenoma 

Alveolaribronchiolar adenoma, 

multiple 

Alvealaribronchiolar carcinoma 

Hepatocellular carcinoma, metastatic, 

liver 

Nose 

Trachea 

Special Senses System 

Eye

Carcinoma, metastatic, harderian 

gland 

Harderian gland 

Adenoma 

Carcinoma 

Bilateral, adenoma 

7777777777777777777777777 

3333333333333333333333333 

0000000000000000000000000 

0000000000000000000000000 Total 

0011112222333444444555555 Tissues/ 

7804672347379012367014679 Tumors 

......................... 50 

......................... 50 

......................... 49 

1 

......................... 49 

......................... 50 

......................... 50 

+ 2 

1 

+MM+++++++++++M++++++++++ 44 

......................... 48 

1 

......................... 49 

1 

+M+M++++++M+M+++++++++M++ 43 

1 

MMMMMMMMMMMMMMMMMMMMMMMMM 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

X X X X 9 

X 2 

X 2 

1 

......................... 50 

......................... 50 

2 

1 

3 

1 

1 

1 
146 Oxazepam, NTP TR 443 

TABLEC2 

Individual Animal Tumor Pathology of Male B6C3F, Mice in the 2-Year Feed Study of Oxazepam: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

~ ~~ 

Urinary System 

Kidney 

Histiocytic sarcoma 

Urinary bladder 

Histiocytic sarcoma 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant mixed 

1566111111111111111111111 

1822022222222222222222223 

I641199999999999999999990 

0000000000000000000000000 

2205400001112233334455560 

1033812561296802455925804 

......................... X 

M++++++++++++++++++++++++ 

X 

.......................... X 

X 
Lesions in Male B6C3F, Mice 147 

TABLEC2 

Individual Animal Tumor Pathology ol Male B6C3F, Mice in the 2-YearFeed Study of Oxazepam: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Urinary System 

Kidney 

Histiocytic Sarcoma 

Urinary bladder 

Histiocytic Sarcoma 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant mixed 

7111111111111111711111111 

3333333333333333333333333 

0000000000000000000000000 

0000000000000000000000000 Total 

0011112222333444444555555 Tissues/ 

7804612347319012367014619 Tumors 

......................... 50 

1 

......................... 49 

I 

......................... 50 

1 

X 2 
148 Oxazepam, NTP TR 443 

TABLEC2 

Individual Animal Tumor Pathology of B6C3Fl Male Mice in the 2-Year Feed Study of Oxazepam: 125 ppm -

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Histiocytic sarcoma 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Carcinoma 

Intestine small, ileum 

Liver 

Hemangiosarcoma, multiple 

Hepatoblastoma 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma 

Mesentery 

Histiocytic sarcoma 

Pancreas 

Histiocytic sarcoma 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Blood vesse.1 

Heart 

Endocrine System 

Adrenal cortex 

Capsule, adenoma 

Adrenal medulla 

Pheochromocytoma benign 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

None 

Genital System 

Epididymis 

Preputial gland 

Prostate 

Seminal vesicle 

Testes 

4556777777777777777777777 

5022012222222222222222222 

3875159999999999999999999 

0100000000000000000001111 

8189676677778889999990002 

0028432945673490145671280 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

X X 

X 

X X 

X xx X X X 

X X xx xx 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... X 

......................... 

......................... 

+++M+++++++++++++++++++++ 

......................... 

......................... X 

......................... 

......................... 

......................... 

......................... 

......................... 
Lesions in Male B6C3Fl Mice 149 

TABLEC2 

Individual Animal Tumor Pathology of Male B6C3Fl Mice in the 2-Year Feed Study of Oxazepam: 125 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

~~ ~~~ 

Alimentary System 

Esophagus 

Gallbladder 

Histiocytic sarcoma 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Carcinoma 

Intestine small, ileum 

Liver 

Hemangiosarcoma, multiple 

Hepatoblastoma 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma 

Mesentery 

Histiocytic sarcoma 

Pancreas 

Histiocytic sarcoma 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Capsule, adenoma 

Adrenal medulla 

Pheochromocytoma benign 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

None 

Genital System 

Epididymis 

Preputial gland 

Prostate 

Seminal vesicle 

Testa 

7777777777777777777777777 

3333333333333333333333333 

0000000000000000000000000 

0000000000000111111111111 Total 

6666677888999000001111111 Tissues/ 

1356718158239034572345679 Tumors 

......................... 50 

......................... 50 

X 1 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

X 1 

......................... 50 

......................... 50 

X 1 

X X 3 

2 

X X X 9 

X X X 9 

X 1 

+ + 2 

X 1 

......................... 50 

X 1 

......................... 50 

......................... 50 

......................... 50 

......................... SO 

......................... 50 

......................... 50 

X X X 4 

......................... 50 

X 1 

......................... 50 

++++++++++++++M++++++++++ 48 

......................... 50 

......................... 50 

1 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 
150 Oxazepam, NTP TR 443 

TABLEC2 

Individual Animal Tumor Pathology of Male B6C3F, Mice in the 2-Year Feed Study of Oxazepam: 125 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Hemangiosarcoma 

Lymph node 

Popliteal, histiocytic sarcoma 

Lymph node, mandibular 

Carcinoma, metastatic, harderian 

gland 

Histiocytic sarcoma 

Lymph node, mesenteric 

Histiocytic sarcoma 

Lymph node, mediastinal 

Histiocytic sarcoma 

Spleen 

Hemangiosarcoma 

Histiocytic sarcoma 

Thymus 

Histiocytic sarcoma 

Integumentary System 

Mammary gland 

Skin 

Subcutaneous tissue, hemangiosarcoma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Hemangiosarcoma 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

Respiratory System 

~ ~~ 

Lung 

Alveolarbronchiolar adenoma 

Alveolarbronchiolar adenoma, 

multiple 

Alveolarbronchiolar carcinoma 

Alveolarbronchiolar carcinoma, 

multiple 

Carcinoma, metastatic, harderian 

gland 

Hemangiosarcoma 

Hepatoblastoma, metastatic, liver 

Hepatocellular carcinoma, metastatic, 

liver 

Histiocytic sarcoma 

Nose 

Trachea 

4556111111111111111111111 

5022012222222222222222222 

3815159999999999999999999 

0100000000000000000001111 

8189616611118889999990002 

0028432945613490145611280 

......................... X 

........................ 

X 

+M+++++++++++++++++++++++ 

......................... X 

+++M+++++++++++++MM++++++ 

MMMMMMMMMMMMMMMMMMMMMMMMM 

......................... X 

......................... 

......................... X 

......................... + 

+ 

......................... xx xxx x X 

A

v 

X 

X 

X 

X 

X X 

......................... 

......................... 
Lesions in Male B6C3Fl Mice 151 

TABLEC2 

Individual Animal Tumor Pathology of Male B6C3F, Mice in the 2-YearFeed Study of Oxazepam: 125 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Hemangiosarcoma 

Lymph node 

Popliteal, histiocytic Sarcoma 

Lymph node, mandibular 

Carcinoma, metastatic, harderian 

gland 

Histiocytic Sarcoma 

Lymph node, mesenteric 

Histiocytic Sarcoma 

Lymph node, mediastinal 

Histiocytic Sarcoma 

Spleen 

Hemangiosarcoma 

Histiocytic Sarcoma 

Thymus 

Histiocytic Sarcoma 

Integumentary System 

Mammary gland 

Skin 

Subcutaneous tissue, hemangiosarcoma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Hemangiosarcoma 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Alveolaribronchiolar adenoma 

Alveolaribronchiolar adenoma, 

multiple 

Alveolaribronchiolar carcinoma 

Alveolaribronchiolar carcinoma, 

multiple 

Carcinoma, metastatic, harderian 

gland 

Hemangiosarcoma 

Hepatoblastoma, metastatic, liver 

Hepatocellular carcinoma, metastatic, 

liver 

Histiocytic Sarcoma 

Nose 

Trachea 

1111111111111111111111111 

3333333333333333333333333 

0000000000000000000000000 

0000000000000111111111111 Total 

6666611888999000001111111 Tissues/ 

1356118158239034512345619 Tumors 

......................... 50 

1 

+ 1 

X 1 

++M++++++++++++++++MM++++ 45 

1 

X 1 

......................... 49 

X 1 

+ 1 

X 1 

......................... 50 

1 

X 1 

++M+++++++++M+++M+++++M++ 43 

X 1 

MMMMMMMMMMMMMMMMMMMMMMMMM 

......................... 50 

1 

......................... 50 

......................... 50 

1 

......................... 50 

1 

1 

......................... 50 

x x X X 11 

x xxx x 6 

X 1 

1 

1 

1 

1 

X 3 

X 1 

......................... 50 

......................... 50 
152 Oxazepam, NTP TR 443 

TABLE C2 

Individual Animal Tumor Pathology of Male B6C3F, Mice in the 2-Year Feed Study of Oxazepam: 125 ppm (continued) 

4556777777777777777777777 

Number of Days on Study 

3875159999999999999999999 

5022012222222222222222222 

Carcass ID Number 

0028432945673490145671280 

8189676677778889999990002 

0100000000000000000001111 

~~ ~~ ~ ~ 

Special Senses System 

Ear 

~ ~ 

External ear, fibrosarcoma 

Eye

Carcinoma, metastatic, harderian 

gland 

Ciliary body, adenoma 

Harderian gland 

Adenoma 

Carcinoma 

~~ ~~ 

Urinary System 

Kidney ......................... Histiocytic sarcoma 

Urinary bladder ......................... 

Systemic Lesions 

Multiple organs ......................... Histiocytic sarcoma 

Lymphoma malignant mixed 
Lesions in Male B6C3Fl Mice 153 

TABLEC2 

Individual Animal Tumor Pathology of Male B6C3FlMice in the 2-YearFeed Study of Oxazepam: 125 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Special Senses System 

Ear 

External ear, fibrosarcoma 

Eye

Carcinoma, metastatic, harderian 

gland 

Ciliary body, adenoma 

Harderian gland 

Adenoma 

Carcinoma 

Urinary System 

Kidney 

Histiocytic sarcoma 

Urinary Bladder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant mixed 

7777777777777777777777777 

3333333333333333333333333 

0000000000000000000000000 

0000000000000111111111111 Total 

6666677888999000001111111 Tissues/ 

1356718158239034572345679 Tumors 

+ 1 

X 1 

+ 2 

1 

X 1 

+ 2 

X 1 

1 

......................... 50 

x 1 

......................... 50 

......................... 50 

X 1 

X 1 
154 Oxazepam, NTP TR 443 

TABLE C2 

Individual Animal Tumor Pathology of Male B6C3F, Mice in the 2-Year Feed Study of Oxazepam: 2,500 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hemangiosarcoma, metastatic, skin 

Hepatoblastoma 

Hepatoblastoma,multiple 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellularadenoma 

Hepatocellularadenoma, multiple 

Sarcoma, metastatic, kidney 

Mesentery 

Fat, hepatocellularcarcinoma, 

metastatic, liver 

Pancreas 

Hepatoblastoma, metastatic,liver 

Salivary glands 

Stomach, forestomach 

Squamous cell papilloma 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adenoma 

Capsule, hepatoblastoma,metastatic, 

liver 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

None 

4555556666666666666666666 

8246990112334446666777789 

6307488568892565679339927 

1111111111111111111111111 

2533244366855624642356777 

1928212575042948396674846 

......................... 

++++++++MM+++++M+++++++M+ 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

.......................... X 

xx x xx xxx xx x x 

X 

X X 

xxxxx xxxxxx xxxxxx xxx 

X X xx 

xxxxxxx X xxxx xx x 

+ 

X ......................... X 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

X ......................... 

......................... 

+++++++++++M+++++++++++++ 

++M+M++M++MM++M+M++++++++ 

......................... 
Lesions in Male B6C3F, Mice 155 

TABLEC2 

Individual Animal Tumor Pathology of Male B6C3F, Mice in the 2-Year Feed Study of Oxazepam: 2,500 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hemangiosarcoma, metastatic, skin 

Hepatoblastoma xx 

Hepatoblastoma, multiple 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Sarcoma, metastatic, kidney 

Mesentery 

Fat, hepatocellular carcinoma, 

metastatic, liver 

Pancreas 

Hepatoblastoma, metastatic, liver 

Salivary glands 

Stomach, forestomach 

Squamous cell papilloma 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adenoma 

Capsule, hepatoblastoma, metastatic, 

liver 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

None 

7777777777777777777777777 

,0011222222222222222222222 

0304000227999999999999999 

1111111111111111111111111 Total 

3356777356223444555667777 Tissues/ 

9080359766383034135120127 Tumors 

......................... 50 

M++++++M+++++++++++++++++ 44 

......................... 50 

......................... 50 

......................... 50 

......................... 49 

......................... 50 

......................... 50 

......................... 50 

X xxx 

xxxxxxxxxxxx 

X 

X xx x 

X 

+ 

1 

x x 20 

1 

X 3 

x xxxxxxxxx 42 

X 6 

xxxx x x xx x 28 

1 

2 

1 

......................... 50 

1 

......................... 50 

......................... 50 

X 1 

......................... 50 

......................... 50 

......................... 50 

++++++++ ++++++++++++++++ 49 

X 1 

1 

......................... 50 

......................... 50 

+++++MM+++++++++++M++++++ 46 

+++++++++M+++++++++++++++ 42 

......................... 50 

X 1 
156 Oxazepam, NTP TR 443 

TABLEC2 

Individual Animal Tumor Pathology of Male B6C3F, Mice in the &Year Feed Study of Oxazepam: 2,500 ppm (continued) 

4555556666666666666666666 

Number of Days on Study 8246990112334446666777789 

6307488568892565679339927 

1111111111111111111111111 

Carcass ID Number 2533244366855624642356777 

1928212575042948396674846 

~ ~~ ~ ~~ ~~ ~~ ~ ~ ~ ~~ ~ ~~ ~ ~~ ~ ~~~~ 

Genital System 

Epididymis ......................... Preputial gland ......................... Prostate ......................... Hepatoblastoma, metastatic, liver X 

Seminal vesicle ......................... Testes ......................... 

Hematopoietic System 

Bone marrow ......................... Lymph node + 

Renal, hepatoblastoma, metastatic, 

liver X 

Lymph node, mandibular +++M++M+++M+++++++++++MM+ 

Lymph node, mesenteric +++++++++M+++++++++M++M++ 

Sarcoma, metastatic, kidney 

Spleen ......................... Hemangiosarcoma, metastatic, skin X 

Thymus +++++++++M+++M++++MMMM++M ~~~~~ ~ ~ ~~ ~ ~ ~~ ~~ ~~ ~ ~ ~ ~~ 

Integumentary System 

Mammary gland MMMMMMMMMMMMMMMMMMMMMMMMM 

Skin ......................... Subcutaneous tissue, hemangiosarcoma X 

Subcutaneous tissue, melanoma NOS 

Musculoskeletal System 

Bone ......................... Skeletal muscle ......................... 

Nervous System 

Brain ......................... Peripheral nerve + + 

Spinal cord + + 

Respiratory System 

Lung ......................... Alveolarbronchiolar adenoma X 

Hepatoblastoma, metastatic, liver xx xx X 

Hepatocellular carcinoma, metastatic, 

liver X X xx 

Sarcoma, metastatic, kidney 

Nose ......................... Trachea ......................... 

Special Senses System 

None 

X 
Lesions in Male B6C3F, Mice 157 

TABLEC2 

Individual Animal Tumor Pathology of Male B6C3F, Mice in the 2-YearFeed Study of Oxazepam: 2,500 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Genital System 

Epididymis 

Preputial gland 

Prostate 

Hepatoblastoma, metastatic, liver 

Seminal vesicle 

Testes 

Hematopoietic System 

Bone marrow 

Lymph node 

Renal, hepatoblastoma, metastatic, 

liver 

Lymph node, mandibular 

Lymph node, mesenteric 

Sarcoma, metastatic, kidney 

Spleen 

Hemangiosarcoma, metastatic, skin 

Thymus 

Integumentary System 

Mammary gland 

Skin 

Subcutaneous tissue, hemangiosarcoma 

Subcutaneous tissue, melanoma NOS 

Musculoskeletal System 

Bone 

Skeletal muscle 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Hepatoblastoma, metastatic, liver 

Hepatocellular carcinoma, metastatic, 

liver 

Sarcoma, metastatic, kidney 

Nose 

Trachea 

Special Senses System 

None 

7777777771777117777771777 

0011222222222222222222222 

0304000227999999999999999 

1111111111111111111111111 Total 

3356777356223444555667777 Tissues/ 

9080359766383034135120127 Tumors 

......................... 50 

......................... 50 

......................... 50 

1 

......................... 50 

......................... 50 

......................... 50 

1 

1 

M++++++++++++++M++++M++++ 42 

+++M+M+++++++++++++++++++ 45 

X 1 

......................... 50 

1 

+MMM+++M+++++++++M++++M++ 37 

MMMMMMMMMMMMMMMMMMMMMMMMM 

......................... 50 

1 

X 1 

......................... 50 

......................... 50 

......................... 50 

2 

2 

......................... 50 

xx X X 5 

X X 7 

xxx X X 10 

X 1 

......................... 50 

......................... 50 
158 Oxazepam, NTP TR 443 

TABLE C2 

Individual Animal Tumor Pathology of Male B6C3Fl Mice in the 2-Year Feed Study of Oxazepam: 2,500 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Urinary System 

Kidney 

Sarcoma 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Lymphoma malignant mixed 

4555556666666666666666666 

8246990112334446666717189 

6301488568892565679339927 

1111111111111111111111111 

2533244366855624642356717 

1928212575042948396674846 

......................... 

......................... 

......................... X 
Lesions in Male B6C3F, Mice 159 

TABLEC2 

Individual Animal Tumor Pathology of Male B6C3F, Mice in the 2-Year Feed Study of Oxazepam: 2,500 ppm (continued) 

Number of Days on Study 

0304000227999999999999999 

0011222222222222222222222 

1171711111111117711111777 

Carcass ID Number 

1111111111111111111111111 

3356711356223444555667777 

9080359766383034135120127 

Total 

Tissues/

Tumors 

Urinary bladder 

Kidney 

Urinary System 

Sarcoma t, 

......................... 

......................... X 

50 

1 

50 

~ 

Systemic Lesions ~~ 

Multiple organs 

Lymphoma malignant mixed 

......................... 1 

50 
160 Oxazepam, NTP TR 443 

TABLEC2 

Individual Animal Tumor Pathology of Male B6C3F, Mice in the 2-Year Feed Study of Oxazepam: 5,000 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatoblastoma 

Hepatoblastoma, multiple 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart ~ ~ ~ ~~ ~~ ~~ ~ 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

None 

Genital System 

Epididymis 

Preputial gland 

Prostate 

Seminal vesicle 

Testes 

4444444445555555555555555 

0135566770133334466666667 

1340114997415792767789995 

222222212121221222221222'1 

3012330818083292320292229 

3034121557434362855651791 

......................... 

+M+M++M+M++M+M+M++M++M++M 

+++++++++++M+++++++++++++ 

+++++++++++M+++++++++++++ 

+++++++++++M+++++++++++++ 

......................... 

......................... 

......................... 

......................... X X X xx 

X 

X X X 

xxxxxxxxxxxxx xxxxxxx xx 

xx 

X xxxxxx xx xxx x xxx 

+ + ......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

MM++MM+++++++++M++M++++++ 

+++++++M+++MIM+M+MM++++++ 

......................... 

......................... 

++++++++++++++++++++M++++ 

......................... 

......................... 

.......................... 
Lesions in Male B6C3FLMice 161 

TABLEC2 

Individual Animal Tumor Pathology of Male B6C3F, Mice in the 2-Year Feed Study of Oxazepam: 5,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatoblastoma 

Hepatoblastoma, multiple 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

HEirt 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

None 

Genital System 

Epididymis 

Preputial gland 

Prostate 

Seminal vesicle 

Testes 

5555555556666666666666666 

7889999990111112222222222 

5793566689245772233445588 

1212212221222222222121212 Total 

9093481319200011313918090 Tissues/ 

4209018029069894517862337 Tumors 

......................... 50 

MM+M+M+MMM+MMMMM+MMM+++M+ 24 

......................... 49 

......................... 49 

......................... 49 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

xx X X xx X 12 

1 

X X 5 

xxx xxxxxxxxxxxxxxx xxxxx 45 

xx X 5 

X xx xxx x xxxx 27 

2 

......................... 50 

........................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

........................ 42 

+M++++++M++M+M++M+M++++++ 37 

......................... 50 

-x 1 

......................... 50 

......................... 49 

......................... 50 

......................... 50 

......................... 50 
162 Oxazepam, NTP TR 443 

TABLE C2 

Individual Animal Tumor Pathology of Male B6C3F, Mice in the 2-Year Feed Study of Oxazepam: 5,000 ppm (continued) 

4444444445555555555555555 

Number of Days on Study 0135566110133334466666661 

1340114991415192167789995 

2222222121212212222212221 

Carcass ID Number 3012330818083292320292229 

3034121557434362855651791 ~ ~~ ~~~~ ~~ ~ 

Hematopoietic System 

Bone marrow ......................... Lymph node ++ 

Lymph node, mandibular ++++M+M+++M++M++M++++M+++ 

Lymph node, mesenteric MM++M+++++MMM++++++++++++ 

Lymph node, mediastinal + 

Spleen ......................... Thymus ++++++M+++++++M++M+M+M+MM 

Integumentary System 

Mammary gland MMMMMMMMMMMMMMMMMMMMMMMMM 

Skin ......................... 

Musculoskeletal System 

Bone ......................... Skeletal muscle ......................... 

Nervous System 

Brain ......................... 

Respiratory System 

Lung ......................... Alveolar/bronchiolar adenoma 

Hepatoblastoma, metastatic, liver X xx 

Hepatocellular carcinoma, metastatic, 

liver X X xx 

NOX ......................... Trachea ......................... 

Special Senses System 

Harderian gland 

Adenoma 

Urinary System 

Kidney ......................... Urinary bladder +++++++++++M+++++++++++++ ~~~~~~ ~ ~ ~ ~ 

Systemic Lesions 

Multiple organs ......................... Lymphoma malignant lymphocytic X 
Lesions in Male B6C3Fl Mice 163 

TABLEC2 

Individual Animal Tumor Pathology of Male B6C3Fl Mice in the 2-Year Feed Study of Oxazepam: 5,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Lymph node, mediastinal 

Spleen 

Thymus 

Integumentary System 

Mammary gland 

Skin 

Musculoskeletal System 

Bone 

Skeletal muscle 

Nervous System 

Brain 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Hepatoblastoma, metastatic, liver 

Hepatocellular carcinoma, metastatic, 

liver 

Nose 

Trachea 

Special Senses System 

Harderian gland 

Adenoma 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Lymphoma malignant lymphocytic 

5555555556666666666666666 

7889999990111112222222222 

5793566689245772233445588 

1212212221222222222121212 Total 

9093481319200011313918090 Tissues/ 

4209018029069894517862337 Tumors 

......................... 50 

+++++++++M+++++++++++M+++ 42 

+M++++++++M+++++M++++++++ 41 

1 

......................... 50 

+MMMMI+++++MMM+++MMM++M++ 31 

MMMMMMMMMMMMMMMMMMMMMMMMM 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

X 1 

X X 5 

X x x x x xx x X x xx 16 

......................... 50 

......................... 50 

+ 1 

X 1 

......................... 50 

......................... 49 

......................... 50 

1 
164 Oxazepam NTP TR 443 

TABLEC3 

Statistical Analysisof Primary Neoplasms in Male B6C3F, Mice in the 2-Year Feed Studyof Oxazepam 

AdrenalCortex:Adenoma 

Overall ratea 

Adjusted rateb 

Terminal rate' 

First incidence (days) 

Life table testd 

Logistic regression testd 

Cochran-Armita e testd 8 Fisher exact test 

HarderianGland:Adenoma or Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Liver: Hepatoblastoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Liver: Hepatocellular Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Liver: Hepatocellular Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

6/49 (12%) 

13.3% 

6/45 (13%) 

729 0 

P-0.456N 

P-0.456N 

P=0.006N 

3/50 (6%) 

6.3% 

1/45 (2%) 

624 

P=O.540 

P=0.154N 

P=0.158N 

0149 (0%) 

0.0% 

0/45 (0%) -

Pc0.001 

P<O.001 

P<O.001 

17/49 (35%) 

37.8% 

17/45 (38%) 

729 

P<O.001 

Pc0.001 

P<O.001 

9/49 (18%) 

18.7% 

6/45 (13%) 

586 

P<O.001 

Pc0.001 

P<O.001 

4/50 (8%) 

9.1% 

4/44 (9%) 

729 (T) 

P=0.384N 

P=0.384N 

P=0.357N 

2/50 (4%) 

4.4% 

1/44 (2%) 

701 

P=0.508N 

P=0.470N 

P=O.S00N 

2/50 (4%) 

4.5% 

2/44(5%) 

729 (T)

P=O.234 

P=O.234 

P=O.253 

18/50 (36%) 

37.4% 

14/44 (32%) 

453 

P-0.472 

P=O.547 

P=O.530 

5/50 (10%) 

10.7% 

3/44 (7%) 

453 

P=0.215N 

P-0.107N 

P=0.183N 

1/49 (2%) 

6.7% 

l/l5 (7%) 

729 (9 P=0.409N 

P=0.409N 

P=0.056N 

0/50 (0%) 

0.0% 

0/15 (0%) -

P=0.251N 

P=O.l13N 

P=0.121N 

21/50 (42%) 

58.3% 

4/15 (27%) 

598 

Pco.001 

Pc0.001 

P<0.001 

34/50 (68%) 

87.3% 

11/15 (73%) 

486 

Pc0.001 

P<O.001 

P<O.001 

45/50 (90%) 

95.7% 

13/15 (87%) 

540 

Pc0.001 

PCO.001 

P<O.001 

Ob0 (0%) 

0.0% 

OD 

-e 

-

-

P=O.O12N 

1/50 (2%) 

5.0% 

010 

596 

P=O.432 

P=0.463N 

P=0.309N 

13/50 (26%) 

52.9% 

OD 

434 

P<0.001 

P=O.O14 

P<O.001 

32/50 (64%) 

95.1% 

OD 

401 

Pc0.001 

P=O.003 

P=O.003 

50/50 (100%) 

100.0% 

o/o

401 

Pc0.001 

Pc0.001 

P<0.001 
Lesions in Male B6C3Fl Mice 165 

TABLEC3 

Statistical Analysisof Primary Neoplasmsin Male B6C3Fl Mice in the 2-YearFeed Study of Oxazepam (continued) 

125 ppm 2,500 ppm 5,000 ppm 

Liver:Hepatoblastoma or Hepatocellular Carcinoma 

Overall rate 9/49 (18%) 

Adjusted rate 18.7% 

Terminal rate 6/45 (13%) 

First incidence (days) 586 

Life table test P<0.001 

Logistic regression test P<O.001 

Cochran-Annitage test P<O.001 

Fisher exact test 

Liver: Hepatoblastoma, Hepatocellular Adenoma, or Carcinoma 

Overall rate 23/49 (47%) 

Adjusted rate 47.9% 

Terminal rate 20/45 (44%) 

First incidence (days) 586 

Life table test P<0.001 

Logistic regression test P<O.ool 

Cochran-Armitage test P<O.OOl 

Fisher exact test 

Lung: Alveolar/bronchiolar Adenoma 

Overall rate 11/50 (22%) 

Adjusted rate 23.9% 

Terminal rate 10/45 (22%) 

First incidence (days) 627 

Life table test P=O.501 

Logistic regression test P=0.001N 

Cochran-Armitage test P<0.001N 

Fisher exact test 

Lung: Alveolar/bronchiolar Adenoma or Carcinoma 

Overall rate 13/50 (26%) 

Adjusted rate 28.2% 

Terminal rate 12/45 (27%) 

First incidence (days) 627 

Life table test P=O.581 

Logistic regression test P<0.001N 

Cochran-Armitage test P<0.001N 

Fisher exact test 

All Organs:Hemangiosarcoma 

Overall rate 1/50 (2%) 

Adjusted rate 2.2% 

Terminal rate 1/45 (2%) 

First incidence (days) 729 (T)

Life table test P=0.407N 

Logistic regression test P=0.016N 

Cochran-Annitage test P=0.046N 

Fisher exact test 

6/50 (12%) 

12.9% 

4/44 (9%) 

453 

P=0.310N 

P=0.184N 

P=O.274N 

19/50 (38%) 

39.5% 

15/44 (34%) 

453 

P=0.315N 

P=0.205N 

P -0.243N 

17/50 (34%) 

36.1% 

14/44 (32%) 

527 

P=O.128 

P=O.133 

P=O.133 

18/50 (36%) 

38.2% 

15/44 (34%) 

527 

P=O.184 

P=O.195 

P=O.194 

5/50 (10%) 

10.7% 

3/44 (7%) 

508 

P=O.l04 

P=O.O90 

P=O.102 

47/50 (94%) 

97.9% 

14/15 (93%) 

540 

P<O.001 

P <0.001 

P<O.001 

50/50 (100%) 

100.0% 

15/15 (100%) 

486 

P<O.001 

P<0.001 

P<O.OOl 

5/50 (10%) 

28.1% 

4/15 (27%) 

486 

P=O.493 

P=0.126N 

P=0.086N 

5/50 (10%) 

28.1% 

4/15 (27%) 

486 

P=O.612 

P=0.063N 

P=0.033N 

1/50 (2%) 

2.6% 

0/15 (0%) 

642 

P=O.620 

P=0.758N 

P=0.753N 

50/50 (100%) 

100.0% 

OlO 

401 

P<0.001 

P<O.001 

P<0.001 

50/50 (100%) 

100.0% 

O/O 

401 

P<0.001 

P<O.001 

P<0.001 

1/50 (2%) 

25.0% 

o/o 

625 

P=O.127 

P=0.570N 

P=0.002N 

1/50 (2%) 

25.0% 

O/O 

625 

P=O.127 

P=0.559N 

P<0.001N 

0/50 (0%) 

0.0% 

o/o

-

-

-

P=0.500N 
166 Oxazepam NTP TR 443 

TABLEC3 

Statistical Analysisof Primary Neoplasms in Male B6C3F, Mice in the 2-Year Feed Study of Oxazepam (continued) 

~ 

0 PPm 125 ppm 2,500 ppm 5,000 ppm 

All Organs: Malignant Lymphoma or Histiocytic Sarcoma 

Overall rate 3/50 (6%) 2/50 (4%) 1/50 (2%) 1/50 (2%) 

Adjusted rate 6.7% 4.5% 2.8% 2.9% 

Terminal rate 3/45 (7%) 2/44 (5%) on5 (0%) o/o

First incidence (days) 729 (T) 729 646 542 

Life table test P=O.246 P=0.510N P=0.637N P=O.433 

Logistic regression test P=0.347N P=0.510N P=0.352N P=0.778N 

Cochran-Armitage test P=O.U)7N 

Fisher exact test P=0.500N P=0.309N P=0.309N 

All Organs:Benign Neoplasms 

Overall rate 32/50 (64%) 31/50 (62%) 37/50 (74%) 32/50 (66%) 

Adjusted rate 68.1% 63.3% 94.0% 100.0% 

Terminal rate 30/45 (67%) 26/44 (59%) 13/15 (87%) o/o

First incidence (days) 627 453 486 401 

Life table test P<0.001 P=0.549N P<O.001 P<O.001 

Logistic regression test P=O.135 P=0.576N P =0.077 P=O.170 

Cochran-Armitage test P=O.o92 

Fisher exact test P=0.500N P=O.101 P =0.339 

All Organs: Malignant Neoplasms 

Overall rate 17/50 (34%) 18/50 (36%) 47/50 (94%) 50/50 (100%) 

Adjusted rate 34.7% 36.7% 97.9% 100.0% 

Terminal rate 13/45 (29%) 13/44 (30%) 14/15 (93%) o/o

First incidence (days) 586 453 540 401 

Life table test P<O.001 P=O.475 P<O.OOl Pco.001 

Logistic regression test PcO.001 P=O.493 Pc0.001 PcO.001 

Cochran-Armitage test PcO.001 

Fisher exact test P=O.500 PcO.001 PCO.001 

All Organs:Benignand Malignant Neoplasms 

Overall rate 37/50 (74%) 35/50 (70%) 50/50 (100%) 50/50 (100%) 

Adjusted rate 75.5% 70.0% 100.0% 100.0% 

Terminal rate 33/45 (73%) 29/44 (66%) 15/15 (100%) o/o

First incidence (days) 586 453 486 401 

Life table test P<O.001 P=0.481N Pc0.001 Pc0.001 

Logistic regression test PCO.001 P=0.409N P<0.001 PCO.001 

Cochran-Armitage test PcO.001 

Fisher exact test P=0.412N P<O.001 PcO.001 

(T)Terminal sacrifice 

a Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for adrenal gland, 

bone marrow, brain, epididymis, gallbladder, heart, kidney, lalynx, liver, lung, nose, pancreas, parathyroid gland, pituitaly gland, preputial gland, 

prostate gland, salivary gland, spleen, testes, thyroid gland, and urinary bladder, for other tissues, denominator is number of animals necropsied. 

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

Observed incidence at terminal kill 

Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding 

to pairwise comparisons between the controls andthat exposed group. The life table analysis regards neoplasms in animals dying prior to terminal 

kill as being (directly or indirectly) the cause of death. The logistic regression test regards these lesions as nonfatal. The Cochran-Armitage and 

Fisher exact tests compare directly the overall incidence rates. For all tests, a negative trend or a lower incidence in an exposure group is indicated 

by N. e Not applicable; no neoplasms in animal group 
Lesions in Male B3C3F, Mice 167 

TABLEC4 

Historical Incidence of Liver Neoplasms in Untreated Male B6C3F, Micea 

Study 

Historical Incidence at Battelle Columbus 

2,4-Dichlorophenol 

4,4-Thiobis(6-r-butyl-mcresol) 

Diphenylhydantoin 

Dowicide EC-7 pentachlorophenol 

Ethylenethiourea 

Firemaster FF-1 polybrominated biphenyl 

Manganese (11) sulfate monohydrate 

Technical grade pentachlorophenol 

Triamterene 

Triamterene 

Tricresyl phosphate 

Overall Historical Incidence 

Total 

Standard deviation 

Range 

a Dataas of 20 August1992 

HepatoblastomaAdenomaCarcinomaAdenoma, 

0/50 

0/50 

0/50 

OB5 

0/49 

0/50 

0/50 

OB2 

0/50 

Ob0 

0/52 

Incidence in Controls 

Hepatocellular Hepatocellular 

4/50 7/50 

17/50 11/50 

19/50 13/50 

5/35 1/35 

11/49 13/49 

9/50 8/50 

30/50 9/50 

5/32 2/32 

17/50 5/50 

21/50 9/50 

18/52 15/52 

312/1,366 223/1,366 (22.8%) (16.3%) 

13.8% 7.2% 

4%-60% 3%-29% 

Hepatoblastoma, 

Hepatocellular 

or Carcinoma 

10/50 

25/50 

29/50 

6/35 

20/49 

16/50 

34/50 

7/32 

20/50 

25/50 

28/52 

485/1,366 (35.5%) 

14.3% 

10%-68% 
168 Oxazepam, NTP TR 443 

TABLEC5 

Summary of the Incidence of Nonneoplastic Lesions in Male B6C3Fl Mice in the 2-Year Feed Studyof Oxazepama 

0 PPm 125 ppm 2,500 PPm 5,000 ppm 

Disposition Summary 

Animals initially in study 60 60 60 60 

1SMd interim cwhaaion 10 10 10 10 

Early deaths 

Moribund 2 4 22 30 

Natural deaths 3 2 13 20 

survivors 

Terminal sacrifice 45 44 15 

Animals examined microscopically 60 60 60 60 

ZSMonth Interim Evaluation 

Alimentary System 

Liver (10) (10) (10) (10)

Basophilicfocus 1 (10%) 

Clear cell focus 1 (10%) 

Eosinophilic focus 1 (10%) 9 (90%) 2 (20%) 

Vacuolization cytoplasmic 7 (70%) 6 (60%) 1 (10%) 1 (10%) 

Hepatocyte, centrilobular, hypertrophy 10 (100%) 10 (100%) 10 (100%) 

Cardiovascular System 

None 

Endocrine System 

Pituitary gland 

Pars distalis,cyst 

Thyroid gland 

Inflammation, chronic active, focal 

Follicular cell, hyperplasia 

General Body System 

None 

Genital System 

Preputial gland 

Inflammation, chronic 

Duct, ectasia 

Hematopoietic System 

None 

Integumentary System 

None 
Lesions in Male B6C3Fl Mice 169 

TABLEC5 

Summary of the Incidence of Nonneoplastic Lesions in Male B6C3Fl Mice in the 2-Year Feed Studyof Oxazepam 

(continued) 

15-Month Interim Evaluatwn (continued) 

Musculoskeletal System 

None 

Nervous System 

None 

Respiratory System 

Lung 

Alveolar epithelium, hyperplasia, focal 

Special Senses System 

None 

Urinary System 

Kidney 

Nephropathy, chronic 

Urinary bladder 

Dilatation 

2-Year Study 

Alimentary System 

Intestine small, jejunum 

Peyer’s patch, hyperplasia, lymphoid 

Liver 

Angiectasis 

Basophilic focus 

Clear cell focus 

Clear cell focus, multifocal 

Eosinophilic focus 

Infarct 

Mixed cell focus 

Necrosis 

Necrosis, multifocal 

Thrombosis 

Vacuolization cytoplasmic 

Artery, angiectasis 

Centrilobular, hypertrophy 

Mesentery 

Congestion 

Fat, inflammation, chronic 

Pancreas 

Acinus, atrophy 

Stomach, forestomach 

Hyperplasia, focal, squamous 

Hyperplasia, multifocal, squamous 

(10)

1 (10%) 

(49)

2 (4%) 

(49) 

2 (4%) 

9 (18%) 

4 (8%) 

18 (37%) 

2 (4%) 

2 (4%) 

1 (2%) 

2 (4%) 

(50)

2 (4%) 

(10) 

(50)

1 (2%) 

(50) 

1 (2%) 

5 (10%) 

1 (2%) 

12 (24%) 

1 (2%) 

2 (4%) 

2 (4%) 

(2) 

1 (50%) 

(50) 

(50)

4 (8%) 

(10) 

1 (2%) 

8 (16%) 

1 (2%) 

1 (2%) 

26 (52%) 

(2) 

8 (16%) 

1 (2%) 

5 (10%) 

1 (2%) 

43 (86%) 

(2)

1 (50%) 

(50)

1 (2%) 

(50) 
170 Oxazepam, NTP TR 443 

TABLEC5 

Summary of the Incidence of Nonneoplastic Lesions in MaleB6C3F, Mice in the 2-Year Feed Study of Oxazepam 

(continued) 

2-Year Stud’ (continued) 

Alimentary System (continued) 

Stomach, glandular 

Necrosis, multifocal 

Tooth 

Gingiva, hyperplasia, focal, squamous 

Cardiovascular System 

Heart 

Angiectasis,focal 

Endocrine System 

Adrenal cortex 

Hypertrophy

Vacuolization cytoplasmic 

Capsule, hyperplasia 

Islets, pancreatic 

Hyperplasia, focal 

Pituitary gland 

Pars distalis, cyst 

Pars distalis, vacuolization cytoplasmic 

Thyroid gland 

Follicle, dilatation, multiple 

Follicular cell, hyperplasia 

General Body System 

None 

Genital System 

Epididymis 

Granuloma sperm 

Infiltration cellular, lymphocyte 

Inflammation, chronic 

Spermatocele 

Preputial gland 

Ectasia 

Infiltration cellular, lymphocyte 

Inflammation, chronic 

Duct, ectasia 

Duct, hemorrhage 

Testes 

Atrophy 

Hematopoietic System 

Lymph node 

Inguinal, hyperplasia, lymphoid 

Lumbar, hyperplasia, lymphoid 

0 PPm 

(50) 

(1)

1 (100%) 

(49)

13 (27%) 

3 (6%) 

(49)

1 (2%) 

(48)

3 (6%) 

(49) 

4 (8%) 

(50)

1 (2%) 

2 (4%) 

2 (4%) 

(50)

2 (4%) 

1 (2%) 

6 (12%) 

35 (70%) 

(50)

1(2%) 

125 ppm 

(50) 

(50)

7 (14%) 

(50) 

22 (44%) 

(50) 

14 (28%) 

7 (14%) 

29 (58%) 

(50) 

2,500 ppm 

(50) 

(49)

1 (2%) 

1 (2%) 

1 (2%) 

(50) 

(42)

3 (7%) 

(501

1 (2%) 

49 (98%) 

(50) 

33 (66%) 

1 (2%) 

8 (16%) 

9 (18%) 

1 (2%) 

(50)

25 (50%) 

5,000 ppm 

(50)

2 (4%) 

(50)

2 (4%) 

(50) 

(37) 

3 (8%) 

(50) 

47 (94%) 

(50) 

21 (42%) 

5 (10%) 

7 (14%) 

(50)

38 (76%) 

(2)

1 (50%) 

1(50%) 
Lesions in Male B6C3F, Mice 171 

TABLEC5 

Summary of the Incidence of Nonneoplastic Lesions in Male B6C3F, Mice in the 2-Year Feed Study of Oxazepam 

(continued) 

2-YeW StUffy (continued) 

Hematopietic System (continued) 

Lymph node, mandibular 

Hemorrhage 

Hyperplasia, lymphoid 

Lymph node, mesenteric 

Angiectasis 

Hematopoietic cell proliferation 

Hemorrhage 

Hyperplasia, lymphoid 

Spleen 

Atrophy 

Hematopoietic cell proliferation 

Hyperplasia, lymphoid 

Thymus 

Atrophy 

Cyst 

Integumentary System 

Skin 

Edema, subacute 

Musculoskeletal System 

Bone 

Cartilage, vertebra, fracture 

Joint, cartilage, fracture 

Vertebra, hyperostosis 

Nervous System 

Peripheral nerve 

Sciatic, axon, degeneration 

Spinal cord 

Nerve, degeneration 

Respiratory System 

Lung 

Hemorrhage, focal 

Infiltration cellular, multifocal, histiocyte 

Alveolar epithelium, hyperplasia 

Special Senses System 

Eye

Bilateral, inflammation, chronic 

0 PPm 

(49) 

3 (6%) 

1(2%) 

(43)

39 (91%) 

(50) 

4 (8%) 

(2)

1(50%) 

ppm ppm1252,500 

(49) 

1 (2%) 

1(2%) 

(50) 

2 (4%) 

(43)

40 (93%) 

(50) 

1 (2%) 

1 (2%) 

(1) 

1 (100%) 

(1) 

1 (100%) 

(50) 

1 (2%) 

2 (4%) 

(45)

1 (2%) 

1 (2%) 

(50) 

6 (12%) 

(37)

35 (95%) 

(2) 

2 (100%) 

(2) 

2 (100%) 

(50) 

1 (2%) 

5,000 ppm 

(42)

1(2%) 

1 (2%) 

(41)

1(2%) 

1 (2%) 

(50) 

1 (2%) 

7 (14%) 

(31)

24 (77%) 

1(3%) 

1 (2%) 

(50)

1 (2%) 
172 Oxazepam, NTP TR 443 

TABLEC5 

Summary of the Incidence of Nonneoplastic Lesions in Male B6C3F, Mice in the 2-Year Feed Studyof Oxazepam 

(continued) 

2-Year Stud’’ (continued) 

Urinary System 

Kidney 

Nephropathy, chronic 

Hydronephrosis (50) 

43 &%) 

1 (2%) 

40 (80%) 3 (6%) 18 (36%) 

cortex, cyst 

Urinary bladder 

Inflammation, chronic active 

Calculus gross obselvation 

2 (4%) 

(49) 

1 (2%) 

(50) (50) (49) 

1 (2%) 

1 (2%) 

a Number of animals examined microscopically at site and number of animals with lesion 
173 

TABLEDl 

TABLED2 

TABLE D3 

TABLED4a 

TABLED4b 

TABLED5 

APPENDIX D 

SUMMARY OF LESIONS 

IN FEMALE B6C3Fl MICE 

IN THE 2-YEAR FEED STUDY 

OF OXAZEPAM 

Summary of the Incidence of Neoplasms in Female B6C3Fl Mice 

in the 2-Year Feed Study of Oxazepam .................................... 175 

Individual Animal Tumor Pathology of FemaleB6C3FlMice 

in the 2-Year Feed Study of Oxazepam .................................... 180 

Statistical Analysis of Primary Neoplasms inFemaleB6C3FlMice 

in the 2-Year Feed Study of Oxazepam .................................... 200 

Historical Incidence of Liver Neoplasms in Untreated Female B6C3Fl Mice ........ 204 

Historical Incidence of Thyroid Gland Neoplasms 

in Untreated Female B6C3Fl Mice ....................................... 205 

Summary of the Incidence of Nonneoplastic Lesions in Female B6C3Fl Mice 

in the 2-Year Feed Study of Oxazepam .................................... 206 
174 Oxazepam, NTP TR 443 
Lesions in Female B6C3Fl Mice 175 

TABLEDl 

Summary of the Incidence of Neoplasms in Female B6C3Fl Mice in the 2-Year Feed Study of Oxazepama 

Disposition Summary 

Is-" iryeim ew"an 

Animals initially in study 

10 

60 

10 

60 

10 

60 

10 

60 

Early deaths 

Moribund 8 7 22 15 

Natural deaths 3 2 26 35 

Survivors 

Terminal sacrifice 39 41 2 

Animals examined microscopically 60 60 60 60 

15-Month Interim Evaluatwn 

Alimentary System 

Liver (10) (10) (10) (10)

Hepatoblastoma 2 (20%) 

Hepatocellular carcinoma 1 (10%) 2 (20%) 3 (30%) 

Hepatocellular carcinoma, multiple 7 (70%) 

Hepatocellular adenoma 1 (10%) 1 (10%) 

Hepatocellular adenoma, multiple 9 (90%) 10 (100%) 

Histiocytic sarcoma 1 (10%) 

Cardiovascular System 

None 

Endocrine System 

Pituitary gland 

Pars distalis, adenoma 

General Body System 

None 

Genital System 

Uterus 

Cervix, histiocytic sarcoma 

Hematopoietic System 

None 

Integumentary System 

None 

Musculoskeletal System 

None 
176 Oxazepam, NTP TR 443 

TABLEDl 

Summary of the Incidence of Neoplasms in Female B6C3Fl Mice in the 2-Year Feed Studyof Oxazepam (continued) 

15-Month Interim Evaluation (continued) 

Nervous System 

None 

Respiratory System 

None 

Special Senses System 

None 

Urinary System 

None 

Systemic Lesions 

Multiple organsb 

Histiocytic sarcoma 

2-Year Study 

Alimentary System 

Esophagus 

Histiocytic sarcoma 

Intestine small, jejunum 

Liver 

Hepatoblastoma 

Hepatoblastoma, multiple 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma 

Mesentery 

Histiocytic sarcoma 

Teratoma malignant, metastatic, ovary 

Fat, lipoma 

Pancreas 

Histiocytic sarcoma 

Salivary glands 

Fibrosarcoma, metastatic, skin 

Stomach, forestomach 

Squamous cell papilloma 

Cardiovascular System 

None 

9 (18%) 

12 (24%) 

13 (26%) 

1 (2%) 

(3) 

1 (33%) 

1 (33%) 

(50) 

(50)

1 (2%) 

(50)

1 (2%) 

(50)

1 (2%) 

(50) 

(50)

1 (2%) 

4 (8%) 

1 (2%) 

16 (32%) 

19 (38%) 

1 (2%) 

(3)

1 (33%) 

(50) 

(50) 

(50)

8 (16%) 

I (14%) 

42 (84%) 

6(12%) 

29 (58%) 

(50) 

(50) 

(50)

I (14%) 

1 (2%) 

1 (2%) 

43 (86%) 

6(12%) 

30 (60%) 
Lesions in Female B6C3Fl Mice 177 

TABLEDl 

Summary of the Incidence of Neoplasms in FemaleB6C3Fl Mice in the 2-Year Feed Study of Oxazepam (continued) 

2-Year Study (continued) 

Endocrine System 

Adrenal cortex 

Capsule, adenoma 

Capsule, histiocytic sarcoma 

Pituitary gland 

Pars distalis, adenoma 

Pars intermedia, adenoma 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

None 

Genital System 

waryCystadenoma 

Granulosa cell tumor malignant 

Granulosa cell tumor benign 

Histiocytic sarcoma 

Luteoma 

Teratoma malignant 

Follicle, adenoma 

Uterus 

Histiocytic sarcoma 

Leiomyosarcoma 

Polyp stromal 

Cervix, histiocytic sarcoma 

Endometrium, carcinoma 

Hematopoietic System 

Bone marrow 

Lymph node 

Pancreatic, histiocytic sarcoma 

Renal, teratoma malignant, metastatic, ovary 

Thoracic, histiocytic sarcoma 

Lymph node, bronchial 

Lymph node, mandibular 

Fibrosarcoma, metastatic, skin 

Histiocytic sarcoma 

Lymph node, mesenteric 

Histiocytic sarcoma 

Teratoma malignant, metastatic, ovary 

Lymph node, mediastinal 

Histiocytic sarcoma 

Spleen 

Histiocytic sarcoma 

Thymus 

Histiocytic sarcoma 

Thymoma NOS 

(50)

1 (2%) 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

1 (2%) 

(50) 

1 (2%) 

1 (2%) 

1 (2%) 

(50)

1 (2%) 

(50) (48) (50)

3 (6%) 1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

2 (4%) 

(50) (50) (49) 

1 (2%) 

1 (2%) 

1 (2%) 

(44) 

(37) 
178 ’ ,Omzqam, NTP TR 443 

TABLEDl 

Summary of the Incidence of Neoplasms in Female B6C3F, Mice in the 2-YearFeed Studyof Oxazepam (continued) 

2-Year Stud’ (continued) 

Integumentary System 

Skin 

Subcutaneous tissue, fibrosarcoma 

Subcutaneous tissue, fibrosarcoma, multiple 

Subcutaneous tissue, hemangiosarcoma 

Subcutaneous tissue, melanoma NOS 

Musculoskeletal System 

Skeletal muscle 

Diaphragm, teratoma malignant, metastatic, 

ovary 

Nervous System 

None 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Alveolarbronchiolar adenoma, multiple 

Alveolarbronchiolar carcinoma 

Fibrosarcoma, metastatic, skin 

Hepatoblastoma, metastatic, liver 

Hepatocellular carcinoma, metastatic, liver 

Histiocytic sarcoma 

Teratoma malignant, metastatic, ovary 

Trachea 

Histiocytic sarcoma 

Special Senses System 

Ear 

External ear, fibrosarcoma 

Harderian gland 

Adenoma 

Carcinoma 

Urinary System 

Kidney 

Histiocytic sarcoma 

Teratoma malignant, metastatic, ovary 

Urinary bladder 

(50) 

1 (2%)

1 (2%) 

1 (2%) 

(50) 

1 (2%) 

(50)

3 (6%) 

1 (2%) 

1 (2%) 

1 (2%) 

3 (6%) 

1 (2%) 

(50) 

(1) 

(3)

1 (33%) 

1 (33%) 

(50) 

1 (2%) 

(50) 

125 ppm 

1 (2%) 

(50) (50)

1 (2%) 1 (2%) 

3 (6%) 1 (2%) 

2 (4%) 2 (4%) 

2 (4%) 8 (16%) 10 (20%) 

1 (2%) 

(50)

1 (2%) 

(3) (1)

3 (100%) 1 (100%) 
Lesions in Female B6C3F, Mice 179 

TABLEDl 

Summary of the Incidence of Neoplasms in Female B6C3F, Mice in the 2-YearFeed Study o f Oxazepam (continued) 

Systemic Lesions 

Multiple organs 

Lymphoma malignant lymphocytic 

Lymphoma malignant 

Histiocytic sarcoma (50) 

2 (4%) 

1(2%) 

(50) 

2 (4%) 

2 (4%) 

(50) 

1(2%) 

1 (2%) 

(501 

Lymphoma malignant mixed 1(2%) 1(2%) 2 (4%) 

Neoplasm Summary 

Total animals with primary neoplasms' 

15-Month interim evaluation 3 2 9 10 

2-Year study 

Total primary neoplasms 

37 43 50 47 

15-Month interim evaluation 3 2 12 22 

2-Year study 60 75 110 96 

15-Month interim evaluation 

Total animals with benign neoplasms 

2 2 9 10 

2-Year study 29 40 38 38 

15-Month interim evaluation 

Total benign neoplasms 

2 2 9 10 

15-Month interim evaluation 

2-Year study 

Total animals with malignant neoplasms 

39 

1 

57 46 

3 

44 

10 

2-Year study 18 15 49 44 

15-Month interim evaluation 

Total malignant neoplasms 

1 3 12 

2-Year study 20 17 63 52 

2-Year study 

Total animals with metastatic neoplasms 

6 2 8 12 

2-Year study 

Total metastatic neoplasms 

12 2 10 12 

Total animals with uncertain neoplasms - benign or malignant 

2-Year study 

Total uncertain neoplasms 

1 1 1 

2-Year study 1 1 1 

a Number of animals examined microscopically at site and number of animals with lesion 

' Primaryneoplasms:allneoplasmsexcept metastatic neoplasms 

Number of animals with any tissue examined microscopically 
180 Oxazepam, NTP TR 443 

TABLED2 

Individual Animal Tumor Pathology of Female B6C3F, Mice in the 2-Year Feed Study of Oxazepam: 0 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma 

Mesentery 

Teratoma malignant, metastatic, ovary 

Fat, lipoma 

Pancreas 

Salivary glands 

Fibrosarcoma, metastatic, skin 

Stomach, forestomach 

Squamous cell papilloma 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

~~ ~ ~~ ~ 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

~ 

General Body System 

None 

Genital System 

Clitoral gland 

ovary

Cystadenoma 

Granulosa cell tumor malignant 

Histiocytic sarcoma 

Luteoma 

Teratoma malignant 

Follicle, adenoma 

3555666666677777777777777 

5179223355733333333333333 

7668240829055555555555555 

2222222222222222222222222 

7848658974644455556677777 

2652798701138912375613679 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... X X X 

X xx X X X 

X xx xx 

X 

+ 

X 

......................... 

......................... X 

......................... X 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

+++++++++M+M+++++++++++++ 

......................... X 

......................... 

-~~~ ~ ~~ ~ ~ ~ 

++M++++++M+++++++++++++++ 

......................... 

X 

X 

X 

X 

+: Tissueexaminedmicroscopically M:Missing tissue X Lesionpresent 

A Autolysis precludes examination I: Insufficienttissue Blank Notexamined 
Lesions in Female B6C3Ft Mice 181 

TABLED2 

Individual Animal Tumor Pathology of Female B6C3FL Mice in the 2-Year Feed Study of Oxazepam: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma 

Mesentery 

Teratoma malignant, metastatic, ovary 

Fat, lipoma 

Pancreas 

Salivary glands 

Fibrosarcoma, metastatic, skin 

Stomach, forestomach 

Squamous cell papilloma 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

General Body System 

None 

Genital System 

Clitoral gland 

ovary

Cystadenoma 

Granulosa cell tumor malignant 

Histiocytic sarcoma 

Luteoma 

Teratoma malignant 

Follicle, adenoma 

7777777777777777777777777 

3333333333333333333333333 

5555556666666666666666666 

2222222222222222222222222 Total 

8888994445555667788999999 Tissues/ 

1479282474568345835013569 Tumors 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

X X xx xx 9 

X xx x X X 12 

x x xx xx xx 13 

1 

++ 3 

1 

X 1 

......................... 50 

......................... 50 

1 

......................... 50 

1 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

+++++M++++++M++++++++++++ 46 

......................... 50 

X 2 

......................... 50 

......................... 48 

......................... 50 

X 1 

1 

1 

X 2 

1 

X 1 
182 Oxazepam, NTP TR 443 

TABLED2 

Individual Animal Tumor Pathology of Female B6C3F, Mice in the 2-Year Feed Study of Oxazepam: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

~~ ~ 

Genital system (cintinued) 

Uterus 

Histiocytic sarcoma 

Polyp stromal 

Cervix, histiocytic sarcoma 

Hematopoietic System 

Bone marrow 

Lymph node 

Renal, teratoma malignant, 

metastatic, ovary 

Lymph node, bronchial 

Lymph node, mandibular 

Fibrosarcoma, metastatic, skin 

Histiocytic sarcoma 

Lymph node, mesenteric 

Teratoma malignant, metastatic, ovary 

Lymph node, mediastinal 

Spleen 

Thymus 

Histiocytic sarcoma 

Thymoma NOS 

Integumentary System 

Mammary gland 

Skin 

Subcutaneous tissue, fibrosarcoma 

Subcutaneous tissue, fibrosarcoma, 

multiple 

Subcutaneous tissue, hemangiosarcoma ~~~~ ~ ~ ~ 

Musculoskeletal System 

Bone 

Skeletal muscle 

Diaphragm, teratoma malignant, 

metastatic, ovary 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

3555666666677777777777777 

5179223355733333333333333 

7668240829055555555555555 

2222222222222222222222222 

7848658974644455556677777 

2652798701138912375613679 

......................... X 

X 

......................... 

+ + + 

X 

+ ......................... X 

X 

+M+++++++++++++++++++++++ 

X 

+ ......................... 

......................... X 

......................... 

......................... X 

X 

X 

......................... 

......................... 

X 

......................... + + 

+ + 
Lesions in Female B6C3Fl Mice 183 

TABLE D2 

Individual Animal Tumor Pathology of Female B6C3Fl Mice in the 2-Year Feed Study of Oxazepam: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Genital System (continued) 

Uterus 

Histiocyticsarcoma 

Polyp stromal 

Cervix, histiocytic sarcoma 

Hematopoietic System 

Bone marrow 

Lymph node 

Renal, teratoma malignant, 

metastatic, ovary 

Lymph node, bronchial 

Lymph node, mandibular 

Fibrosarcoma, metastatic, skin 

Histiocytic sarcoma 

Lymph node, mesenteric 

Teratoma malignant, metastatic, ovary 

Lymph node, mediastinal 

Spleen 

Thymus 

Histiocytic sarcoma 

Thymoma NOS 

Integumentary System 

Mammary gland 

Skin 

Subcutaneous tissue, fibrosarcoma 

Subcutaneous tissue, fibrosarcoma, 

multiple 

Subcutaneous tissue, hemangiosarcoma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Diaphragm, teratoma malignant, 

metastatic, ovary 

Nervous System 

Brain 

Peripheral nelve 

Spinal cord 

7777777777777777777777777 

3333333333333333333333333 

5555556666666666666666666 

2222222222222222222222222 Total 

8888994445555667788999999 Tissues1 

1479282474568345835013569 Tumors 

......................... 50 

1 

X 1 

1 

......................... 50 

3 

1 

1 

+M++++++++++++M++++++++++ 48 

1 

1 

++++++++++++++M++++++++++ 48 

1 

1 

......................... 50 

......................... 50 

1 

X 1 

......................... 50 

......................... 50 

1 

1 

1 

......................... 50 

......................... 50 

1 

......................... 50 

2 

2 
184 Oxazepam, NTP TR 443 

TABLED2 

Individual Animal Tumor Pathology of Female B6C3F, Mice in the 2-YearFeed Study of Oxazepam: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Alveolarbronchiolar adenoma, 

multiple 

Alveolarbronchiolar carcinoma 

Fibrosarcoma, metastatic, skin 

Hepatocellular carcinoma, metastatic, 

liver 

Teratoma malignant, metastatic, ovary 

NOS2 

Trachea 

Special Senses System 

Ear 

Eye

Harderian gland 

Adenoma 

Carcinoma 

Urinary System 

Kidney 

Teratoma metastatic, malignant, ovary 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed 

35556666.66677777771777777 

5179223355733333333333333 

76682,40829055555555555555 

2222222222222222222222222 

7848658974644455556677777 

2652798701138912375613679 

......................... X 

X 

X 

x 

X 

X 

......................... 

......................... 

+ 

+ + 

+ + + 

X 

X 

......................... 

X 

......................... 

......................... X 

X X 
Lesions in Female B6C3F, Mice 185 

TABLED2 

Individual Animal Tumor Pathology of Female B6C3F,Mice in the 2-YearFeed Study of Oxazepam: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Respiratory System 

Lung 

Aiveolaribronchiolar adenoma 

Aiveolaribronchiolar adenoma, 

multiple 

Aveolarbronchiolar carcinoma 

Fibrosarcoma, metastatic, skin 

Hepatocellular carcinoma, metastatic, 

liver 

Teratoma malignant, metastatic, ovary 

N0.W 

Trachea 

Special Senses System 

Ear 

Eye

Harderian gland 

Adenoma 

Carcinoma 

Urinary System 

Kidney 

Teratoma malignant, metastatic, ovary 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed 

5555556666666666666666666 

3333333333333333333333333 

7777777777777777777777777 

2222222222222222222222222 

8888994445555667788999999 

1479282474568345835013569 

Total 

Tissues/ 

Tumors 

......................... X X

50 

3 

1 

1 

1 

......................... 

......................... 

X X 

50 

50 

1 

3 

1 

1 

3 

2 

1 

......................... 

......................... 

50 

50 

1 

......................... 

x 1 

2 

1 

50 
186 ,. , Oxazepam,NTPTR 443 

TABLED2 

Individual Animal Tumor Pathology of Female B6C3F, Mice in the 2-YearFeed Study of Oxazepam: ,125 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Histiocytic sarcoma 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatoblastoma 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma 

Mesentery 

Histiocytic sarcoma 

Pancreas 

Histiocytic sarcoma 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Capsule, adenoma 

Capsule, histiocytic sarcoma 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Pars intermedia, adenoma 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

Tissue NOS 

3444667777777777'77777'7777 

9799460133333333333333333 

1102921425555555555555555 

33333333.33333.333333333333 

4542333110001111222234444 

4850086794570358148940126 

......................... X 

+M+++++++++++++++++++++++ 

......................... 

.......................... 

......................... 

......................... 

.......................... 

.......................... 

......................... X 

X 

'X 

xx x xxx x X 

X xx x x xx 

X 

+ + + 

X 

.......................... X 

......................... 

......................... 

......................... 

.......................... 

......................... 

......................... X 

X 

.......................... 

......................... 

......................... 

+++++++++++M+++++++++++++ 

X X X 

X 

......................... xx 

+ 
Lesions in Female B6C3Fl Mice 187 

TABLED2 

Individual Animal Tumor Pathologyof Female B6C3Fl Mice in the 2-YearFeed Study of Oxazepam: 125 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Histiocyticsarcoma 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatoblastoma 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma 

Mesentery 

Histiocyticsarcoma 

Pancreas 

Histiocytic sarcoma 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Hl%3rt 

Endocrine System 

Adrenal cortex 

Capsule, adenoma 

Capsule, histiocytic Sarcoma 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Pars intermedia, adenoma 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

Tissue NOS 

7777777777777777777777777 

3333333333333333333333333 

5555566666666666666666666 

3333333333333333333333333 Total 

4555500011222333344455556 Tissues/

8023712346237235737915690 Tumors 

......................... 50 

1 

......................... 49 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

1 

X X X 4 

1 

xx X x xx xx 16 

xxxxx xx x x xxx 19 

1 

3 

1 

......................... 50 

1 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

1 

1 ......................... 50 

......................... 50 

++++++++++M++++M++++M++++ 47 

......................... 49 

X xx xx 8 

1 

......................... 50 

X X 4 
188 Oxazepam, NTP TR 443 

TABLED2 

Individual Animal Tumor Pathologyof Female B6C3F, Mice in the 2-YearFeed'Studyof Oxazepam: 125 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Genital System 

Clitoral gland 

ovary

Cystadenoma 

Granulosa cell tumor benign 

Histiocytic sarcoma 

Teratoma malignant 

Uterus 

Leiomyosarcoma 

Endometrium, carcinoma 

Hematopoietic System 

Bone marrow 

Lymph node 

Pancreatic, histiocytic sarcoma 

Thoracic, histiocytic sarcoma 

Lymph node, mandibular 

Lymph node, mesenteric 

Histiocytic sarcoma 

Lymph node, mediastinal 

Histiocytic sarcoma 

Spleen 

Histiocyticsarcoma 

Thymus 

Histiocytic sarcoma 

Thymoma NOS 

Integumentary System 

Mammaly gland 

Skin 

Musculoskeletal System 

Bone 

Skeletal muscle ~~ ____~~ ~ 

Nervous System 

Brain 

Peripheral newe 

Spinal cord 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Alveolarbronchiolar carcinoma 

Hepatmllul& carcinoma, metastatic, 

- liver 

Histiocytic sarcoma 

NOX 

Trachea 

Histiocytic sarcoma 

3444667777777777777777777 

9799460133333333333333333 

1102921425555555555555555 

3333333333333333333333333 

4542333110001111222234444 

4850086794570358148940126 

M++M+++M+++++++++++++++++ 

......................... X 

X 

X 

X ......................... X 

X 

......................... + + 

X 

X 

+++++++++++++++++++MMM+++ 

+M+++++++++++++++++++++++ 

X 

++ 

X ......................... X 

+M+++++++++++++++++++++++ 

X 

+++'++++M+++++++++++++++++ 

......................... 

......................... 

......................... 

......................... + + + 

+ + + 

......................... X 

X 

X X 

X ......................... 

......................... X 
Lesions in Female B6C3F, Mice 189 

TABLED2 

Individual Animal Tumor Pathology of Female B6C3FL Micein the 2-YearFeed Study of Oxazepam: 125 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Genital System 

Clitoral gland 

ovary

Cystadenoma 

Granulosa cell tumor benign 

Histiocytic sarcoma 

Teratoma malignant 

Uterus 

Leiomyosarcoma 

Endometrium, carcinoma 

Hematopoietic System 

Bone marrow 

Lymph node 

Pancreatic, histiocytic sarcoma 

Thoracic, histiocytic sarcoma 

Lymph node, mandibular 

Lymph node, mesenteric 

Histiocytic sarcoma 

Lymph node, mediastinal 

Histiocytic sarcoma 

Spleen 

Histiocytic sarcoma 

Thymus 

Histiocytic sarcoma 

Thymoma NOS 

Integumentary System 

Mammary gland 

Skin 

Musculoskeletal System 

Bone 

Skeletal muscle 

Nervous System 

Brain 

Peripheral nelve 

Spinal cord 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Alveolarbronchiolar carcinoma 

Hepatocellular carcinoma, metastatic, 

liver 

Histiocytic sarcoma 

Nose 

Trachea 

Histiocytic sarcoma 

7777777777777777777777777 

3333333333333333333333333 

5555566666666666666666666 

3333333333333333333333333 Total 

4555500011222333344455556 Tissues/ 

8023712346237235737915690 Tumors 

......................... 47 

......................... 50 

X X 3 

1 

1 

1 

......................... 50 

1 

1 

......................... 50 

2 

1 

1 

......................... 47 

+++++++++++++++++++++MM+M 46 

1 

1 

......................... 50 

1 

......................... 49 

1 

X 1 

......................... 49 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

3 

3 

......................... 50 

1 

X X 3 

2 

1 

......................... 50 

......................... 50 

1 
190 ' , Oxazepam, NTP TR 443

TABLED2 

Individual Animal Tumor Pathology of Female B6C3F, Mice in the 2-YearFeed Study of Oxazepam: 125 ppm (continued) 

3444667777771117111111111 

Number of Days on Study 9199460133333333333333333 

1102921425555555555555555 

3333333333333333333333333 

Carcass ID Number 45423331100011112222344'44 

4850086794510358148940126 

Special Senses System 

Ear + + 

Eye

Harderian gland + + 

Adenoma X X 

Urinary System 

Kidney .......................... Histiocytic sarcoma X X 

Urinary bladder ......................... 

Systemic Lesions 

Multiple organs ......................... Histiocytic sarcoma X X 

Lymphoma malignant lymphocytic X 

Lymphoma malignant mixed X 
Lesions in Female.B6C3F, Mice 191 

TABLED2 

Individual Animal Tumor Pathology of Female B6C3F, Mice in the 2-YearFeed Study of Oxazepam: 125 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Special Senses System 

Ear 

Eye

Harderian gland 

Adenoma 

Urinary System 

Kidney 

Histiocytic sarcoma 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed 

7777777777777777777777777 

3333333333333333333333333 

5555566666666666666666666 

3333333333333333333333333 Total 

4555500011222333344455556 Tissues/ 

So23712346237235737915690 Tumors 

2 

+ 1 

+ 3 

X 3 

......................... 50 

2 

......................... 50 

......................... 50 

2 

X 2 

1 
192 Oxazepam, NTP TR 443 

TABLED2 

Individual Animal Tumor Pathology of Female B6C3F, Mice in the 2-Year Feed Study of Oxazepam: 2,500 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatoblastoma 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Pancreas 

Salivary glands 

Stomach, forestomach 

Squamous cell papilloma 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

None 

Genital System 

Clitoral gland 

ovary

9tadenoma 

Follicle, adenoma 

Uterus 

Polyp stromal 

3345566666666666666666666 

9977911233333444466666789 

3605645204568245834567791 

4343333434433334434333433 

0709677161078981160689179 

1174965376041741463595788 

......................... 

++++++++++++MMM+++M++++++ 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... X xx x x x 

xx X X X X 

xxxxx xx xxxx xxxxx xxx 

xx X X 

x x xx x x xx x xxxx ......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

+++++M++++MM+MM+++++++M++ 

+++M++++++++++M+++++M+M++ 

......................... x x 

++I+++++++++++M++++++++M+ 

++++++++++++++++++++++M++ 

......................... X 
Lesions in Female B6C3Fl Mice 193 

TABLED2 

Individual Animal Tumor Pathology of Female B6C3F, Mice in the 2-Year Feed Study of Oxazepam: 2,500 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatoblastoma 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Pancreas 

Salivary glands 

Stomach, forestomach 

Squamous cell papilloma 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Hart ~~ ~~ ~~~ 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

None 

Genital System 

Clitoral gland 

ovary

Cystadenoma 

Follicle, adenoma 

Uterus 

Polyp stromal 

6677777777777777777777777 

9900000111111111111111133 

2901266011222229999999955 

3443334434333343333333444 Total 

9019772190678906668889001 Tissues/ 

3290090066125182342362455 Tumors 

......................... 50 

+M+++++++M++M++++M+M+++++ 41 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

X X 8 

X 7 

xxxxxxxxxxxxxxxx x xxxxxx 42 

xx 6 

x x xx x x x x xx x x x x x x 29 

......................... 50 

......................... 50 

......................... 50 

X 1 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

+++++++++++++++++M+++M+M+ 41 

I+++++M++++++++++M+++++++ 43 

......................... 50 

xx x 5 

......................... 47 

++++++++M++++++++++++++++ 48 

X 1 

xx 2 

......................... 50 

1 
194 Oxazepam,NTPTR 443 

TABLED2 

Individual Animal Tumor Pathologyof Female B6C3F, Mice in the 2-Year Feed Studyof Oxazepam: 2,500 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Lymph node, mediastinal 

Spleen 

Thymus 

Integumentary System 

Mammary gland 

Skin 

Subcutaneous tissue, melanoma NOS 

Musculoskeletal System 

Bone 

Skeletal muscle 

NervousSystem 

Brain 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Alveolarbronchiolar carcinoma 

Hepatoblastoma, metastatic, liver 

Hepatocellular carcinoma, metastatic, 

liver 

Nose 

Trachea 

Special Senses System 

Ear 

External ear, fibrosarcoma 

Harderian gland 

Adenoma 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Lymphoma malignant 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed 

3345566666666666666666666 

9977911233333444466666789 

3605645204568245834567791 

4343333434433334434333433 

0709677161078981160689179 

1174965376041741463595788 

......................... 

+ 

++++++M++++++++++++++++++ 

+++M+++++++M++M++++++++M+ 

+ ++ ......................... 

+++++MMMMMM+++M++++M+++++ 

++++++++M++++++++++++++++ 

......................... X 

......................... 

......................... 

......................... ++ 

+ 

......................... X 

X 

X X 

......................... 

......................... 

+ 

X 

......................... 

......................... 

......................... X 

X 

X 
Lesions in Female B6C3Fl Mice 195 

TABLED2 

Individual Animal Tumor Pathologyof Female B6C3Fl Mice in the 2-Year Feed Study of Oxazepam: 2,500 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Lymph node, mediastinal 

Spleen 

Thymus 

Integumentary System 

Mammary gland 

Skin 

Subcutaneous tissue, melanoma NOS 

Musculoskeletal System 

Bone 

Skeletal muscle 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Alveolar/bronchiolar carcinoma 

Hepatoblastoma, metastatic, liver 

Hepatocellular carcinoma, metastatic, 

liver 

NW 

Trachea 

Special Senses System 

Ear 

External ear, fibrosarcoma 

Harderian gland 

Adenoma 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Lymphoma malignant 

Lymphoma malignant lymphocytic 

Lymphomamalignantmixed 

6677777777777777777777777 

9900000111111111111111133 

2901266011222229999999955 

3443334434333343333333444 Total 

9019772190678906668889001 Tissues/ 

3290090066125182342362455 Tumors 

......................... 50 

1 

+++++M+++++++++++++++++++ 48 

M+++MMM+MM+++++++MM++++++ 38 

3 

......................... 50 

M+++++M+++M+M+++++MM++MM+ 34 

++++++M++++++++M+++++++++ 47 

......................... 50 

1 

......................... 50 

......................... 50 

......................... 50 

2 

+ 2 

......................... 50 

1 

X 2 

xxx X X X 8 

......................... 50 

......................... 50 

+ 

X 

......................... 50 

......................... 50 

......................... 50 

1 

1 

X 2 
196 Oxazepam, NTP TR 443 

TABLED2 

Individual Animal Tumor Pathology of Female B6C3F, Mice in the 2-Year Feed Study of Oxazepam: 5,000 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatoblastoma 

Hepatoblastoma, multiple 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Hart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

None 

Genital System 

Clitoral gland 

@sly

Uterus 

Hematopoietic System 

Bone marrow 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

0004444444444444445555555 

0000111155511119990233445 

6113024504701891494635114 

4444444444444444444444444 

1564151236435156111254564 

0301376324482108857143619 

......................... 

+++++M++++++++++++++++M+M 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... X X 

xxxxx xxxxxxxxxxxxx x 

X X 

x xxxxxxxxxxxxxxx X ......................... 

......................... 

......................... 

......................... 

M++++++++++++++++++++++++ 

......................... 

......................... 

......................... 

......................... 

++++MM++++M+++++MM+++++MM 

......................... 

......................... X X 

+++M+M++++++MM+++++++++++ 

......................... 

+++++M+++++++++++++++++++ 

......................... 

++MM+++++++++++M++++++M++ 

+M+MMM+++++++++++M+M+++++ 

+++A+++++++++++++++++++++ 

+++++++++++++M++++MMM++++ 
Lesions in Female B6C3Fl Mice 197 

TABLED2 

Individual Animal TumorPathologyof Female B6C3F, Mice inthe 2-Year Feed Study of Oxazepam: 5,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatoblastoma 

Hepatoblastoma, multiple 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

None 

Genital System 

Clitoral gland 

ovary

Uterus 

Hematopoietic System 

Bone marrow 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

5555555555555555566666666 

6666666777888999901111111 

2367899007239456602344777 

4444444444444444444444444 Total 

6624437582666533354423235 Tissues/ 

7241044905935505986297231 Tumors 

......................... 50 

MMM++++++++M++M+++++++MM+ 40 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

X X X X X 7 

X 1 

X 1 

x x x x x xx x x x x x x x x x x x x x x x x x 43 

X xx X 6 

x xxxx x xxxx x xx 30 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 49 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

+++++M+++++++M++++++M++++ 40 

++++++++++++++++M++++++++ 49 

......................... 50 

X X xx 6 

......................... 46 

......................... 50 

......................... 49 

......................... 50 

++M+++++M++++++++++++++++ 44 

M+M++++M+++++M+++++M++M+M 37 

......................... 49 

++MMM+MMM++++M+++M+MM+MM+ 34 
198 Oxazepam, NTP TR 443 

TABLED2 

Individual Animal Tumor Pathologyof Female B6C3F, Mice in the 2-Year Feed Studyof Oxazepam: 5,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Integumentary System 

Mammary gland 

Skin 

Musculoskeletal System 

Bone 

Skeletal muscle 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord ~~~~ ~ 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Hepatoblastoma, metastatic, liver 

Hepatocellular carcinoma, metastatic, 

liver 

Nose 

Trachea 

Special Senses System 

Harderian gland 

Adenoma 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

0004444444444444445555555 

0000111155577779990233445 

6773024504701891494635174 

4444444444444444444444444 

7564757236435756777254564 

0307376324482108857143619 

MMMM+++M+++++++++++++++++ 

......................... 

......................... 

......................... 

......................... 

......................... 

X 

X X X ......................... 

.......................... 

+ 

X 

......................... 

......................... 

......................... 
Lesions in Female B6C3F, Mice 199 

TABLED2 

Individual Animal Tumor Pathologyof Female B6C3F, Mice in the 2-Year Feed Study of Oxazepam: 5,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Integumentary System 

Mammary gland 

Skin 

Musculoskeletal System 

Bone 

Skeletal muscle 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Alveolarbronchiolar adenoma 

Hepatoblastoma, metastatic, liver 

Hepatocellular carcinoma, metastatic, 

liver 

Nose 

Trachea 

Special Senses System 

Harderian gland 

Adenoma 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

5555555555555555566666666 

6666666777888999901111111 

2361899001239456602344777 

4444444444444444444444444 Total 

6624437582666533354423235 Tissues/ 

I241044905935505986297231 Tumors 

++++++++M++++++++++M+++++ 43 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

+ 1 

+ 1 

......................... 50 

X 1 

X 2 

X xx x X xx 10 

......................... 50 

......................... 50 

1 

1 

......................... 50 

......................... 50 

......................... 50 
200 Oxazepam, NTP TR 443 

TABLED3 

Statistical Analysisof Primary Neoplasms in Female B6C3Fl Mice in the 2-Year Feed Study of Oxazepam 

HarderianGland:Adenoma 

Overall ratea 

Adjusted rateb 

Terminal rate' 

First incidence (days) 

Life table testd 

Logistic regression testd 

Cochran-Armita e testd I Fisher exact test 

HarderianGland:Adenoma or Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Liver: Hepatoblastoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Liver: Hepatocellular Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Liver: Hepatocellular Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

0 PPm 

1/50 (2%) 

2.0% 

0/39 (0%) 

516 

P=O.u)2 

P=0.300N 

P=0.320N 

2/50 (4%) 

4.2% 

OB9 (0%) 

516 

P=O.304 

P=0.159N 

P=0.214N 

0/50 (0%) 

0.0% 

0/39 (0%) -e

P<O.001 

P<O.001 

P<O.001 

25/50 (50%) 

59.3% 

22B9 (56%) 

598 

P<0.001 

P<0.001 

P=O.o64 

9/50 (18%) 

21.6% 

7/39 (18%) 

598 

P<O.001 

P<0.001 

P<O.001 

125 ppm 

3/50 (6%) 

7.1% 

2/41 (5%) 

732 

P=O.318 

P=O.301 

P=O.309 

3/50 (6%) 

7.1% 

2/41 (5%) 

732 

P=O.512 

P=O.495 

P=O.500 

1/50 (2%) 

2.3% 

0/41 (0%) 

714 

P=O.519 

P=O.502 

P-0.500 

35/50 (70%) 

79.5% 

32/41 (78%) 

471 

P=O.o62 

P ~0.037 

P-0.033 

5/50 (10%) 

11.9% 

4/41 (10%) 

714 

P=0.173N 

P=0.185N 

P=O.i94N 

2,500 ppm 

1/50 (2%) 

5.6% 

on (0%) 

710 

P=O.673 

P=0.706N 

P=0.753N 

1/50 (2%) 

5.6% 

o n (0%) 

710 

P=0.599N 

P=0.407N 

P=0.500N 

8/50 (16%) 

31.7% 

o n (0%) 

614 

P<0.001 

P=O.007 

P=O.003 

35/50 (70%) 

96.7% 

1/2 (50%) 

393 

P<O.001 

P=O.O14 

P=O.O33 

49/50 (98%) 

100.0% 

2/2 (100%) 

393 

P<0.001 

P<O.001 

P<0.001 

5,000 ppm 

1/50 (2%) 

2.8% 

OD 

479 

P=O.673 

P=0.619N 

P=0.753N 

1/50 (2%) 

2.8% 

010 

479 

P=O.673 

P=0.305N 

P=0.500N 

8/50 (16%) 

57.8% 

OD 

471 

P<0.001 

P=0.003 

P=O.003 

36/50 (72%) 

100.0% 

OD 

403 

P<O.001 

P=O.001 

P=O.O20 

44/50 (88%) 

100.0% 

o/o 

410 

P<O.001 

P<0.001 

P<0.001 
Lesions in Female B6C3F, Mice 201 

TABLE D3 

Statistical Analysisof Primary Neoplasms in Female B6C3F, Mice in the 2-Year Feed Study of Oxazepam (continued) 

0 PPm 

~~ ~ 

125 ppm 2,500 ppm 5,000 ppm 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Liver:Hepatoblastoma, Hepatocellular Adenoma, or Carcinoma 

66.5% 

23/50 (56%) 

P<O.001 

P<O.001 

P<O.001 

598 

25/39 (64%) 

P=O.O84 

P=O.126 

471 

33/41 (80%) 

81.8% 

36/50 (72%) 

P=O.O72 

zi2 (100%) 

50/50 (100%) 

100.0% 

P<O.001 

P<0.001 

393 

PcO.001 

P<0.001 

P<0.001 

403 

OD 

100.0% 

47/50 (94%) 

P<O.001 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Lung: Alveolar/bronchiolar Adenoma 

10.3% 

4/50 (8%) 

735 (TI 

4/39 (10%) 

P=0.152N 

P=0.638N 

P=O.111 

1/50 (2%) 

P=0.165N 

P=0.165N 

1/41 (2%) 

2.4% 

735 (TI 

P=0.181N 

o n (0%) 

0/50 (0%) 

0.0% 

-

P=0.769N 

P=0.769N 

P=0.059N 

P=0.665 

P=O.197 

596 

o/o 

10.0% 

1/50 (2%) 

P=0.181N 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Lung: Alveoladbronchiolar Carcinoma 

1/39 (3%) 

2.6% 

1/50 (2%) 

P=0.116N 

P=0.578N 

P=O.390 

735 (T> 

3/41 (7%) 

7.3% 

3/50 (6%) 

P=O.309 

P=O.323 

P=O.323 

735 (TI 

o n (0%) 

1/50 (2%) 

2.9% 

P=O.762 

P=O.502 

645 

P=0.753N 

0/50 (0%) 

o/o 

0.0% 

-

-

-

P=0.500N 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Lung: Alveolar/bronchiolar Adenoma or Carcinoma 

5/39 (13%) 

12.8% 

5/50 (10%) 

P=0.038N 

P=0.458N 

P=O.o42 

735 (TI 

4/41 (10%) 

9.8% 

4/50 (8%) 

P=0.500N 

P=0.468N 

P=0.468N 

'35 (T> 

o n (0%) 

1/50 (2%) 

2.9% 

P=0.247N 

P=O.615 

645 

P=0.102N 

P=O.662 

P=O.197 

596 

o/o 

10.0% 

1/50 (2%) 

P=0.102N 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Ovary: Cystadenoma 

1/39 (3%) 

2.6% 

1/50 (2%) 

P=0.118N 

P=O.708 

P=O.328 

735 Q 

P=O.319 

P=O.331 

701 

2/41 (5%) 

7.0% 

3/50 (6%) 

P=0.309 

50.0% 

1/48 (2%) 

735 (T) 

1n (50%) 

P=O.742 

P=O.O90 

P=O.O90 

0/50 (0%) 

o/o 

0.0% 

-

-

-

P=0.500N 
202 Oxazepam, NTP TR 443 

TABLED3 

Statistical Analysis of Primary Neoplasms in Female B6C3F, Mice in the 2-Year Feed Study of Oxazepam (continued) 

125 ppm 2,500 ppm 5,000 ppm 

Pituitary Gland (Pars Distalis): Adenoma 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

735 (T) 

2/39 (5%) 

2/50 (4%) 

5.1% 

P=0.813N 

8/40 (20%) 

20.0% 

8/49 (16%) 

P=O.O51 

735 (r) 

0/43 (0%)

0.0% 

o n (0%) -

P=0.910N 

0149 (0%)

0.0% 

o/o

-

-

Logistic regression test P=0.813N P=O.O51 P=0.910N -

Cochran-Armitage test P=O.OOSN 

Fisher exact test P=0.043 P=0.286N P=0.253N 

Thyroid Gland (Follicular Cell): Adenoma 

Overall rate 0/50 (0%) 4/50 (8%) 5/50 (10%) 6/50 (12%) 

Adjusted rate 0.0% 9.8% 34.3% 46.1%

Terminal rate OB9 (0%) 

First incidence (days) - Life table test PeO.001 

Logistic regression test P=0.007 

Cochran-Armitage test P=0.042 

Fisher exact test 

All Organs (Malignant Lymphoma): Lymphocytic and Mixed 

Overall rate 3/50 (6%) 

Adjusted rate 7.2% 

Terminal rate 2/39 (5%) 

First incidence (days) 622 

Life table test P=O.309 

Logistic regression test P=0.072N 

Cochran-Armitage test P=0.090N 

Fisher exact test 

All Organs: Malignant Lymphoma or Histiocytic Sarcoma 

Overall rate 4/50 (8%) 

Adjusted rate 9.3% 

Terminal rate 2/39 (5%) 

First incidence (days) 622 

Life table test P=O.475 

Logistic regression test P=0.017N 

Cochran-Armitage test P=0.025N 

Fisher exact test 

All Organs:Benign Neoplasms 

Overall rate 29/50 (58%) 

Adjusted rate 67.2% 

Terminal rate 25/39 (64%) 

First incidence (days) 516 

Life table test P<O.001 

Logistic regression test P=O.ool 

Cochran-Armitage test P=O.170 

Fisher exact test 

4/41 (10%) o n (0%) o/o 

735 (r)

P=O.O70 

P=O.O70 P=O.O17 

P=O.O59 

3/50 (6%) 

7.0% 

2/41 (5%) 

662 

P=0.636N 

P=O.662 

P=0.661N 

5/50 (10%) 

11.4% 

3/41 (7%) 

662 

P=O.536 

P=O.497 

P=O.500 

40/50 (80%) 

88.9% 

36/41 (88%) 

471 

P=O.O41 

P=O.O15 

P=O.O15 

663 

P=O.OOl 

P=O.O19 

P=0.028 

3/50 (6%) 

13.9% 

on (0%) 

393 

P=O.233 

P=0.563N 

3/50 (6%) 

13.9% 

o n (0%) 

393 

P=O.415 

P=0.378N 

P=0.500N 

38/50 (76%)

100.0% 

2t2 (100%) 

393 

P<0.001 

P=O.O22 

P=O.O44 

470 

P<O.ool 

P=O.O13 

0/50 (0%) 

0.0% 

o/o 

-

-

=0.457N P

0/50 (0%) 

0.0% 

o/o

-

-

P=0.134N 

=0.059N P

38/50 (76%)

100.0% 

o/o 

403 

P<O.ool 

P=O.oo2 

P=0.044 
Lesions in Female B6C3Fl Mice 203 

TABLED3 

Statistical Analysis of Primary Neoplasms in Female B6C3Fl Mice in the 2-Year Feed Study of Oxazepam (continued) 

0 PPm 125 ppm 2,500 ppm 5,000 ppm 

AllOrgans: Malignant Neoplasms 

Overall rate 18/50 (36%) 15/50 (30%) 49/50 (98%) 44/50 (88%) 

Adjusted rate 38.4% 34.0%100.0% 

Terminal rate 11/39 (28%) 12/41 (29%) 2i2 (100%) OD 

First incidence (days) 357 662 393 410 

Life table test PcO.001 P=0.296N P<0.001 P<O.001 

Logistic regression test Pc0.001 P=0.344N PCO.001 P<0.001 

Cochran-Armitage test PCO.001 

Fisher exact test P=0.335N Pc0.001 PcO.001 

AllOrgans: BenignandMalignant Neoplasms 

Overall rate 37/50 (74%) 43/50 (86%) 50/50 (100%) 47/50 (94%) 

100.0% 

Adjusted 100.0% rate

Terminal rate 29B9 (74%) 38/41 (93%) 2i2 (100%) 

100.0% 93.5% 78.5% 

o/o 

First incidence (days) 471 357 393 403 

Life table test PcO.001 PCO.001PCO.001P=0.266 

Logistic regression test P=O.107 PCO.001 PcO.001 P=O.O48 

Cochran-Armitage test P=0.002 

Fisher exact test P=O.105 Pco.001 P=O.o06 

(T)Terminal sacrifice 

a Number of neoplasm-bearing animaldnumber of animals examined. Denominator is number of animals examined microscopically for adrenal gland, 

bone marrow, brain, clitoral gland, gallbladder, heart, kidney, larynx, liver, lung, nose, ovary, pancreas, parathyroid gland, pituitary gland, salivaly 

gland, spleen, thyroid gland, and urinary bladder; for other tissues, denominator is number of animals necropsied. 

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

Observed incidence at terminal kill 

Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding 

to painvise comparisons between the controls and that exposed group. The life table analysis 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 Notapplicable;noneoplasms in animalgroup 
204 Oxazepam, NTP TR 443 

TABLED4a 

Historical Incidence of Liver Neoplasms in Untreated Female B6C3F, Micea 

Incidence in Controls 

Study Hepatoblastoma, 

Hepatocellular Hepatocellular Hepatocellular 

Hepatoblastoma Adenoma Carcinoma Adenoma, or Carcinoma 

Historical Incidence at Battelle Columbus 

2,4-Dichlorophenol 0/50 0/50 2/50 2/50 

4,4-Thiobis(6-t-butyl~~re~ol)~ 0151 17/51 4/5 1 20/51 

Diphenylhydantoin' 0/48 5/48 0/48 5/48 

Dowicide EC-7 pentachlorophenol 0/34 1/34 0/34 1/34 

Ethylene thiourea' 0/50 2/50 2/50 4/50 

Firemaster FF-1 polybrominated biphenyl' 0/50 4/50 1/50 5/50 

Manganese (11) sulfate monohydrate 0/5 1 12/51 3/51 13/51 

Technical grade pentachlorophenol OB3 3/33 OB3 3/33 

Triamtereneb 0/50 10/50 4/50 13/50 

Triamtereneb 0/50 7/50 5/50 10/50 

Tricresyl phosphateb 0/50 12/50 10/50 21/50 

Overall Historical Incidence 

Total 1/1,363 (0.1%) 159/1,363 (11.7%) 80/1,363 (5.9%) 223/1,363 (16.4%) 

Standard deviation 0.4% 8.3% 5.5% 10.7% 

Range 0%-2% 0%-33% 0%-20% 3%-42% 

a Data as of 20 August1992 

Mice housed individually ' Micehousedindividually after becomingpregnant 
Lesions in Female B3C3F, Mice 205 

TABLED4b 

Historical Incidence of Thyroid Gland Neoplasms in Untreated Female B6C3F, Micea 

Study 

Historical Incidence at Battelle Columbus 

2,4-Dichlorophenol 

4,4-Thiobis(6~-butyl~~-l)~ 

Diphenylhydantoin' 

Dowicide EC-7 pentachlorophenol 

Ethylenethiourea' 

Firemaster FF-1 polybrominated biphenyl' 

Manganese (11) sulfate monohydrate 

Technical grade pentachlorophenol 

Triamtereneb 

Triamtereneb 

Tricresyl phosphateb 

Overall Historical Incidence 

Total 

Standard deviation 

Range 

a Data as of 20 August 1992 

Mice housed individually ' Micehousedindividually after becoming pregnant 

Follicular Cell 

Adenoma 

1/49

o/s1 

4/47 

3f34 

0/50 

0/49 

2/50 

Of33 

1/49 

0/50 

1/49 

32/1,348 (2.4%) 

2.8% 

0%-9% 

Incidence in Controls 

Follicular Cell 

Carcinoma 

0/49 

0/5l 

0/47 

OM 

o/so

0/49 

0/50 

Of33 

1/49 

0/50 

0149 

2/1,348 (0.1%) 

0.5% 

0%-2% 

Follicular Cell 

Adenoma or Carcinoma 

1/49

o/s1 

4/47 

3/34

o/so 

0/49 

2/50 

Of33 

2/49 

0/50 

1/49 

34/1,348 (2.5%) 

2.9% 

0%-9% 
206 Oxazepam, NTP TR 443 

TABLED5 

Summary of the Incidence of Nonneoplastic Lesions in Female B6C3F, Mice in the 2-Year Feed Studyof Oxazepama 

Disposition Summary 

Animals initially in study 

IS" interim cwhruion 

Early deaths 

Moribund 

Natural deaths 

Survivors 

Terminal sacrifice 

Animals examined microscopically 

15-Month Interim Evaluation 

Alimentary System 

Liver 

Eosinophilic focus 

Infarct 

Hepatocyte, centrilobular, hypertrophy 

Pancreas 

Ectopic liver 

Infarct, focal 

Acinus, atrophy 

Stomach, glandular 

C$t epithelial inclusion 

Cardiovascular System 

None 

Endocrine System 

Adrenal cortex 

Hyperplasia 

Capsule, accessory adrenal cortical nodule 

Pituitary gland 

Pars distalis, hyperplasia 

Thyroid gland 

Inflammation, chronic active, focal 

Follicular cell, hyperplasia 

General Body System 

None 

Genital System 

waryFollicle, cyst 

Uterus 

Endometrium, hyperplasia, cystic, glandular 

0 PPm 

60 

10 

8 

3 

39 

60 

(10) 

(10) 

(10) 

(10) 

2 (20%) 

(10) 

10 (100%) 

125 ppm 

60 

10 

7 

2 

41 

60 

(10) 

2 (20%) 

(10) 

10 (100%) 

2,500 ppm 

60 

10 

22 

26 

2 

60 

(10) 

1 (10%) 

(10) 

10 (100%) 

5,000 ppm 

60 

10 

15 

35 

60 

(10) 

(10)

8 (80%) 
Lesions in Female B6C3F, Mice 207 

TABLED5 

Summary of the Incidence of Nonneoplastic Lesions in Female B6C3F, Mice in the 2-Year Feed Studyof Oxazepam 

(continued) 

0 PPm 125 ppm 2,500 ppm 5,000 ppm 

15-Month Interim Evaluation (continued) 

Hematopoietic System 

Spleen (10)

Developmental malformation 

Integumentary System 

None 

Musculoskeletal System 

None 

Nervous System 

None 

Respiratory System 

Lung 

Alveolar epithelium, hyperplasia, focal 

Arteriole, infiltration cellular, lymphocyte 

Special Senses System 

None 

Urinary System 

Kidney 

Nephropathy, chronic 

2-Year Study 

Alimentary System 

Intestine small, duodenum 

Inflammation, granulomatous 

Intestine small, jejunum 

Peyer's patch, hyperplasia, lymphoid 

Liver 

Basophilic focus 

Clear cell focus 3 (6%) 

Congestion 1 (2%) 

Developmental malformation 1 (2%) 

Eosinophilic focus 9 (18%) 19 (38%) 2 (4%) 5 (10%) 

Hematopoietic cell proliferation, focal 1 (2%) 

Hemorrhage 1 (2%) 1 (2%) Infiltration cellular, lymphocyte 1 (2%) 

Mixed cell focus 1 (2%) 

Necrosis, multifocal 2 (4%) 1 (2%) 

Thrombosis 2 (4%) 
208 Oxazepam, NTP TR 443 

TABLEDS 

Summary of the Incidence of Nonneoplastic Lesions in Female B6C3F, Mice in the 2-Year Feed Study of Oxazepam 

(continued) 

Alimentary System (continued) 

Liver (continued) 

Vacuolization cytoplasmic 

Bile duct, hyperplasia, focal 

Centrilobular, hypertrophy 

Mesentery 

Artery, inflammation, chronic 

Fat, inflammation, chronic 

Pancreas 

Inflammation, granulomatous 

Stomach, forestomach 

Dysplasia 

Hyperplasia, diffuse, squamous 

Hyperplasia, focal, squamous 

Hyperplasia, multifocal, squamous 

Ulcer 

Stomach, glandular 

Congestion, multifocal 

Blood vessel 

Aorta, mineralization 

Artety, inflammation, chronic 

Heart 

Mineralization 

Atrium, inflammation, chronic 

Endocrine System 

Adrenal cortex 

Cyst

Hyperplasia 

Hypertrophy

Vacuolization cytoplasmic 

Pituitaly gland 

Pars distalis, angiectasis 

Pars distalis, cyst 

Pars distalis, hyperplasia 

Thyroid gland 

Infiltration cellular, lymphocyte 

Follicular cell, hyperplasia 

General Body System 

None 

Genital System 

Clitoral gland 

Duct, ectasia 

0 PPm 

(50)

1 (2%) 

(50) 

1 (2%) 

2 (4%) 

2 (4%) 

(50)

2 (4%) 

2 (4%) 

6 (12%) 

(50) 

16 (32%) 

125 ppm 

1 (2%) 

2 (4%) 

(3)

1 (33%) 

1 (33%) 

(50) 

(50) 

1 (270) 

1 (2%) 

(50) 

(50)

1 (2%) 

1 (2%) 

(49)

2 (4%) 

8 (16%) 

(50) 

34 (68%) 

2,500 ppm 

11 (22%) 

(43) 

1(2%) 

4 (9%) 

(50) 

49 (98%) 

5,000 ppm 

29 (58%) 

2 (4%) 

(49) 

1 (2%) 

(50)

1 (2%) 

44 (88%) 
Lesions 209 in Female B6C3Fl Mice 

TABLED5 

Summary of the Incidence of Nonneoplastic Lesions in Female B6C3F, Mice in the 2-Year Feed Studyof Oxazepam 

(continued) 

ZYeur Study (continued) 

Genital System (continued) 

ovary

Atrophy 

erst 

Mineralization 

Bilateral, follicle, cyst 

Follicle, cyst 

Periovarian tissue, cyst 

Uterus 

Endometrium, hyperplasia, cystic 

Hematopoietic System 

Bone marrow 

Myelofibrosis 

Calvarium, myelofibrosis 

Myeloid cell, erythroid cell, atrophy 

Lymph node 

Bronchial, hyperplasia, lymphoid 

Lymph node, mandibular 

Atrophy 

Hyperplasia, plasma cell 

Lymph node, mesenteric 

Atrophy 

Hematopoietic cell proliferation 

Hemorrhage 

Hyperplasia, lymphoid 

Lymph node, mediastinal 

Angiectasis 

Spleen 

Hematopoietic cell proliferation 

Hyperplasia, focal, reticulum cell 

Hyperplasia, lymphoid 

Capsule, mineralization 

Lymphoid follicle, atrophy 

Lymphoid follicle, degeneration 

Red pulp, atrophy 

Thymus 

Atrophy 

Hyperplasia, focal, lymphoid 

Integumentary System 

Mammaly gland 

Hyperplasia, cytic.

Skin 

Ulcer, multifocal 

Dermis, sebaceous gland, hyperplasia 

0 PPm 

(50)

39 (78%) 

2 (4%) 

13 (26%) 

(50)

46 (92%) 

1(2%) 

(1) 

(50)

13 (26%) 

1(2%) 

2 (4%) 

1 (2%) 

2 (4%) 

2 (4%) 

(50)

41 (82%) 

1(2%) 

(50)

1(2%) 

(50) 

125 ppm 

(50)

39 (78%) 

1(2%) 

14 (28%) 

1(2%) 

(50)

50 (100%) 

1 (2%) 

1(2%) 

(2) 

(47) 

(46) 

1 (2%) 

1(2%) 

1 (2%) 

(49)

44 (90%) 

(49) 

(50)

1 (2%) 

1 (2%) 

2,500 ppm 

(48)

44 (92%) 

1(2%) 

3 (6%) 

1(2%) 

(50)

19 (38%) 

(3)

1 (33%) 

(50)

13 (26%) 

(34)

30 (88%) 

(47) 

(50) 

5,000 ppm 

(50)

38 (76%) 

5 (10%) 

(49)

14 (29%) 

1(3%) 

(49)

4 (8%) 

(34)

22 (65%) 

(43) 

(50) 
210 Oxazepam,NTPTR 443 

2-Year Study (continued) 

Musculoskeletal System 

Bone 

Femur, hyperostosis, focal 

NervousSystem 

Brain (50) (50) Compression 1(2%) 

Peripheral nerve (2) (3)

3 (100%) 

(2) (1) Sciatic, axon, degeneration 2 (100%) 1(50%) 1 (100%) 

Nerve, degeneration 

Degeneration 

Spinal cord (2) 

1(50%) 

1(50%) 

(3)

1 (33%) 

3 (100%) 

(2) 

1(50%) 

1(50%) 

1(100%) 

Lung 

Respiratory System 

Alveolar epithelium, hyperplasia 

Infiltration cellular, lymphocyte 

Foreignbody 

(50) 

1(2%) 

(50) 

1(2%) 

(50) 

1 (2%) 

1(2%) 

1(2%) 

(50) 

Eye 

Special Senses System 

Harderian gland 

Phthisis bulbi 

cyst 

Kidney 

Urinary System 

Renal tubule, necrosis, acute, diffuse 

Artery, pelvis, inflammation, chronic 

Nephropathy, chronic 

Inflammation, subacute 

Ulcer 

Inflammation, chronic 

Urinary bladder 

(50) 

14 (28%) 

(50) 

(50) 

12 (24%) 

1(2%) 

1(2%) 

(50) 

(50) 

9 (18%) 

(50) 

1(2%) 

3 (6%) 

(50) 

1(2%) 

1(2%) 

a Number of animals examined microscopically at site and number of animals with lesion 
211 

APPENDIX E 

GENETIC TOXICOLOGY 

SALMONELLA TYPHIMURIUM MUTAGENICITY TEST PROTOCOL . . . . . . . . . . . . . . . .. . . . . . .... . . . .. 212

CHINESE HAMSTER OVARYCELLCYTOGENETICS PROTOCOLS...... . ..... . . . . . . . . . .... . . . . . . 212 

MOUSEPERIPHERAL BLOOD MICRONUCLEUS TESTPROTOCOL . . . . ... . ...... . . . . .... . . . . . . .. 213

RESULTS ..................................................................... 213 

TABLE El Mutagenicity of Oxazepam in Salmoneh Qphimuriurn .... . .. l . . l . . .... . . . . . . . . 214 

TABLE E2 Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells 

by Oxazepam . . . . . . . . . ... . . . . ... . . . ...... . .... . . ... . . . . . .. . .. . . . . ... 216 

TABLEE3 Induction of ChromosomalAberrationsinChinese Hamster Ovary Cells 

by Oxazepam . . . ... . . . . . . . . . .. . . . . . . . .. . . . . .. . . . . . . . . . . . . . . . . . . . . . .. 217 

TABLEE4 Frequency of Micronucleiin B6C3Fl Mouse Peripheral Blood Erythrocytes 

Following Administration of Oxazepam in Feed for 14 Weeks . . . . .... . . . . . . . . . . . 218 
212 Oxazepam, NTP TR 443 

GENETIC TOXICOLOGY 

sAL.MONELLA TYPHIMURIUM MUTAGENICITYTESTPROTOCOL 

Testing was performed as reported by Zeiger et al. (1992). Oxazepam was sent to the laboratory as a 

coded aliquot from Radian Corporation (Austin, TX). Oxazepam was incubated with the Salmonella 

fyphinzuriunz tester strains TA97, TA98, TA100, TA102, and TA1535 either in buffer or S9 mix (metabolic 

activation enzymes and cofactors from Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster 

liver) for 20 minutes at 37" C. Top agar supplemented with 1-histidine and d-biotin was added, and the 

contents of the tubes were mixed and poured onto the surfaces of minimal glucose agar plates. Histidineindependent mutant colonies arising on these plates were counted following incubation for 2 days at 37" C. 

Each trial consisted of triplicate plates of concurrent positive and negative controls and at least five doses 

of oxazepam. The high dose was limited by solubility and toxicity. All assays were repeated. 

In this assay, a positive response is defined as a reproducible, dose-related increase in histidineindependent (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 determinationof mutagenicity. A negative response is obtained when no increase 

in revertant colonies is observed following chemical treatment. No minimum percentage or fold increase 

is required for a chemical to be judged positive or weakly positive. 

CHINESEHAMSTER OVARY CELL CYTOGENETICS PROTOCOLS

Testing was performed as reported by Galloway er al. (1987). Oxazepam was sent to the laboratory as a 

coded aliquot by Radian Corporation. Oxazepam was tested in cultured Chinese hamster ovary (CHO) 

cells for induction of sister chromatid exchanges (SCEs) and chromosomal aberrations (Abs), both in the 

presence and absence of Aroclor 1254-induced male Sprague-Dawley rat liver S9 and cofactor mix. 

Cultures were handled under gold lights to prevent photolysis of bromodeoxyuridine-substituted DNA. 

Each test consisted of concurrent solvent and positive controls and of at least three doses of oxazepam. 

The high dose was limited by toxicity. A single flask per dose was used, and tests yielding positive results 

were repeated. 

Sister Chromatid Exchange Test: In the SCE test without S9, CHO cells were incubated for 26 hours with 

oxazepam in McCoy's 5A medium supplemented with fetal bovine serum, I-glutamine, and antibiotics. 

Bromodeoxyuridine (BrdU) was added 2 hours after culture initiation. After 26 hours, the medium 

containing oxazepam 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 oxazepam, serum-free 

medium, and S9 for 2 hours. The medium was then removed and replaced with medium containing serum 

and BrdU and no oxazepam 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 SCEs/cell from each dose level. Because significant chemicalinduced cell cycle delay was seen at the 50 p@mL dosewithout S9, 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 than 0.001. An increase of 20% or greater at any single dose was considered weak evidence 
Genetic Toxicology 213 

of activity; increases at two or more doses resulted in a determination that the trial was positive. A 

statistically significant trend (P10.05) in the absence of any responses reaching 20% above background led 

to a call of equivocal. 

ChromosomalAberrations Test: In the Abs test without S9, cells were incubated in McCoy’s 5A medium 

with oxazepam for 12 hours; Colcemid was added and incubation continued for 2 hours. The cells were 

then harvested by mitotic shake-off, fHed, and stained with Giemsa. For the Abs test with S9, cells were 

treated with oxazepam and S9 for 2 hours, after which the treatment medium was removed and the cells 

were incubated for 10 hours in fresh medium, with Colcemid present for the final 2 hours. Cells were 

harvested in the same manner as for the treatmentwithout S9. 

Cells were selected for scoring on the basis of good morphology and completeness of karyotype 

(21 k 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. Statistical analyses 

were conducted on both the dose-response curve and individual dose points. For a single trial, a 

statistically significant (P10.05) difference for one dose point and a significant trend (P10.015) were 

considered weak evidence for a positive response; significant differences for two or more doses indicated 

the trial was positive. A positive trend test in the absence of a statistically significant increase at any one 

dose resulted in an equivocal call (Galloway et af., 1987). 

MOUSE PERIPHERAL BLOOD MICRONUCLEUS TEST PROTOCOL 

A detailed discussion of this assay can be found in MacGregor et af. (1990). Peripheral blood samples 

were obtained from 10 male and 10 female B6C3Fl mice from each dose group at the end of the 14-week 

toxicity study. Smears were immediately prepared and fixed in absolute methanol, stained with a 

chromatin-specific fluorescent dye mixture of Hoechst 33258/pyronin Y (MacGregor et al., 1983), and 

coded. Slides were scanned to determine the frequency of micronuclei in 10,OOO normochromatic 

erythrocytes (NCEs) per animal. The criteria of Schmidt (1976) were used to define micronuclei, with the 

additional requirement that the micronuclei exhibit the characteristic fluorescent emissions of DNA (blue 

with 360 nm and orange with 510 nm UV illumination); the minimum size limit was approximately onetwentieth the diameter of the NCE cell. The frequency of micronucleated cells among NCEs was analyzed 

by a statistical software package (ILS, 1990), which employed a one-tailed trend test across dose groups 

and a t-test for pairwise comparisons of each dose group to the concurrent control. 

RESULTS 

Oxazepam (3 to 3,333 puplate) did not induce mutations in Safnzoneffaryphinwiunz strains TA102, 

TA100, TA1535, TA97, or TA98 when tested in a preincubation protocol with or without Aroclor 

1254-induced male Sprague-Dawley rat or Syrian hamster liver S9 (Table El). In cytogenetic tests with 

Chinese hamster ovary cells, oxazepam did not induce sister chromatid exchanges (Table E2) or 

chromosomal aberrations (Table E3), with or without S9. Cell cycle delay was noted at the 50 pg/mL dose 

in the SCE test without S9; harvest time was extended to allow accumulation of sufficient second-division 

metaphase cells for analysis. Peripheral blood samples obtained from B6C3F, mice in the 14-week toxicity 

study were analyzed for frequency of micronucleated NCEs; no increase in micronucleated NCEs was 

observed in any of the dose groups (Table E4). 
214 Oxazepam, NTP 443 TR

TABLEEl 

Mutagenicity of Oxazepam in Salmonella typhimuriurna 

Revertants/plete b 

Strain Dose -s9 + hamster S9 + rat s9 

(PdPla9 10% 30% 10% 30% 

TA102 

0 131 f 2.6 213 f 5.7 341 f 23.3 197 f 11.5 443 * 24.8 

3 136 f 5.7 

10 128 f 10.7 

33 139 f 1.8 223 f 15.0 295 f 8.1 216 f 9.5 403 f 17.1 

100 130 f 8.3 196 f 10.0 340 f 24.6 208 f 6.4 436 f 38.4 

333 43 f 3.9c 170 f 2.7 358 f 25.0 204 f 14.3 413 f 22.0 

i,m 155 f 12.4 318 f 1.3 166 f 28.6 375 sr: 19.9 

1,666 103 f 21.5' 102c 

3,333 172 f 1.2' 133 f 17.3' 

Trial summary Negative Negative Negative Negative Negative 

Positive controld 676 f 9.5 879 f 17.9 2,127 f 104.3 1,291 f 49.3 1,104 f 125.0 

TAlOO 

0 159 f 8.0 146 f 1.9 145 f 8.1 161 f 10.0 149 f 2.5 

3 118 f 4.6 

10 128 f 5.5 144 f 2.3 160 f 9.0 

33 137 f 14.0 159 f 17.2 145 f 9.1 157 f 8.9 137 f 7.4 

100 139 f 8.9 137 f 15.4 146 f 2.1 156 f 8.9 127 f 1.2 

333 115 f 9.7 141 f 2.9 113 f 8.7 133 f 7.0 134 f 4.8 

1,000 102 f 5.8 93 f 2.8 126 f 2.5 134 f 4.1 

1,666 100 f 14.9 135 f 13.2 

Trial summary Negative Negative Negative Negative Negative 

Positive control 316 * 3.5 445 f 11.7 462 f 31.2 599 f 23.1 244 f 6.7 

TA1535 

0 20 f 1.5 8 f 0.9 12 f 2.9 12 f 1.7 14 f 2.1 

3 20 f 2.3 

10 19 f 1.5 

33 19 f 2.9 8 f 3.0 10 f 2.0 10 f 2.0 14 f 0.3 

100 15 f 4.2 11 f 2.0 11 f 3.4 11 f 1.5 15 f 1.5 

333 18 f 2.0 8 f 1.0 9 f 2.5 11 f 0.6 15 f 1.9 

' 1,Ooo 9 f 1.3 8 f 0.3 9 f 0.7 12 f 2.7 

8 -c 1.2e 7 f 1.9 

3,333 5 f 0.7e 9 f 0.6e 1,666 

Trial summary Negative Negative Negative Negative Negative 

Positive control 332 f 10.2 180 f 18.4 340 f 22.8 214 f 27.7 206 f 4.7 
Genetic Toxicology 215 

TABLEEl 

Mutagenicity of Oxazepam in SahoneUa typhimurium (continued) 

Revertantdplate 

Strain Dose -s9 +hamster S9 +rat s 9

bdPlaW 10% 30% 10% 30% 

TA97 

0 184 f 6.1 179 f 3.0 164 f2068.3 f 1.5 209 f 6.1 

3 193 f 7.4 

10 182 f 2.6 204 163 f 

180 f 10.5 163 f 5.0 183 2 

8.2 f 4.2 

33 6.9203 f 0.9191 f 9.1

f 

333 148 f 12.7 175 f 11.0 174 f 14.0 198 f 1623.0 f 17.4 

1,Ooo 172 f 17.3 168 f 168 9.1 f 184 5.8 f 16.8

100 160 f 7.3 167 f1879.0 f 200 12.4 1503.1 f 6.2 

3,333 139 107 f 5.7' f 11.2' 

Trial summary Negative Negative Negative Negative Negative

Positive control 403 f 11.1 463 f 15.8 368 f 24.9 449 f 10.1 454 f 25.6 

TA98 

18 f 2.3 28 f 2.0 19 f 0.9 19 f 1.5 23 f 1.2 

15 f 2.2 

16 f 2.0 

19 f 2.3 18 f 0.6 27 f 4.3 24 f 3.0 28 f 2.2 

20 f 0.7 21 f 2.1 27 f 4.1 22 f 3.7 33 f 2.6 

17 f 1.7 20 f 0.3 23 f 3.3 23 f 4.6 31 f 4.7 

20 f 2.3 34 f 2.3 14 f 0.7 23 f 1.7 

17 f 0.6e 27 f 1.5 22 f 3.9 21 f 2.3 

Trial summary Negative Negative Negative Negative Negative 

Positive control 423 f 27.7 523 f 10.6 495 f 21.7 202 f 10.9 104 f 5.9 

a Study performed at SRI, International. A detailed description of the protocol is presented in Zeiger et al. (1992). 

Revertants are presented as mean f standard error from three plates. 

Slight toxicity 

2-aminoanthracene was used on all strains in the presence of S9; in the absence of S9, 4-nitro+phenylenediamine was tested on 

TA98, sodium azide was tested on TAlOO and TA1535.9-aminoacridine was tested on TA97, and mitomycin-C was tested on 

TA102. 

e Precipitateonplate 
216 Oxazepam, NTP TR 443 

TABLEE2 

Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells by Oxazepama 

No. of sc4 Relative SC4 

Dose Total Chromo- No. of Chromo- SCW Hrs Chromosome 

Compound (bg/mL) somes Cells SCES some Cell in BrdU m b

-s9 

Summary:Negative 

Dimethylsulfoxide 50 1,049 424 0.40 8.5 26.0 

50 1,045 393 0.37 7.9 31.0' 

Mitomycin-C 0.001 50 1,048 579 0.55 11.6 26.0 46.91 

0.004 10 210 223 1.06 22.3 26.0 182.37 

Oxazepam 5 50 1,048 448 0.42 9.0 26.0 13.67 

17 50 1.048 428 0.40 8.6 26.0 8.59 

50 50 1,048 449 0.42 9.0 31.Oc 13.92 

P=0.061d 

+s9 

Summary:Negative 

Dimethylsulfoxide 50 1,046 402 0.38 8.0 26.0 

Cyclophosphamide 50 1,043 579 0.55 11.6 26.0 44.44 0.125 

0.500 10 209 205 0.98 20.5 26.0 155.22 

Oxazepam 5 50 1,048 452 0.43 9.0 26.0 12.22 

17 50 1,050 460 0.43 9.2 26.0 13.99 

50 50 1.048 445 0.42 8.9 26.0 10.48 

P=O.O75 

a Studyperformed at SitekResearch Laboratories.SCE = sisterchromatidexchange;BrdU = bromodeoxyuridine. A detailed 

description of the protocol is presented in Galloway er d . (1987). 

SCFdchromosome of culture exposed to oxazepam relative to those of culture exposed to solvent 

Because oxazepam induced a delay in the cell division cycle, harvest time was extended to maximize the number of second-division 

metaphase cells available for analysis. 

Significance of relative SCEs/chromosome testedby the linear regression trend test vs. log of the dose 
Genetic Toxicology 217 

TABLEE3 

Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells by Oxazepama 

-s9 +s9 

Dose Total No. of Abs/ Percent DoseTotal No. of Abs/ Percent 

(pglmL) Cells Abs CellCells (pglmL) Cells Abs CellCells 

with Abs with Abs 

Harvest time: 14.0 hours Harvest time: 12.0 hours 

Summary: Negative Summary: Negative 

Dimethylsulfoxide Dimethylsulfoxide 

200 5 0.03 1.o 200 0.51 

12 

0.01 

Mitomycin-C Cyclophosphamide 

9 36.0 0.48 25

Oxazepam Oxazepam 

25 200 1 0.01 0.5 43 200 0 0.00 0.0 

54 200 3 0.02 1.o 93 200 3 0.02 1.5 

116 200 0 0.00 0.0 200 200 2 0.01 1.o 

P=0.842b P=O.135 

a Study performed at Sitek Research Laboratories. Abs = aberrations. A detaileddescription of the protocol is presented in 

Galloway er of. (1987). 

Significance of percent cells with aberrations tested by the linear regression trend test vs. log of the dose 

0.4 25 0.36 20 32.0 
218 Oxazepam, NTP TR 443 

TABLEE4 

Frequency of Micronuclei in B6C3F, Mouse Peripheral Blood Erythrocytes Following Administration 

of Oxazepam in Feed for 14 Weeksa 

Dose Percent Micronucleated 

(PP@ NCE Cells 

Male 0 0.082 f 0.008 

625 0.081 f 0.009 

1,250 0.078 f 0.007 

2,500 0.085 f 0.010 

5,0@) 0.074 f 0.008 

10,Ooo 0.069 f 0.007 

P=0.899b 

Female 0 0.042 f 0.006 

625 0.039 f 0.005 

1,250 0.034 f 0.007 

2,500 0.031 f 0.005 

5,Ooo 0.042 f 0.005 

10,Ooo 0.043 f 0.007 

P=O.194 

a NCE = nonnochromatic erythrocyte. A minimum of 10,OooNCEs scored per animal, 10 animals per dose group; data presented 

as mean 2 standard error. A detailed description of the protocol is presented in MacGregor et uf. (1990). 

One-tailed trend test 
219 

APPENDIX F 

ORGAN WEXGHTS 

AND ORGAN-WEXGHT-TO-BODY-'WEIGHTRATIOS 

TABLEF1 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Swiss-Webster Mice 

in the 14-Week Feed Study of Oxazepam ................................... 220 

TABLE F2 Organ Weights and Organ-Weight-to-Body-Weight Ratios for B6C3F, Mice 

in the 14-Week Feed Study of Oxazepam ................................... 221 

TABLEF3 Organ Weights and Organ-Weight-to-Body-Weight Ratios for B6C3FL Mice 

at the 15-Month Interim Evaluation in the 2-Year Feed Study of Oxazepam ......... 222 
220 Oxazepam, NTP TR 443 

TABLEF1 

Organ Weights and Organ-Weight-to-Bodyweight Ratios for Swiss-Webster Mice in the 14-Week Feed Study 

of Oxazepama ~~ ~ ~~ 

0 PPm 625 ppm 1,250 ppm 2,500 ppm 5,000 ppm 10,000 ppm 

Male 

n 10 10 10 10 10 9 

Necropsy body wt 35.8 f 0.8 38.5 f 0.9 35.2 f 0.7 38.2 f 35.5 11f35.410 f 0.7 

Heart 

Absolute 0.179 f 0.009 0.174 f 0.007 0.144 f 0.004'. 0.159 f 0.006 0.162 f 0.008 0.141 f 0.005** 

Relative 5.00 f 0.24 4.53 f0.23 4.10 f 0.12.. 4.19 f 0.20. 4.59 f 0.243.98 f 0.10'. 

R. Kidney 

Absolute 0.292 f 0.010 0.294 f0.006 0.280 f 0.012 0.280 f 0.012 0.270 f 0.008 0.269 f 0.012 

Relative 8.18 f 0.23 7.66 f 0.21 7.94 f 0.27 7.35 f 0.28 7.62 f 0.26 7.59 f 0.25 

Liver 

Absolute 1.808 f 0.080 2.354 f 0.074.* 2.275 f 0.112. 2.761 f 0.134** 3.035 f 0.115.' 3.528 f 0.137** 

Relative 50.43 f 1.35 61.23 f 1.88 64.60 f 2.75.. 72.61 f 3.92'. 85.52 f 2.61.. 99.98 f 4.27** 

Lungs 

Absolute 0.251 f 0.009 0.293 f 0.020 0.266 f 0.012 0.283 f 0.011 0.290 f 0.014 0.245 f 0.008 

Relative 7.04 f 0.26 7.63 f 0.56 7.59 f 0.37 7.43 f 0.26 8.14 f 0.32 6.95 f 0.32 

R. Testis 

Absolute 0.097 f0.003 0.099f 0.004 0.095 f 0.004 0.100 f 0.004 0.093 f 0.002 0.096 f 0.004

Relative 2.71 f 0.10 2.57 f 0.10 2.71 f 0.14 2.65 f 0.12 2.64 f 0.07 2.72 f 0.10 

Thymus 

Absolute 0.037 f 0.003 0.045 f 0.002 0.045 f 0.003 0.049 f 0.004*0.048 f 0.0030.043 f 0.003 

Relative 1.03 f 0.08 1.16 f 0.05 1.28 f 0.08 1.26 f 0.08 1.34 f 0.06' 1.23 f 0.08 

Female 

n 10 10 10 10 10 10 

Necropsy body wt 29.8 f0.6 32.0 f 0.7 31.3 f 0.5 32.2 f 0.8* 31.0 f30.70.6 f 0.4

Heart 

Absolute 0.135 f 0.005 0.137 f 0.004 0.143 f 0.008 0.142 f 0.004 0.136 f 0.003 0.140 f 0.005 

Relative 4.54 f 0.17 4.31 f 0.13 4.59 f 0.29 4.43 f 0.17 4.41 f 0.154.56 f 0.12 

R. Kidney 

Absolute 0.204 f 0.007 0.223 f 0.006 0.229 f 0.007* 0.229 f 0.006.0.240 f0.005*.0.233 f 0.007.. 

Relative 6.90 f 0.31 6.97 f 0.13 7.30 f 0.13 7.14 f 0.20 7.74 f 0.19' 7.60 f 0.19 

Liver 

Absolute 1.405 f 0.028 1.731 f 0.049** 1.910 f 0.052.. 2.328 f 0.109*. 2.610 f 0.064** 3.084 f 0.070** 

Relative 47.32 f 1.14 54.28 f 1.47 61.16 f 2.12.. 72.42 f 2.77** 84.16 f 1.93.. 100.63 f 1.95.' 

Lungs 

Absolute 0.268 f 0.015 0.256 f 0.008 0.257 f 0.008 0.273 f 0.013 0.260 f 0.010 0.243 f 0.010 

Relative 9.04 f 0.57 8.03 f 0.25 8.22 f 0.27 8.59 f 0.55 8.39 f 0.34 7.92 f 0.28 

Thymus 

Absolute 0.050 f 0.004 0.049 f 0.004 0.049 f 0.002 0.052 f 0.002 0.050 f 0.003 0.047 f 0.003 

Relative 1.68 f 0.11 1.52 f 0.10 1.57 f 0.06 1.62 f 0.07 1.63 f 0.10 1.52 f 0.11 

. Significantly different (PSO.05) from the control group by Williams' or Dunnett's test 

PSO.01 

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weight/g body weight 

(mean fstandard error) 
Organ Weight Analyses 221 

TABLEF2 

Organ Weights and Organ-Weight-to-Bodyweight Ratiosfor B6C3F, Mice in the 14-Week Feed Study of Oxazepama 

0 PPm 625 ppm 1,2502,500 ppmpm 5,000 10,000 ppm ppm

Male 

n 10 10 9 10 10 

Necropsy body wt 34.6 f 0.7 36.3 f 0.735.0 f 0.635.0 f 0.534.0 f 0.633.7 f 0.6 

Heart 

Absolute 0.170 f 0.009 0.162 f 0.003 0.172 f 0.007 0.151 f 0.004 0.163 f 0.005 0.165 f 0.003 

Relative 4.90 f 0.21 4.49 f 0.13 4.90 f 0.17 4.33 f 0.12' 4.79 f 0.14 4.90 f 0.10 

R. Kidney 

Absolute 0.280 f 0.008 0.285 f 0.0070.296 f 0.007.249 f 0.005.0.259 f 0.007 0.266 f 0.008 

Relative 8.08 f 0.13 7.86 f0.178.45 f 0.147.12 f 0.10..7.62 f 0.167.89 f 0.15 

Liver 

Relative 

Absolute 

47.24 f 0.96 

1.634 f0.052 1.919 f 0.044'. 

52.91 f 0.67.. 

2.150 f 0.037.. 

61.42 f 0.47.. 

2.186 f 0.038't 

62.50 f 0.94.. 

2.545 f 0.065.. 

74.83 * 1.09.. 

2.966 2 0.057.. 

88.09 f 1.19.. 

Lungs 

Absolute 0.227 f 0.009 0.229 f 0.009 0.242 f 0.010 0.272 f 0.013. 0.222 2 0.009 0.271 f 0.020 

Relative 6.55 f 0.18 6.34 f 0.30 6.94 2,0.31 7.75 f 0.31 6.54 f 0.30 8.04 f 0.57. 

R. Testis 

Absolute 0.124 f 0.003 0.127 f 0.003.126 f 0.002.129 f 0.002.125 f 0.002.129 & 0.003 

Relative 3.60 f 0.08 3.50 & 0.093.59 f 0.073.69 f 0.073.69 f 0.07385 f 0.10 

Thymus 

Absolute 0.040 f 0.002 0.054 f 0.002.0.052 f 0.003.0057 f 0.004..0050 f 0.0020.057 f 0.005.. 

Relative 1.16 f 0.07 1.50 f 0.061.49 f 0.08 1.64 f 0.12..1.47 f 0.051.71 f 0.15** 

Female 

n 10 10 9 9 

Necropsy body wt 29.9 f 0.6 31.5 f 0.830.4 f 0.630.1 f 0.530.9 f 0.630.3 f 0.4 

Heart 

Absolute 0.135 f 0.003 0.145 f 0.003 0.155 f 0.005'. 0.140 f 0.005 0.144 2 0.004 0.146 f 0.003 

Relative 4.55 f 0.14 4.62 f 0.14 5.11 f 0.22. 4.67 f 0.13 4.67 f 0.09 4.81 f 0.09 

R. Kidney 

Absolute 0.202 f 0.006 0.241 f 0.005'.0.247 f 0.004'.0.222 f 0.006.0.228 f 0.005..0.232 f 0.002" 

Relative 6.77 f 0.13 7.67 f 0.16..8.15 f 0.18'.7.38 f 0.15.7.39 f 0.13.7.68 f 0.08.. 

Liver 

Absolute 1.387 f 0.021 1.743 f 0.031.. 1.871 f 0.046.. 1.898 f 0.044.' 2.391 2 0.072.. 2.837 f 0.061*' 

Relative 46.59 f 1.01 55.44 f 1.02" 61.54 2 0.76.. 63.20 f 1.19.. 77.35 f 1.16.. 93.64 f 1.03.. 

Absolute 

Lungs 

0.235 f 0.010 0.258 f 0.009.240 f 0.0110.230 2 0.0100.245 f 0.0150.240 f 0.010 

Relative 7.91 f 0.35 8.21 f 0.337.90 f 0.347.66 f 0.297.94 f 0.43 7.91 f 0.27 

Thymus 

Absolute 0.056 f 0.002 0.059 k 0.002.058 f 0.0030.064 f 0.0040.064 f 0.004.056 f.O.001 

Relative 1.86 f 0.07 1.88 f 0.051.90 2 0.092.13 2 0.132.07 & 0.121.84 f 0.03 

Significantly different (PSO.05) from the control group by Williams' or Dunnett's test 

I* PSO.01 

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratiosare given as mg organ weight/g body weight 

(mean f standard error) 

10 
222 Oxazepam, NTP TR 443 

TABLEF3 

Organ Weights and Organ-Weight-to-Bodyweight Ratiosfor B6C3F, Mice 

at the 15-Month Interim Evaluation in the 2-Year Feed Studyof Oxazepama 

0 PPm 125 ppm 2,500ppm 5,000ppm 

n 10 10 10 10 

Male 

Necropsy body wt 48.9 f 1.2 50.0 f 1.2 40.1 f 0.6'. 35.5 f 0.7.. 

R. Kidney 

Absolute 0.388 f 0.007 0.335 f 0.010'. 0.279 f 0.005+f 0.257 f 0.007.' 

Relative 7.96 f 0.17 6.71 f 0.13.. 6.95 f 0.13.' 7.23 f 0.10.. 

Liver 

Absolute 2.264 f 0.120 2.561 f 0.1% 3.450 f 0.157. 7.162 f 0.605.. 

Relative 46.02 f 1.35 51.30 f 3.93 86.39 f 4.85.. 201.34 f 16.73.. 

Female 

Necropsy body wt 53.3 f 2.0 47.3 * 1.9'. 40.2f 1.3.' 35.8 f 0.6" 

R. Kidney 

Absolute 0.258 f 0.008 0.248 2 0.006 0.239f 0.007 0.242 f 0.006 

Relative 4.87 f 0.14 5.29 2 0.15' 5.95 f 0.101. 6.75 f 0.12.. 

Liver 

Absolute 2.008 f 0.079 1.874 2 0.044 3.262f 0.312' 6.980 f 0.729.. 

Relative 37.76 f 0.94 39.95 & 0.99 80.14 * 5.22'. 195.30 f 20.16.. 

. Significantly different (PsO.05) from the control group by Williams' or Dunnett's test 

I +P10.01 

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weight/g body weight 

(mean f standard error). 
223 

APPENDIX G 

NEUROBEHAVIORAL STUDIES 

INTRODUCTION................................................................. 224 

METHODS..................................................................... 224 

RESULTS..................................................................... 225 

DISCUSSION................................................................... 230 

TABLEG1- TABLE622 .......................................................... 232 
224 Oxazepam, NTP TR 443 

NEUROBEHAVIORAL STUDIES 

INTRODUCTION 

Although the acute and short-term neurobehavioral effects of benzodiazepines have been fairly extensively 

investigated in animals, few studies of repeated measures have been performed throughout the course of a 

prolonged chronic treatment regimen. Therefore, a battery of neurobehavioral assays including forelimb 

and hindlimb grip strength, thermal sensitivity, motor activity, and startle response were performed on 

groups of Swiss-Webster and B6C3Fl mice during weeks 2 and 12 of the 14-week studies; before study 

initiation and during months6 and 12 of the 57-week study in Swiss-Webster mice; and before study 

initiation and during months 6, 12, 18, and 24 in the 2-year study in B6C3Fl mice. 

METHODS 

In the 14-week studies, 10 male and 10 female mice of each strain and exposure group (0, 625, 1,250, 

2,500, 5,OOO, and 10,OOOppm) were tested. Ten male and 10 female mice of each strain and exposure 

group [0, 125 (B6C3Fl mice only), 2,500, and 5,OOO ppm] were also tested in the chronic studies. Thirty 

animals per day underwent neurobehavioral testing; each testing period occurred over 4 consecutive days. 

To reduce extraneous variation, animals designated for neurobehavioral testing were placed on a separate 

cage rack in the study room and were undisturbed for at least 12 hours prior to testing. This rack of 

animals was moved to the behavior laboratory for acclimation at least 1 hour before testing began. For 

the most part, testing was limited to the hours between 8:30 a.m. and 1 p.m. 

Gdp strength. Forelimb and hindlimb grip strength were measured using a device similar to that described 

by Meyer et al. (1979), and data were entered directly into a Xybion@ electronic data collection system. 

The animal was allowed to grip a triangular ring with its forepaws and was pulled back along a platform 

until its grip was broken. As the backward motion continued, the animal's hindpaws reached a T-shaped 

hindlimb grip bar, which it was allowed to grasp and then was forced to release by continued pulling. 

Chatillon push-pull strain gauges (Kew Gardens, NY) were used to record the maximum strain required to 

break the forelimb and hindlimb grip. Each animal was given five trials with less than 1 minute between 

trials so that a measure of degree of habituation or fatigue could be observed. The body weight of each 

animal was recorded. 

Thermal sensitivity (analgesia test). A model 550 Analgesia Meter (Omnitech Electronics Inc., 

Columbus, OH) was used for this test and data were entered directly into Xybion@. The device consisted 

of a square acrylic plate arena with a clear acrylic plate cover mounted on a heat source to ensure 

maximum experimenter visibility. The animal was placed on the heat source and the arena covered. The 

dependent variable was rodent reaction time to the heat stimulus (55" C). The response monitored was 

characteristically a vigorous licking of the hind paws. Latency was measured manually using a built-in 

timer. Measurement began when the animal touched the plate and ended at the onset of the pain-sensing 

response. Animals failing to make the response in 30 seconds were removed and assigned an arbitrary 

maximum score of 30 seconds. 

Motor activity. Motor activity was measured using a photocell movement detection procedure under stressfree conditions. Chambers were sound insulated and darkened, and each contained an individual acrylic 

plate test cage. A ventilation fan with baffled air intake and exhaust system was mounted in each cubicle, 

and a 4-inch speaker was used for delivery of 75 dB white noise. A U-shaped photocellAight source holder 

was placed under the test cages and photo beam detector units were inserted so that infrared photo beams 

6 cm apart passed through the test cage just above the cage floor. Animal movement inside the cage 

interrupted these photo beams and was translatcd into activity counts by means of modular signal 

processing equipment (Coulburn Instruments, Lehigh Valley, PA). Motor activity was determined over 
Neurobehavioral Studies 225 

five continuous 3-minute periods, with the totals at the end of each period printed on a microprocessorbased, 10-channel printer. This gave an indication of habituation of activity. 

Startle response. Startle response is measured using an SR-LAB Startle Response System (San Diego 

Instruments, San Diego, CA). It is composed of four isolation cabinets, a computer control unit and 

connection box, four startle chambers, and four test station control boxes. Response measurement takes 

place within a sound isolated cubicle (Coulburn Instruments Isolation Cubicle Model E10-20) equipped 

with a light, a ventilation fan, a small viewing port, and a thermometer. The computer unit controls the 

presentation of all stimuli in four chambers simultaneously. The startle chamber in which the test animal 

is enclosed is constructed of transparent acrylic. The sensitivity of each chamber may be individually and 

reproducibly adjusted. A calibration routine is performed prior to each testing session to detect significant 

sensitivity differences among the chambers. Each test station control box contains a complete sound 

generation system to produce high frequency noise stimuli up to 120 dB(A), an adjustable background 

noise level [70 dB(A)], and an AC relay for tactile (air-puff) stimuli. 

In the 14-week studies, startle response was examined by measuring the interaction between prepulse 

inhibition and the habituation of the startle reflex over SO repeated trials. An acclimation period of 

3 minutes was followed by 10 startle trials with a tactile airpuff (15 to 20 psi, 20 msec duration per trial) 

as the main startle stimulus (block 1). This was followed by 60 prepulse trials in which an SO to 90 dB(A) 

white noise prepulse preceded the tactile stimulus by 100 msec (block 2). The final 10 trials were identical 

to the first 10 trials (block 3). In the chronic studies, each block consisted of 20 trials, for a total of 

60 repeated trials. Startle response for each trial was recorded 20 msec after the main startle stimulus was 

turned off in order to demonstrate a response in which there was no stimulus interference. The 20 msec 

wait time was added to all latencies. All trials were separated by an 8 second inter-trial interval. A 

background noise level of 70 dB(A) prevailed when the prepulse or main stimulus was off. Each session 

took approximately 15 minutes to complete. Inclusion of a prepulse and measurement of the startle 

response over repeated trials provided data for the initial reactivity, habituation, and prepulse inhibition of 

the startle response in the same test. 

Multiple aspects of the startle response were studied simultaneously. Initial reactivity to an air-puff tactile 

stimulus is determined by values obtained for block 1 (mean of tactile trials 1-10 or 1-20). Startle 

amplitude can be modified by presenting very brief, low intensity stimuli (prepulses) shortly before an 

intense, startle eliciting stimulus. Prepulse inhibition occurs using prepulses at the threshold of audibility 

which, by themselves, do not elicit startle responses. The inhibitory effect of an auditory prepulse was 

determined by the responses of block 2 (mean of trials 11-70 or 21-40), and comparison with those of 

block 1. A general decrease in response amplitude over repeated stimulus presentations was also shown 

and was referred to as habituation. The extent of habituation was determined by comparison of block 3 

(mean of trials 71-80 or 41-60) to block 1. The startle response-dependent variable that was statistically 

analyzed was the average amplitude across the entire response window of each trial. 

Statistical methods. Neurobehavioral data were analyzed by analysis of variance and Dunnett’s test 

(Dunnett, 1955). 

RESULTS 

Swiss-Webster Mice 

14-Week Study 

Grip strength. No significant differences in forelimb grip strength were observed in male or female mice 

during prestudy testing. One week of exposure to oxazepam led to a significant reduction in mean 

forelimb grip strength in male [F(5,54) = 2.82, P10.0251 and female [F(5,54) = 3.11, P10.0151 mice (the 

two numbers in parentheses represent the degrees of freedom for the F test). Results of an analysis of 

variance followed by Dunnett’s test for comparison of individual means revealed that the 5,000 and 
226 Oxazepam, NTP TR 443 

10,OOO ppm groups were different from controls during week 2 (Table Gl). For males in these groups, the 

deficits represented a 15% decrease compared to controls. Similar decreases were seen in 5,000 (12%) and 

10,OOOppm (14%) females. Forelimb grip strength deficits of the same order of magnitude 

[F(5,53) = 4.33, P10.002] were still evident at week 12 in 625, 5,000, and 10,000 ppm males, but not in 

females. There were no consistent differences in habituation between exposed and control groups of either 

sex. 

No significant differences in prestudy hindlimb grip strength were observed in male or female mice. 

Significant deficits in mean hindlimb grip strength were evident in males [F(5,54) = 4.32, P10.0021 and 

females [F(5,54) = 6.19, P10.0001] during week 2 (Table G2). The 5,OOO ppm females and 10,OOO ppm 

males and females had significantly lower grip strength scores compared to those of the controls at 

2 weeks. The extent of the hindlimb grip strength deficit was slightly greater than that of the forelimb grip 

strength. Decreases for those groups significantly different from control were between 20% and 24%. 

Hindlimb grip strength deficits had disappeared in female mice at week 12, but were still evident to a 

somewhat lesser degree (8%) in male mice [F(5,53) = 2.37, P10.0511 compared to controls. Overall, the 

degree of habituation of hindlimb grip strength across trials was similar in exposed and control mice of 

each sex at weeks 2 and 12. Decreases in grip strength could not be related to decreases in body weight as 

all exposed groups had similar or greater body weights than those of control groups. 

Thermal sensitivity. No evidence of chemical-related changes in analgesia or pain sensation were indicated 

in either sex at week 2. However, at week 12, significant decreases in paw lick latency were demonstrated 

in males [F(5,53) = 4.13, P10.0031 and females [F(5,53) = 3.10,P10.0161. Paw lick latency was reduced 

in male mice in the 625, 5,000, and 10,000ppm groups, the same groups with grip strength deficits. The 

625, 1,250, 2,500, and 5,000 ppm groups of female mice also had decreased paw lick latency (Table G3). 

Motor activity. Chemical-related increases in motor activity were evident in all groups of male and female 

mice at week 2 [male, F(5,54) = 5.10, P10.0007; female, F(5,54) = 4.53, P10.0016; Table G4]. Although 

there appeared to be no dose-related effects, exposed males (46%) showed an overall greater increase in 

motor activity compared to controls than did females (35%). Motor activity also increased from prestudy 

to week 2. The facilitatory effect of oxazepam on motor activity was no longer evident in male mice at 

week 12. Change from prestudy to week 12 was similar in all groups of male mice. In contrast, increased 

motor activity in female mice persisted to week 12 [F(5,53) = 2.50, P10.04151. Females in the 1,250 and 

10,OOO ppm groups showed significantly greater activity counts than did controls at 12 weeks. 

Startle response. Table G5 shows the startle response profiles of each exposure group. The mean initial 

reactivities (block 1)of male and female mice collectively were 59 f 3 and 59 f 4 amplitude units, 

respectively. Mean prestudy startle responses of males and females collectively during block 2 were 30 k 1 

and 34 f 3 amplitude units, respectively. Mean startle responses for male and female mice collectively 

during block 3 were 51 f 3 and 45 f 3 amplitude units, respectively. Thus, a 13% habituation of the 

startle response was observed overall in male mice whereas a 24% habituation occurred in females at study 

initiation. 

A group (dose) by block design was used to analyze startle response data. An analysis of variance on 

startle response data from males at week 2 (Table G6) indicated significant overall effects of group, 

F(5,4782) = 56.35, P10.0001; block, F(2,4782) = 303.60, P10.0001; and group x block, F(10,4782) = 8.98, 

P1O.OOO1. The group by block interaction suggested differences between exposure groups in startle 

response across trial blocks. Separate analyses of each trial block provided additional evidence of 

chemical-related effects on startle behavior. Significant F-ratios were demonstrated for all trial blocks 

[block 1,F(5,594) = 9.38, P10.0001; block 2, F(5,3594) = 48.27, P10.0001; block 3, F(5,594) = 11.02, 

P1O.OOOll. Initial reactivity (block 1) of males to the startle eliciting stimulus was reduced for all exposed 

groups compared to that of the controls; this general tendency was also prevalent in blocks 2 and 3. This 

reduction was significant in the 625, 5,000, and 10,000 ppm groups during block 1and all exposed groups 
Neurobehavioral Studies 227 

during block 2. For the most part, no consistent changes in the inhibitory effectof a prepulse were 

evident in males at week 2. The effect of the prepulse on control animals at week2 was identical to that 

during prestudy. Compared to prestudy the 625 and 10,000ppm groups showed decreases while the 1,250, 

2,500, and 5,OOO ppm groups demonstrated increases in prepulse inhibition of the startle response. During 

block 3, all exposed groups, except the 1,250 ppm group, had startle responses significantly less than those 

of controls. The 2,500 and 5,000 ppm groups also showed the highest percent habituation. 

For female mice at week2 (Table G6), significant effects were indicated for group, F(5,4782) = 27.42, 

P1O.OOO1; block, F(2,4782) = 390.98,P1O.OOO1; and group x block, F(10,4782) = 5.05, P1O.OOOl. 

Separate analyses for each trial block indicated significant differences between groups [block1, 

F(5,594) = 2.39, P10.0367; block 2, F(5,3594) = 22.43, P10.0001; block 3 F(5,594) = 8.47,P1O.O001]. 

The average startle response for females at week2 was reduced for all groups compared to their respective 

scores at study initiation. The overall chemical-related reduction of the startle response that was clearly 

demonstrated in males was not evident in females. The prepulse inhibition was similar among groups at 

week 2 and was similar to that exhibited during prestudy. Habituation was generally reduced from 

prestudy to 2 weeks for all female mice. Habituation seemed to be impaired to a greater extent in all 

exposed groups, except in the 10,000 ppm group, compared to controls. 

Analysis of variance of startle behavior for male mice during week 12 (Table G7) revealed significant 

effects for group, F(5,4702) = 45.67, P10.0001; block, F(2,4702) = 554.61,P1O.OOOl; and group x block, 

F(10,4702) = 18.42,P1O.OOOl. Individual analyses indicated significant effects at each trial block [block 1, 

F(5,584) = 12.48,P1O.OOOl; block 2, F(5,3534) = 41.60,P10.0001; block 3, F(5,584) = 9.33,P<O.OOOl]. 

Compared to prestudy and week 2, initial startle responses were lower in all males at week 12. Repeated 

testing may have resulted in an overall dampening of the response. Nonetheless, exposure led to a 

reduction in startle behavior throughout the studies. During block 1, the 1,250, 2,500, 5,000, and 

10,000 ppm groups had startle responses significantly lower than those of the controls. All exposed males 

had startle responses significantly lower than those of the controls during block 2. The prepulse inhibition 

was similar to that of the controls for all groups except the2,500 ppm group, which showed decreased 

sensitivity to the effects of the auditory prepulse. During block 3, the 2,500, 5,000, and 10,000 ppm groups 

demonstrated decreased startle responses. The degree of habituation was similar to that reported for 

prestudy with the exception of the2,500 ppm group, which showed no habituation. 

Significant effects of group [F(5,4702) = 45.67, P10.0001],block [F(2,4702) = 554.61, P10.0001], and 

group x block [F(10,4702) = 18.42, P10.0001] were indicated by analysis of variance performed on 

week 12 data for females (Table G7). Separate analyses of each trial block suggested significant 

differences between exposure groups [block 1, F(5,584) = 12.48, P1O.OOO1; block 2, F(5,584) = 41.60, 

P10.0001; block 3, F(5,584) = 9.33,P10.0001)]. All scores were lower at week 12 compared to prestudy 

or week 2. Initial startle reactivity of 1,250 ppm females was significantly less than that of the controls. 

While the percent prepulse inhibition was higher overall at week 12 than at week 2, again, no differences 

between exposed and control females were observed. Startle response of the 5,000 and 10,000ppm groups 

was significantly less than that of controls during block 3. The 5,000 and 10,OOO ppm groups demonstrated 

the greatest habituation of the startle response as well. The 625, 1,250, and 2,500 ppm groups showed 

little or no habituation comparedto control mice. 

57-Week Study 

Grip strength. During the 6-month evaluation in the 57-week study of Swiss-Webster mice, forelimb grip 

strength was significantly decreased in 5,000 ppm male mice (Table GS). Hindlimb grip strength was not 

affected by oxazepam exposure (Table G9). At 12 months, neither forelimb nor hindlimb grip strength in 

either sex was altered by oxazepam exposure. Although for males, forelimb decreases were demonstrated 

in the 2,500 (18%) and 5,000 ppm (24%) groups, the reduction in the number of animals in these groups 

may have influenced the statistical analysis and resulting lack of difference between these groups and 

controls. 
228 Oxazepam, NTP TR 443 

Thermal Sensitivity. Results of the thermal sensitivity test revealed decreases in paw lick latency in the 

exposed mice at 6 months (Table G10). Paw lick latencies in the 2,500 and 5,000 ppm female groups were 

significantly different from those of the controls. Decreases in paw lick latencies were observed in males 

in the 2,500 (29%) and 5,OOO ppm (48%) groups. However, this effect was significant only in the 

5,OOO ppm group compared to the control group. At 12 months, significant differences in paw lick latency 

scores were not evident, although the5,000 ppm female group showed a 38% decrease compared to the 

control group. 

Motor activity and startle response. Motor activity at 6 months was not affected by oxazepam exposure, but 

motor activity was reduced by 53% in 5,000 ppm females at 12 months (Table G11). Startle response was 

not altered by oxazepam at either 6 or 12 months (data not shown). 

B6C3Fl Mice 

14-Week Study 

Grip sfrengfh. One week of oxazepam exposure led to a significant reduction in mean forelimb grip 

strength in males [F(5,54) = 5.51, P10.001] but not in females. Results of an analysis of variance followed 

by Dunnett’s test for comparison of individual means showed that all male exposure groups were 

significantly different from controls during week 2 (Table G12). No effect of oxazepam was observed 

during week 12 in male or female mice. For male mice there were no consistent differences between 

exposed and control groups in performance across trials. However, exposed females showed a slightly 

greater degree of habituation than controls. This might have been due, in part, to the lack of any 

difference in performance of the control animals, across five trials. 

Significant decrements in mean hindlimb grip strength were evident in male [F(5,54) = 12.45, P10.001] 

and female [F(5,54) = 3.93, P10.0041 mice. All exposed mice, except females receiving 625 ppm, had 

lower hindlimb grip strength scores compared to controls (Table G13). Males were slightly more sensitive 

to the chemical-related grip strength deficit than females. The mean decreases for exposed males and 

females were 20% and 13%, respectively. During week 12, all hindlimb grip strength deficits had 

disappeared in all mice. For most exposed groups, the degree of habituation of hindlimb grip strength 

performance was greater than controls during week 2, but not during week 12. 

Body weights of exposed animals were similar to or greater than those of the controls. Decreases in grip 

strength were not considered related to body weights. 

Thernzaf sensitivity. No evidence of chemical-related changes in analgesia or pain sensation were indicated 

at week 2. However, at week 12, significant decreases in paw lick latency were demonstrated in female 

mice [F(5,54) = 3.06, P10.0171. The 625, 1,250, and 10,000ppm groups had latency scores that were 

lower than controls (Table G14). 

Motor activity. Chemical-related increases in motor activity were evident in both male and female mice at 

weeks 2 and 12 [male,week 2: F(5,54) = 13.37,P10.0001;female, week 2: F(5,54) = 6.31, P10.0001; 

male,week 12: F(5,54) = 3.98, P<O.O038; female, week 12: F(5,54) = 6.16, P10.0001].Significant 

differences in motor activity between exposed and control groups of each sex were observed at each time 

period (Table G15). In exposed males, the changes in motor activity from prestudy to week 2 and from 

prestudy to week 12were similar. Exposed females showed greater changes in motor activity from 

prestudy to week 12 than from prestudy to week 2. 

Startle response. Examination of data from the prestudy period provided various indices of the startle 

response (Table G16). The mean initial reactivities of male and female mice collectively were 67 2 1and 

61 +- 2 amplitude units, respectively. Mean startle responses collectively during block 2 were 52 It- 2 and 

45 +- 1 amplitude units for males and females, respectively. Thus, the inclusion of an auditory prepulse 
Neurobehnviornl Studies 229 

caused an inhibition of the startle response, overall, in male (22%) and female (26%) mice. Mean startle 

responses for male and female mice collectively during block 3 were 58 -+ 2 and 59 -+ 2 amplitude units, 

respectively. A 13% habituation of the startle response was observed overall in male mice, whereas only a 

3% habituation occurred in females. 

An analysis of variance on startle data from male mice at week 2 (Table G17) indicated significant overall 

effects of group, F(5,4782) = 62.95, P10.0001; block, F(2,4782) = 331.42, P10.0001; and group x block, 

F(10,4782) = 4.41,P1O.OOOl. The group x block interaction suggested that there were differences 

between exposure groups in startle performance across trial blocks. Separate analyses for each trial block 

provided additional evidence for chemical-related effects on startle behavior. Significant F-ratios were 

demonstrated for all trial blocks [block 1, F(5,594) = 9.20, P10.0001; block 2, F(5,3594) = 49.08, 

PSO.0001; block 3, F(5,594) = 10.86,P<O.OOOl]. Initial reactivity (block 1) to the startle eliciting stimulus 

was significantly greater (P10.05) in the 625 and 1,250 ppm groups of males. During block 2, the 625, 

2,500, 5,000, and 10,000 ppm groups had average startle responses that were significantly less than those of 

the controls while the 1,250 ppm group had responses greater than controls. In general, the inhibiting 

effects of a prepulse were greater for exposed mice than controls. Comparison of blocks 1 and 3 revealed 

no habituation of the startle response in male controls. For the most part, exposed groups showed a 

greater degree of habituation. 

Analysis of variance of startle response behavior for female mice at week 2 (Table G17) revealed 

significant effects for group, F(5,4782) = 57.45, P10.0001; block, F(2,4782) = 276.50, P10.0001; and 

group x block, F(10,4782) = 4.95, P10.0001. Individual analyses indicated significant dose-related effects 

at each trial block [block 1, F(5,594) = 6.64, P10.0001; block 2, F(5,3594) = 63.08, P50.0001; block 3, 

F(5,594) = 4.79,PSO.O003]. Compared to presludy, little change in initial reactivity to the startle stimulus 

was observed at week 2 except in the 2,500 ppm group, which had scores greater than controls. This is 

different from the male 625 and 1,250 ppm groups in which startle amplitudes were significantly greater 

than those of the controls at week 2. During block 2, the 1,250, 5,000, and 10,000 ppm groups had average 

startle responses less than controls while the 2,500 ppm group had greater scores. Similar to males at 

week 2, prepulse inhibition was generally greater in exposed females than controls. This can be compared 

to an overall 26% prepulse inhibition for females at prestudy. During block 3, the 625 ppm group had 

startle scores greater than controls. Repeated trial presentation resulted in a greater degree of habituation 

of the startle response in the 1,250 and 2,500ppm groups. 

At week 12, significant effects were indicated in males for group, F(5,4782) = 16.52, P10.0001; block, 

F(2,4782) = 772.01, P<O.OOOl; and group x block, F(10,4782) = 9.30, P50.0001 (Table GB). Separate 

analyses for each trial block indicated significant differences between groups [block 1, F(5,594) = 2.41, 

P10.0354; block 2, F(5,3594) = 21.37, P10.0001; block 3, F(5,594) = 7.72, P50.0001]. The average startle 

response for the 625, 2,500, and 10,000 ppm males was lower than their responses at prestudy or week2. 

This may have been due to the fact that repeated testing caused an overall reduction in response, or 

alternatively, the increase in age may have made the animals less hyperactive. During block 2, the 625, 

2,500, and 10,OOO ppm males had startle scores significantly lower than controls. Similar to week 2, 

prepulse inhibition was, for the most part, greater in exposed males than in controls. Overall, the effects 

of an auditory prepulse on tactile startle response were greater at week 12 than at week 2. Startle 

responses of all but the 625 and 2,500 ppm males were greater than those of the controls during block 3. 

In general, startle response habituation was greater in controls than in exposed mice. Compared to the 

degree of habituation at week 2, exposed males showed little change, whereas control males showed 

greater habituation at week 12 (-34%) than at week 2 (+4%). 

Significant effects of group [F(5,4782) = 11.10, P<O.OOOl], block [F(2,4782) = 612.92, P10.0001], and 

group X block [F(10,4782) = 7.85, P10.0001] were indicated by analysis of variance performed on females 

at week 12 (Table GB). Separate analyses of each trial block suggested significant differences between 

exposure groups [block 1, F(5,594) = 4.48, P10.0005;block 2, F(5,3594) = 13.13, P10.0001; block 3, 
230 Oxazepam, NTP TR 443 

F(5,594) = 3.52, P<O.O038]. The initial startle response of the 10,000ppm females was significantly less 

than that of the controls. Similar to male mice, the startle behavior of all female groups at week 12 was 

less, overall, than at prestudy or week 2. Startle responses for the 2,500, 5,000, and 10,000 ppm females 

were less than those of the controls during block 2. There appeared to be no differences between 

exposure groups with regard to the inhibitory effect of a prepulse. The lack of any group differences may 

have been due to the greater prepulse inhibition occurring in control females at week12 (-50%) than at 

week 2 (-28%). The 625, 1,250, and 10,000 ppm groups showed greater startle response than controls 

during block 3. Similar to their male counterparts, habituation was greater for control than exposed 

females at week 12. 

2-Year Study 

Grip strength. At the 6-month neurobehavioral evaluation in the 2-year study of B6C3Fl mice, mean 

forelimb grip strength appeared to be reduced in exposed mice, but there was no statistically significant 

difference between exposure groups (Table G19). Males receiving 5,000 ppm showed increased hindlimb 

grip strength compared to control animals (Table G20). No significant changes in forelimb or hindlimb 

grip strength were observed in females. 

At the 12-month evaluation, decreases in forelimb grip strength were evident for males in all exposure 

groups (Table G19). This decreased forelimb grip strength did not appear to be an indirect effect (i.e., 

due to body weight changes), based on further analysis of covariance results. However, it was uncertain if 

the decreased forelimb grip strength was a direct chemical effect because a dose-related trend was not 

apparent. Control data were similar to the 6-month values. Hindlimb grip strength in male mice was not 

affected by exposure. No significant changes in forelimb or hindlimb grip strength were observed in 

females at 12 months. 

The 18-month evaluation revealed decreases in forelimb grip strength in both sexes in the 2,500 and 

5,OOO ppm groups (Table G19); these were not thought to be related to body weight changes. The 

18-month data provided somewhat more certainty that the decreased forelimb grip strength was a 

chemical-related effect because a dose-related trend was more apparent than at 12 months. On the other 

hand, the abdominal swelling and neoplasia seen in many of the animals in these exposure groups may 

have contributed, in part, to the grip strength deficit. Oxazepam exposure did not affect hindlimb grip 

strength in either sex (Table G20). 

Motor activity. At 6 and 12 months, motor activity in both sexes was increased compared to controls 

(Table G21). Motor activity of 5,000 ppm males and females was significantly reduced at 18months, 

probably indicating the debilitating effect of the drug or of the drug-induced neoplasia at this point in 

time. 

Thermal sensitivity and starfle response. Paw lick latencies (Table G22) and startle response (data not 

shown) were relatively unchanged by oxazepam at 6 and 12 months. At the 18-month interval, paw lick 

latencies were decreased in the 125 ppm group while startle response remained relatively unchanged. 

No neurobehavioral changes were noted at the 24-month testing interval. Due to high mortality in the 

2,500 and 5,OOO ppm groups, testing was limited to the controls and 125 ppm groups. Neurobehavioral 

study mice were observed daily for clinical signs of withdrawal during an eight-day observation period after 

completion of dosing. No symptoms of withdrawal were observed. 

DISCUSSION 

Neurobehavioral results suggest two pharmacologically distinct actions of oxazepam. A nonspecific muscle 

relaxant or depressant effect was particularly evident as reflected by deficits in grip strength. These were 

more prevalent in males than females of each strain, and only temporary as differences in grip strength 
Neurobehavioral Studies 231 

tended to diminish by week 12 of the 14-week studies. This is consistent with published reports that 

repeated dosing leads to an attenuation of the depressant effects of the benzodiazepines. A disinhibitory 

action of oxazepam was indicated by a facilitatory effect on motor activity. Increases in motor activity may 

be due to the anxiety-reducing effect of oxazepam. These effects were seen at weeks 2 and 12 of exposure 

with each sex and strain, although they were diminished somewhat at week12 in exposed male SwissWebster mice. As reported in the literature, repeated dosing often leads to an enhancement of the 

stimulatory effects of low-dose benzodiazepine treatment. In all exposed female B6C3Fl groups, especially, 

motor activity was increased to a greater extent at week 12 than at week2. 

The tactile startle response assesses somatosensory integrity, the abilityof an animal to transform sensory 

input to motor output. In general, when increases in initial startle response were reported, they were 

evident at week 2 in low- to middle-dose groups. This could represent the presence of a hyperarousal 

state, or perhaps reflect a disinhibitory action as was proposed to account for the increase in motor 

activity. Continued exposure led to significant reductions in initial startle reactivity. 

The inhibitory effect of an auditory prepulse on tactile startle responding was generally augmented in 

exposed B6C3Fl mice, but not in the Swiss-Webster mice, compared to controls. Alone, an auditory 

startle response is usually 3 to 4 times weaker than a tactile one. Thus, greater prepulse inhibition seen in 

exposed mice may be due to increased sensitivity to acoustic stimuli. Alternatively, exposed mice may be 

better able to distinguish acoustic and tactile stimuli. For whatever reason, these findings suggest that 

effects of oxazepam on the sensory componentof the startle reflex circuit may account for the differences 

observed. Habituation of the startle response appeared to be variably affected by oxazepam. 

Effects of oxazepam on the sensory system, in general, and arousal mechanisms, specifically, may also be 

involved in the changes observed during the thermal sensitivity measurements. An increased sense of 

awareness by exposed mice may have contributed to decreased paw lick latencies seen in some exposure 

groups. 

The purpose of long-term neurobehavioral studies was to determine if chronic exposure caused a change in 

the types or severity of neurobehavioral signs from those observed in the 14-week studies. The chronic 

studies were designed to include neurobehavioral evaluations at study initiation and at 6-month intervals, 

using the same animals at each time point. Because of high mortality in the 2,500 and 5,000 ppm groups 

in both strains, a complete assessment was not possible and replacement animals had to be used. 

There was evidence of decreased forelimb grip strength in dosed Swiss-Webster mice at 6 months, and in 

B6C3Fl mice at 12 and 18 months. As with other measures, it was difficult to determine if the changes 

seen in B6C3Fl mice at 18months were a direct effect of oxazepam exposure or an indirect effect due to 

the deteriorating condition of animals that weighed less and were developing liver neoplasia. Mice in the 

125 ppm group that survived to 24 months did not develop deficits in forelimb or hindlimb grip strength. 

Dosed Swiss-Webster mice showed decreased paw lick latencies at 6 months, as was noted in the 13-week 

studies, but consistent changes were not seen in the B6C3Fl mice at any time. 

In contrast, increased motor activity was noted in B6C3F, mice, but not in Swiss-Webster mice. Decreased 

motor activity in dosed mice of both strains late in the study was attributed to their poor condition. Thus, 

the direct effects of oxazepam exposure on motor activity appeared to be strain dependent, as SwissWebster mice showed an increase in activity only at week 2 in the 13-week study, while the effect in 

B6C3Fl mice persisted for at least a year. 

Although observation of noticeable behavioral changes in response to chronic oxazepam treatment was 

diminished by poor survival, no novel behavioral effects or significant enhancement of behavioral effects 

were apparent with 57-week and 2-year oxazepam exposure. 
232 Oxazepam, NTP TR 443 

TABLEGI 

Mean Forelimb Grip Strength of Swiss-Webster Mice in the 14-Week Feed Study of Oxazepama 

Dose Prestudy Week 2 Week 12 

(PPm) (9) (9) (9) 

Male 

0 103.16f 6.72 125.50f 4.86 147.72f 6.15 

625 109.38f 5.67 118.84f 3.21 126.78f 3.69.' 

1,250 107.60f 4.24 117.22f 2.82 136.50f 3.22 

2,500 111.36f 4.21 117.64f 5.50 140.46f 4.37 

5,m 105.30f 3.68 107.10f 4.16' 126.92f 2.95'' 

10,m 105.90f 2.67 106.92f 4.75' 129.06f 3.10'' 

Female 

0 91.38f 3.37 109.10f 2.83 119.22f 5.08 

625 85.54k 3.77 106.76f 3.07 124.642 3.55 

1,250 89.16f 5.17 108.70f 4.38 126.93f 8.91 

2,500 88.84f 4.05 103.00f 4.30 110.50f 6.62 

5,m 89.24f 4.23 93.76f 4.64. 113.62f 4.92 

10,Ooo 90.76f 5.17 95.62f 3.09' 109.04k 6.43 

* Significantly different (PSO.05) from the control group by Dunnett's test; data are given as grams of force needed to break grip 

*' PSO.01 

a Mean f standard error; n=10 except for 625 ppm males and 1,250ppm females in Week 12,where n=9 

TABLE G2 

Mean Hindlimb Grip Strength of Swiss-Webster Mice in the 14-Week Feed Study of Oxazepama 

Dose Prestudy Week 2 Week 12 

(PPm) (9) (9) (9) 

Male 

0 77.14f 4.59 88.96f 3.05 106.42? 3.31 

625 78.52f 3.76 85.44f 1.39 102.13f 3.16 

1,250 80.28f 3.50 84.36f 2.21 106.06f 2.37 

2,500 82.08f 2.64 85.52f 3.09 105.96f 2.63 

5,000 74.92f 3.72 79.10f 3.19 98.06f 1.79 

10,000 74.82f 2.80 71.38f 4.39" 98.22f 1.96 

Female 

0 67.46f 2.08 77.52f 3.17 90.32f 3.17 

625 64.40k 3.00 78.14f 1.96 95.08f 2.29 

1,250 66.50 f 3.08 74.36f 1.94 94.40f 4.24 

2,500 65.48f 3.41 69.70f 4.15 83.76f 4.65 

5,000 66.64f 3.36 59.22f 4.24" 86.44f 3.43 

10,000 67.44f 2.71 62.06f 3.06" 84.52f 4.75 

** Significantly different (PSO.01)from the control group by Dunnett's test; data are given as grams of force needed to break grip 

a Mean f standard error; n=10 except for 625 ppm males and 1,250ppm females in Week 12,where n=9 
NeurobehavioralStudies 233 

TABLE63 

Mean Paw Lick Latency of Swiss-Webster Mice in the 14-Week Feed Study of Oxazepama 

Dose Prestudy Week 2 Week 12 

(PPm) (sec.) (sec.) (sec.) 

Male 

0 14.62 f 1.94 13.58 f 1.49 17.29 f 2.47 

625 11.81 f 0.72 9.98 f 1.22 7.34 * 0.55'. 

1,250 13.46 f 1.28 12.78 2 1.68 12.73 f 1.76 

2,500 15.10 f 1.63 12.25 f 1.92 11.72 f 2.34 

5,OOO 15.56 f 1.55 16.30 f 2.45 9.94 f 0.73' 

10.000 15.29 * 1.71 17.81 f 2.62 9.88 * 0.67* 

Female 

0 13.29 f 1.06 11.55 f 0.53 13.04 f 1.83 

625 13.36 f 1.27 10.26 f 0.80 8.35 f 0.77' 

1,250 13.01 f 1.52 10.94 2 0.96 8.43 f 0.79. 

2,500 12.18 f 0.95 10.52 f 0.98 8.57 f 0.91' 

5,OOO 12.34 f 0.88 12.79 f 1.45 8.47 f 0.72. 

10,000 13.41 f 0.90 16.34 f 2.69 11.33 f 1.32 

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

** PSO.01 

a Mean f standard error; n=10 except for 625 ppm males and 1,250 ppm females in Week 12, where n=9 

TABLE64 

Mean Total Motor Activity Count of Swiss-Webster Mice in the 14-Week Feed Study of Oxazepama 

Dose Prestudy Week 2 Change from Prestudy Week 12 Change from Prestudy 

(PP4 (%) (%I 

Male 

0 380 f 19 310 f 30 -3 463 f 32 +22 

625 352 f 14 555 f 32** +58 449 f 12 +28 

1,250 310 f 23 545 f 49" +47 472 f 43 +28 

2,500 354 f 19 508 f 23* +44 424 f 38 +20 

5,ooO 359 * 21 545 f 21.. +52 447 f 19 +25 

10.000 369 f 15 551 f 27** +49 478 f 26 +30 

Female 

0 386f 17 392 f 14 +2 428 f 27 +11 

625 311 f 25 503 f 35. +33 513 f 27 +36 

1,250 364 f 12 539 f 29** +48 564 f 16* +55 

2,500 382 f 11 538 f 28** +41 504 f 35 +32 

5,OOO 352 f 18 504 * 25. +43 489 f 38 +39 

10,OOO 366f 17 558 f 33** +52 548 f 30* +50 

l Significantly different (PSO.05) from the control group by Dunnett's test 

** PSO.01 

a Mean f standard error; n=10 except for 625 ppm males and 1,250 ppm females in Week 12, where n=9 
234 Oxazepam, NTP TR 443 

TABLEG5 

Startle Response Profiles Prior to Exposure of Swiss-Webster Mice in the 14-Week Feed Study 

of Oxazepama 

Dose Block lb Block 2' Prepulse Inhibitiond Block 3e Habituation' 

(PPm) (%) (%) 

Male 

0 55 rt 3 28rtl -49 56 2 3 +2 

625 67 f 5 3022 -5 5 47 f 3 -30 

1,250 72 f 5 35 rt 2. -5 1 61 rt 4 -1 5 

2,500 60+6 26 f 1 -51 46 rt 4 -23 

5,000 54 f 4 31 f 2 -43 58 rt 4 +7 

10,Ooo 47 rt 4 28+2 -40 37 2 2.. -21 

Females 

0 56 f 3 34f3 -39 46 rt 3 -1 8 

625 44 rt 2. 2821 -36 35 rt 2. -20 

1,250 60+3 26 rt 2. -5 I 44 2 3 -27 

2,500 79 f 4" 48 2 2.. -39 58 rt 4' -27 

5,Ooo 54 rt 2 31 f 1 -43 43 rt 3 -20 

10,Ooo 63 k 2 35 f 2 -44 45 rt 2 -29 

l Significantly different (PSO.05) from the control group by Dunnett's lest 

** PSO.01 

a Mean f standard error of average responses (amplitudes) for each trial; n=10 

Block 1 = mean of first 10 trials (tactile stimulus without auditory prepulse) 

Block 2 = mean of trials 11-70 (tactile stimulus preceded by auditory prepulse); 80-90 dB 

Percent prepulse inhibition = block 1 - block 2 x 100 

block 1 e

' Block 3 = mean of last 10 trials (tactile stimulus without auditory prepulse) 

Percent habituation = block I - block 3 x 100 

block 1 
Neurobehavioral Studies 235 

TABLEG6 

Startle ResponseProfiles of Swiss-Webster Mice at Week 2 in the 14-Week Feed Study of Oxazepama 

Dose Block lb Block 2' Inhibitiond Prepulse Block 3e Habituationf

(PPm) (W (94 

Male 

0 65 f 4 33 f 1 -49 59 f 5 -9 

625 39 f 3** 24 f 1** -38 37 f 2** -5 

1,250 54 f 6 26 f 1.. -52 53 f 6 -2 

2,500 55 f 5 22 f 1** -60 44 f 3' -20 

5,Ooo 47 f 5. 19 f 0'. -60 31 f 2** -34 

10,Ooo 29 f 2** 19 f 0" -34 27 f 2'. -7 

Female 

0 36+3 22 f 1 -39 35 f 2 -3 

625 37 f 4 22 f 1 -41 39 f 3 +5 

1,250 37 2 2 20 f 0 -46 45 f 2* +22 

2,500 3822 24 f 1 -37 46 f 3* +21 

5,Ooo 36+2 21 2 1 -42 42 f 3 +17 

10,Ooo 28f2 16 f 0** -43 26 f I* -7 

l Significantly different (PsO.05) from the control group by Dunnett's test 

** PdO.01 

a Mean f standard error of average responses (amplitudes) for each trial; n=10 

Block 1 = mean of first 10 trials (tactile stimulus without auditory prepulse) 

Block 2 = mean of trials 11-70 (tactile stimulus preceded by auditory prepulse); 80-90 dB 

Percent prepulse inhibition = block 1 - block 2 x 100 

block 1 

e Block 3 = meanoflast 10 trials (tactile stimulus without auditory prepulse) 

Percent habituation = block 1 - block 3 x 100 

block 1 
236 Oxazepam, NTP TR 443 

TABLEG7 

Startle Response Profilesof Swiss-Webster Mice at Week 12 in the 14-Week Feed Study of Oxazepama 

Dose Block lb Block 2' Prepulse Inhibitiond Block 3e Habituationf 

(PPW (%) (%) 

Male 

0 36f3 15 f 0 -58 30 f 2 -17 

625 31 f 3 12 f 01' -61 27 f 2 -13 

1,250 28 f 2. 12 f 0'. -57 26 f 2 -7 

2,500 14 f 1** 11 f 0** -21 16 f 1** +14 

5,Ooo 25 f 2** 11 f 0** -56 22 f 2.1 -12 

10,Ooo 20 f I** 11 2 O*' -45 17 f 1** -15 

Female 

0 3022 14 f 0 -53 21 f 1 -30 

625 25 f 2 13 f 0* -48 23 f 1 -8 

1,250 22 f 2' 11 f 0** -50 24 f 2 +9 

2,500 23 f 2 13 f O* -43 25 f 2 +9 

5,000 3of3 13 f 0' -57 16 f 1' -47 

10,Ooo 2322 12 f o* -48 15 f 0' -35 

l Significantly different (PSO.05) from the control group by Dunnett's test 

** PSO.01 

a Mean f standard error of average responses (amplitudes) for each trial; n=10 

Block 1 = mean of first 10 trials (tactile stimulus without auditoly prepulse) 

Block 2 = mean of trials 11-70 (tactile stimulus preceded by auditory prepulse); 80-90 dB 

Percent prepulse inhibition = block 1 - block 2 x 100 

block 1 

e Block 3 = mean of last 10 trials (tactile stimulus without auditory prepulse) 

Percent habituation = block 1 - block 3 x 100 

block 1 
Neurobehavioral Studies 237 

TABLEGS 

Mean Forelimb Grip Strength of Swiss-Webster Mice in the 57-Week Feed Study of Oxazepama 

6 Months 12 Months 

(9) (9) 

Male 

0 89 & 3 122 k 5b 103 f 7' 

2,500 86 k 3 108 f 4 84 f gd 

5,Ooo 88k4 102 k 5. 78 k lod 

Female 

0 70 f 6 106 f 4 84 f gb 

2,500 75 f 4 93 f 5b 78 f 3' 

5,000 69 k 3b 93 rt 4b 74 f 7d 

* Significantly different (PSO.05) from the control group by Dunnett's test a Mean k standard error; n=10 except where indicated; data are given as grams of force needed to break grip strength 

n=9 

n=7 

n=4 

TABLEG9 

Mean Hindlimb Grip Strength of Swiss-Webster Mice in the 57-Week Feed Study of Oxazepama 

Dose Prestudy 6 Months 12 Months 

(PPm) (9) (9) (9) 

Male 

0 108 f 4 105 f 4b 96 f 4' 

2,500 108 k 4 115 f 4 86 f 8d 

5.000 110 f 5 115 f 3 102 f 2d 

Female 

0 81 f 4 100 rt 4 85 f 3b 

2,500 77 k 3 95 k 3b 78 f 3' 

5.000 77 k 3b 100 k gb 77 rt 3d 

a Mean k standard error; n=10 except where indicated; data are given as grams of force needed to break grip strength; differences 

from the control group are not significant by Dunnett's tat. 

n=9 

n=7 

n=4 
238 Oxazepam, NTP TR 443 

TABLEG10 

Mean Paw Lick Latency of Swiss-Webster Mice in the 57-Week Feed Study of OxaZepama 

Dose Prestudy 6 Months 12 Months 

(PPW (seconds) (seconds) (seconds) 

Male 

0 15.0 f 1.4 17.0 f 2.8b 14.1 f 2.9' 

2,500 13.5 f 2.1 12.1 f 2.5 7.2 f 1.7d 

5,000 13.2 f 1.2 8.9 & 0.9' 17.8 f 4.4d 

Female 

0 13.2 fi 2.1 14.6 f 2.5 14.1 f 2.6b 

2,500 12.8 f 1.2 7.1 f 0.6**b 10.9 f 1.8' 

5,OOo 11.2 f 1.2b 6.8 f 8.7 ? l.6d 

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

** PSO.01 

a Mean & standard error; n=10 exceptwhereindicated 

n=9 

n=I 

n=4 

TABLEG11 

Mean Total EIorizontal Activity Count of Swiss-Webster Mice in the 57-Week Feed Study of OxaZepama 

Dose Prestudy 6 Months 12 Months 

(PPW 

Male 

0 278 fi 11 279 & 13b 266 f 22' 

2,500 233 f 14 234 f 15 166 f 21d 

5,OOO 261 f 19 273 f 27 279 & 47d 

Female 

0 265 f 14 287 f 18 266 f 29b 

2,500 264 % 17 294 & 27b 218 & 31' 

5,m 277 f 17 314 f 18b 125 f 32*d 

l Significantly different (PSO.05) from the control group by Dunnett's test a Mean fi standard error; n=10 except where indicated 

n=9 ' n=I 

n=4 
Neurobehaviorol Studies 239 

TABLEG12 

Mean Forelimb Grip Strength of B6C3F, Mice in the 14-Week Feed Study of Oxazepama 

Week 2 Week 12 

(8) (9) 

Male 

0 114.28 f 3.66 135.48 f 3.62 164.60 f 3.79 

625 115.14 f 3.55 120.30 f 2.96. 160.58 f 3.39 

1,250 111.72 f 3.46 122.00 f 3.93. 157.04 f 4.73 

2,500 112.10 f 2.65 117.24 f 3.48'. 156.48 f 4.79 

5,000 112.32 f 3.25 116.28 f 2.90.. 152.48 f 3.14 

1o.Ooo 113.76 f 3.31 112.38 f 3.48'; 156.90 f 1.98 

Female 

0 103.48 f 2.59 108.54 f 2.08 145.98 f 2.14 

625 99.10 f 3.18 107.12 f 1.91 143.90 f 1.82 

1,250 95.84 f 2.06 103.80 f 3.23 143.80 f 3.38 

2,500 102.54 f 2.79 102.60 f 3.44 145.76 f 2.52 

5,Ooo 99.02 f 2.66 104.14 f 3.33 140.06 f 1.58 

10,Ooo 100.10 f 2.76 99.02 f 2.67. 141.60 f 2.34 

l Significantly different (PSO.05) from the control group by Dunnett's test 

** PSO.01 

a Mean f standard error; n=lQ data are given asgrams of force needed to break grip strength 

TABLE613 

Mean Hindlimb Grip Strength of B6C3F, Mice in the 14-Week Feed Study of Oxazepama 

Week 2 Week 12 

(8) (9) 

Male 

0 80.18 f 3.43 91.78 f 1.65 101.76 f 2.01 

625 79.28 f 3.80 79.18 f 2.49" 107.82 f 1.44 

1,250 82.86 f 3.26 75.42 f 2.42.. 102.62 f 1.36 

2,500 81.88 f 2.82 72.54 f 2.89" 104.96 f 2.36 

5,m 81.16 f 2.89 68.58 f 2.08.. 106.54 f 2.20 

10,Ooo 80.90 f 3.70 71.72 f 2.38.' 98.84 f 1.82 

Female 

0 70.10 f 2.36 73.20 f 2.08 96.28 f 3.10 

625 67.34 f 3.33 67.14 f 1.20 99.08 f 1.85 

1,250 70.20 f 2.36 64.18 f 1.56' 94.68 f 2.11 

2,500 72.26 f 2.60 64.20 f 1.81. 97.82 f 1.75 

5,Ooo 67.42 f 1.91 62.36 f 3.26.' 96.36 f 1.31 

10,Ooo 68.44 f 2.68 61.50 f 2.42" 96.24 f 2.04 

l Significantly different (PSO.05) from the control group by Dunnett's test 

** PSO.01 

a Mean f standard error; n=1@ data are given asgrams of force needed to break grip strength 
240 Oxazepam, NTP TR 443 

TABLEG14 

Mean Paw Lick Latency of B6C3Fl Mice in the 14-Week Feed Study of Oxazepama 

Dose Prestudy Week 2 Week 12 

(PPm) (9) (8) (g) 

Male 

0 12.22 f 0.80 14.91 f 2.07 12.07 f 1.17 

625 10.24 f 0.76 11.38 f 1.20 10.58 & 1.06 

1,250 14.01 f 1.53 15.87 f 2.32 11.05 f 1.22 

2,500 14.40 f 1.96 17.00 f 1.91 13.47 f 1.05 

5,000 15.09 f 2.09 15.27 f 1.90 13.35 f 1.37 

10,000 13.47 f 1.09 17.71 2 2.15 15.64 f 2.11 

Female 

0 10.66 f 0.95 13.46 f 2.30 13.29 2 1.22 

625 10.60 f 1.25 13.22 f 1.47 8.92 f 0.48* 

1,250 12.12 2 0.44 13.63 f 1.72 9.71 f 1.07. 

2,500 11.89 f 1.11 13.82 f 1.73 11.23 f 0.82 

5,ooo 9.29 f 0.59 17.94 f 2.32 12.01 2 1.12 

10,000 12.24 f 0.83 15.22 f 2.21 9.61 It 0.84* 

l Significantly different (PsO.05) from the control group by Dunnett’s test a Mean f standard error; n=10; data are given as grams of force needed lo break grip strength 
Neurobehavioml Studies 241 

TABLEG15 

Mean Total Motor Activity Count of B6C3F, Mice in the 14-Week Feed Study of Oxazepama 

Dose Prestudy Week 2 

(PPm) 

Male 

0 168 f 22 316 f 12 

625 207 f 12 451 f 22** 

1,250 185 f 11 467 f 16'. 

2,500 205 f 10 499 f 22'. 

1o.Ooo 

5,m 

192 f 11 

184 2 11 

501 f 20.. 

507 f 25" 

Females 

0 176 f 6 325 f 19 

625 195 f 6 467 f 22.. 

1,250 187 f 15 434 f 29* 

2,500 189 f 11 500 f 28" 

5,000 202 f 12 479 f 33" 

10,m 18Of 6 495 f 22'. 

Change Prestudyb fromWeek

(W 

+88 

+118 

+ 152 

+ 143 

+ 176 

+161 

+ 85 

+ 139 

+ 132 

+ 165 

+137 

+175 

12 

378 f 14 

459 * 21. 

449 f 19' 

473 f 19.. 

460 f 9. 

477 f 23.' 

386 f 21 

521 f 29** 

545 2 20** 

512 f 24** 

533 f 41*' 

571 f 15** 

Change Prestudy' from

(94 

+ 125 

+122 

+ 143 

+131 

+150 

+ 148 

+119 

+ 167 

+191 

+171 

+ 164 

+217 

l Significantlydifferent(PSO.05)fromthecontrolgroup by Dunnett's test 

** PSO.01 

a Mean f standarderror; n=10 

Percent change from prestudyfor Week 2 = Week 2 (activity count) - Prestudy (activity count) x 100 

Prestudy (activity count) 

Percent change from prestudy for Week 12 = Week 12 (activity count) - Prestudy (activity count) X 100 

Prestudy (activity count) 
242 Oxazepam, NTP TR 443 

TABLE616 

Startle Response Profiles Prior to Exposure of B6C3F, Mice in the 14-Week Feed Study of Oxazepama 

Dose Block lb Block 2' Prepulse Inhibitiond Block 3e 

(PPW (%) 

Male 

0 63 f 4 57 f 1 -10 54 2 3 

625 71 f 4 58 f 2 -18 6424 

1,250 65 f 4 47 f 2" -28 52 f 3 

2,500 70 f 4 50 f 2. -29 61 2 4 

5,Ooo 64f3 44 f 1** -31 54 2 3 

10,Ooo 71 f 4 56 2 1 -21 63 f 4 

Female 

0 55 f 3 40 f 1 -27 53 2 3 

625 57 2 3 41 f 1 -28 60 f 4 

1,250 62 f 3 48 f 1'. -23 64 f 4 

2,500 68 * 31 48 & 1** -29 53 2 2 

5,Ooo 63 f 4 49 f 1'. -22 61 f 3 

10,Ooo 6of3 44 f 1' -27 60 f 4 

* Significantlydifferent(PSO.05)fromthecontrolgroup by Dunnett's test 

** PSO.01 

a Mean f standard error of average responses (amplitudes) for each trial; n=10 

Block 1 = mean of first 10 trials (tactile stimulus without auditory prepulse) ' Block 2 = mean of trials 11-70 (tactile stimulus preceded by auditory prepulse); 80-90dB 

Percent prepulse inhibition = block 1 - block 2 x 100 

block 1 

e Block 3 = meanoflast 10 trials (tactile stimulus without auditory prepulse) 

Percent habituation = block 1 - block 3 x 100 

block 1 

Habituationf 

(W 

-14 

-10 

-20 

-13 

-16 

-1 1 

-4 

+5 

+3 

-22 

-3 

0 
Neurobehavioral Studies 243 

TABLEG17 

Startle Response Profilesof B6C3F, Mice at Week 2 in the 14-Week Feed Study of Oxazepama 

Dose Block lb Block 2' Prepulse Inhibitiond Block 3e Habituationf 

(PPW (W (W 

Male 

0 67 f 4 51 1 -24 70 f 5 +4 

625 82 f 3* 45 f 1** -45 62 1?: 3 -24 

1,250 92 f 4** 58 f 1** -37 85 f 4* -8 

2,500 69 f 3 41 f 1'. -41 52 f 3** -25 

5,000 71 f 3 45 f 1** -37 63 f 3 -1 1 

10,OoO 

Female 

66f4 38 f 1.' -42 53 f 3** -20 

0 6of4 43f 1 -28 52 f 3 -1 3 

625 62 f 4 43f 1 -31 64 f 4* +3 

1,250 70 f 4 34 f 1'1 -5 1 49 f 3 -30 

2,500 82 f 4** 50 f 1** -39 60 f 4 -27 

5,OoO 60 f 3 39 f 1'. -35 49 f 3 -1 8 

1o.OoO 55 f 4 30 f 1** 45 46 f 2 -16 

l Significantly different (PSO.05) from the control group by Dunnett's test 

** PSO.01 

a Mean f standard error of average responses (amplitudes) for each trial; n=10 

Block 1 = mean of first 10 trials (tactile stimulus withoul auditory prepulse) 

Block 2 = mean of trials 11-70(tactile stimulus precededby auditory prepulse); 80-90dB 

Percent prepulse inhibition = block 1 - block 2 x 100 

block 1 

e Block 3 = mean oflast 10 trials (tactile stimulus without auditory prepulse) 

Percent habituation = block 1 - block 3 x 100 

block 1 
244 Oxazepam, NTP TR 443 

TABLEG18 

Startle Response Profilesof B6C3FLMice at Week 12 in the 14-Week Feed Study of Oxazepama 

Dose Block lb Block 2' Prepulse Inhibitiond Block 3e

(PPW (%I 

Male 

0 38f2 19-C 1 -50 25 f 2 

625 35 f 2 14 f 0** -60 27 f 2 

1,250 46 f 2 18 f 0 -61 43 f 3.' 

2,500 39 f 3 15 f 0.. -62 32 f 2 

5,000 39 f 3 185 1 -54 34 f 3' 

10,Ooo 42 f 3 15 f o** -64 34 f 31 

Female 

0 40 f 3 20 f 0 -50 28f2 

625 38f2 18 f 0 -53 39 f 3. 

1,250 42 f 3 18 f 0 -57 40 f 3'. 

2,500 33 f 2 17 f o* -48 32 f 2 

5,000 32 f 2 15 f 0.. -53 31 -t 2 

1o.Ooo 29 f 2** 16 f O** -45 38 f 3. 

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

** PSO.01 

a Mean f standard error of average responses (amplitudes) for each trial; n=10 

Block 1 = mean of first 10 trials (tactile stimulus without auditory prepulse) 

Block 2 = mean of trials 11-70 (tactile stimulus preceded by auditory prepulse); 80-90 dB 

Percent prepulse inhibition = block 1 - block 2 x 100 

block 1 

e Block 3 = mean of last 10 trials (tactile stimulus without auditory prepulse) 

Percent habituation = block 1 - block 3 x 100 

block 1 

Habituationf 

(W 

-34 

-23 

-7 

-18 

-13 

-19 

-30 

+3 

-5 

-3 

-3 

+31 
Neurobehavioral Studies 245 

TABLEG19 

Mean Forelimb Grip Strength of B6C3Fl Mice in the 2-Year Feed Study of Oxazepama 

Dose Prestudy 6 Months 12 Months 18 Months 24 Months 

(PPm) (9) (9) (6) (6) (9) 

Male 

0 86 f 5 128 f 6 130 f 3 104 f 4 88 f 3b 

125 83 f 4 115 f 4 111 2 3'* 97 f 2 86 f 5b 

2,500 87 f 4 111 f 5 108 f 2" 77 f 4** 

5,c@o 85 f 5 118 f 4 114 f 3** 65 f 2**' 

Female 

0 79 f 4 116 f 2 110 f jb 102 f 4b 

125 80 f 3 109 f 2 110 f 3 98 f 4 

2,500 81 f 3 105 2 2 100 f 2 86 f 3* 

5,c@o 80 f 2 113 f 4 102 f 3 78 f 6**e 

l Significantly different (PsO.05)from the control group by Dunnett's test 

*' PSO.01 

a Mean f standard error; n=10 except where indicated; data are given asgramsofforceneededtobreakgripstrength 

n=9 

n=7 

n=8 

e n=4 

TABLEG20 

Mean Hindlimb Grip Strength of B6C3Fl Mice in the 2-Year Feed Study of Oxazepama 

Dose Prestudy 6 Months 12 Months 18 Months 24 Months 

(PPm) (9) (6) (6) (9) (6) 

Male 

0 59 -c 3 103 rt 5 83 f 4 80 f 4 65 f 3b 

125 59 f 2 107 f 3 92 f 4 83 f 5 71 f gb 

2,500 61 f 3 101 f 4 89 f 2 73 f 2 

5.Ooo 63 f 5 116 f 3 96 f 4 70 f 5' 

Female 

0 65 f 4 98 k 4 94 f 4b 80 f 3b 

125 70 f4 104 f 4 98 f 4 85 f 7 

2,500 67 f 3 98 f 3 84 f 4 75 f 4 

5,000 66 f 3 101 f 4 8824 68 f 7e 

a Mean f standard error; n=10 except where indicated; data are given asgrams offorceneededtobreakgrip strength; 

differences from the control group are not significant by Dunnett's test. 

n=9 ' n=7 

n=8 

e n=4 
246 Oxazepam, NTP 443 TR

TABLEG21 

Mean Total Motor Activity Count of B6C3F, Mice in the 2-Year Feed Study of Oxazepama 

Dose Prestudy 18 12 6 MonthsMonthsMonths 24 Months

(PPW 

Male 

0 183 f 15 164 f 12 154 f 10 146 f 13 153 f 12b 

125 180 f 14 194 f 12 162 f 7 151 f 9 151 f 13b 

2,500 190 f 16 286 f 11" 219 f 13'. 168 f 10 -

5,OOO 176 f 18 246 f 13" 217 k 13'' 62 f 14*" -

Female 

0 189 f 24 148 f 14 155 f lob 158 f gb 159 f lld 

125 189 f 16 231 2 10'' 171 f 9 173 f 14 173 f gb 

2,500 201 f 19 270 f 10'' 215 f 8" 171 f 9 - 5,000 178 f 19 291 f 11" 223 f 13" 86 f 17'" -

l Significantly different (PSO.05)from the control group by Dunnett's test 

** PSO.01 

a Mean f standard error; n=10 exceptwhereindicated 

n=9 

n=7 

n=8 

TABLE622 

Mean Paw Lick Latency of B6C3F, Mice in the 2-Year Feed Study of Oxazepama 

Dose Prestudy 6 Months 12 Months 18 Months 24 Months

(PP@ (seconds) (seconds) (seconds) (seconds) (seconds) 

Male 

0 11.8 f 0.7 13.7 k 2.2 17.0 f 3.0 19.2 f22.42.8 k 2.4b 

125 9.4 f 0.5 10.2 f 0.9 10.9 f 1.0 10.6 f17.30.9' f 2.3b 

- 2,500 12.3 f 0.8 11.4 f 0.9 12.5 f 0.8 13.2 k 1.6 

5,m 11.5 k 0.8 11.2 k 0.7 14.0 k 2.1 14.2 f 1.7' -

Female 

0 11.4 f 0.9 13.4 f 2.2 10.7 2 1.4b 11.2 f 2.4b18.0 f 2.9d 

125 9.0 f 0.7 7.9 f 0.4 8.4 f 0.6 12.8 f 1.8 17.5 f 3.1b 

- 2,500 11.3 f 1.1 11.0 f 2.1 9.4 f 0.7 15.4 +. 2.3 

11.6 2 1.1 9.8 f 0.5 9.8 f 0.9 14.4 f l.Ge - 5,ooo 

l Significantlydifferent (PSO.05) from the control group by Dunnett's test a Mean f standard error; n=10 except where indicated 

n=9 

n=7 

n=8 

e n=4 
247 

APPENDIX H 

CHEMICAL CHARACTERIZATION AND 

DOSE FORMULATION STUDIES 

PROCUREMENT AND CHARACTERIZATION OF OXAZEPAM l l l . . l . . . l . . . l . . . . . . .. . . . . . . . . . . . . . 248 

PREPARATION AND ANALYSISOF DOSEFORMULA'rIONS . . . .. . . . . . . . . . . . . .. . ... . . . ... . . . . . . . 248 

FIGUREH1 Infrared Absorption Spectrum of Oxazepam . . . ..... . . . . . .. . . . . . . . . . . . . . ... . 250 

FIGUREH2 Nuclear Magnetic Resonance Spectrum of Oxazepam ... . . . . . . . . . ... . . . . . . .. . . 251 

TABLE H1 Preparation and Storage of Dose Formulations inthe Feed Studies 

ofoxazepam ........................................................ 252 

TABLE H2 Results of Analysis of Dose Formulations Administered to Swiss-Webster 

and B6C3F, Mice in the 14-Week Feed Studies of Oxazepam . . . . . .... . . . . . . . . . . 253 

TABLE H3 Results of Analysis of Dose Formulations Administered to Swiss-Webster 

and B6C3F, Mice in the Chronic Feed Studies of Oxazepam ..... .. . . . . . . . . . . . .. 255 

TABLE H4 Results of Referee Analysis of Dose Formulations in the 14-Week 

and Chronic Feed Studies of Oxazepam . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . , 257 
248 Oxazepam, NTP TR 443 

CHEMICAL CHARACTERIZATION AND 

DOSE FORMULATION STUDIES 

PROCUREMENT AND CHARACTERIZATION OF OXAZEPAM 

Oxazepam was obtained from Rouse1 Corporation (Englewood Cliffs, NJ) in one lot (86017.01), which was 

used throughout the studies. Identity, purity, and stability analyses were conducted by the analytical 

chemistry laboratory, Midwest Research Institute (Kansas City, MO). The reports on analyses performed 

in support of the oxazepam studies are onfile at the National Institute of Environmental Health Sciences. 

The chemical, a white, powdered solid, was identified as oxazepam by infrared, ultravioletbisible, and 

nuclear magnetic resonance spectroscopy. All spectra were consistent with those expected for the structure 

and with the literature spectra (Florey, 1974) of oxazepam (Figures H1 and H2). The observed melting 

point of 204.5 "C was consistent with the literature reference (Merck, 1983). 

The purity of oxazepam was determined by elemental analyses, Karl Fischer water analysis, functional 

group titration, thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC). 

Functional group titration was performed by dissolving a sample in dimethylformamide and titrating with 

0.1 N tetrabutylammonium hydroxide. TLC was performed on Silica Gel 60 F-254 plates with two solvent 

systems: A) ch1oroform:methanol (1O:l) and B) ethyl acetate:methanol:glacial acetic acid (80:20:10). 

One pL of a 5 mg/mL solution of anthracene in methanol was used as an internal standard. Visualization 

was accomplished with ultraviolet light (254 and 366 nm) and a spray of 37% formaldehyde solution in 

concentrated sulfuric acid. HPLC was performed with a Hewlett-Packard RP-18 column (200 x 4.6 mm 

ID) using a solvent system consisting of 1)water containing 1%(vh) glacial acetic acid and 2) methanol 

containing 1% (vh) glacial acetic acid, with a solvent ratio of 5050, at a flow rate of 1mWminute. 

Detection was with ultraviolet light at 254 nm. 

Elemental analyses for carbon, hydrogen, nitrogen, and chlorine were in agreement with the theoretical 

values for oxazepam. Karl Fischer analysis indicated less than 0.03% water. Functional group titration 

indicated a purity of 101.4% f 0.5%. TLC analysis using system A indicated a major spot and onetrace 

impurity; using system B, a major spot was observed. HPLC resolved a major peak with no impurity 

peaks with areas 0.1% or greater relative to the major peak. Major peak comparison between this lot and 

a United States Pharmacopeia XXI (USP) standard indicated a relative purity of 103% f 1%. The overall 

purity was determined tobe greater than 99%. 

Stability studies were performed by the analytical chemistry laboratory. HPLC was performed using the 

system described above except with a solvent ratio of 3565. These studies indicated that oxazepam was 

stable for 2 weeks when stored protected from light at temperatures up to 60" C. The stability of the bulk 

chemical was monitored periodically at the study laboratory using HPLC as described above. No 

degradation of the bulk chemical was observed. 

PREPARATION AND ANALYSISOF DOSEFORMULATIONS 

The dose formulations were prepared by mixing oxazepam and feed (Table Hl). The mixture was stored 

in sealed, labeled, plastic buckets for up to 3 weeks at 5" C. 

Homogeneity and dose formulation stability analysesof the 500 ppm concentration were performed by the 

analytical chemistry laboratory. Aliquots were mixed with 5 mL of internal standard solution 

(acetophenone, 0.03 mg/mL in methanol) and 15 mL of methanol, then diluted to 50 mL with deionized 

water. After mixing, HPLC analysis was performed using the following system: a Burdick and Jackson C,, 

column (250 x 4.6 mm ID) and a mobile phase solvent system consisting of water:methanol:glacial acetic 
Chemical Characterization and Dose Formulations 249 

acid (43:57:1) (vbb),at a flow rate of 1mL/minute. Detection was with a Waters 440 detector at 254 nm. 

Homogeneity was confirmed, and the stability of the dose formulations was confirmed for at least 3 weeks 

when stored protected from light at 5" C. 

Periodic analyses of the dose formulations of oxazepam were conducted at the study laboratory using 

HPLC. Periodic analyses of the dose formulations of oxazepam were conducted at the analytical chemistry 

laboratory using HPLC. During the 14-week studies, all of the dose formulations for Swiss-Webster and 

B6C3Fl mice were within 10%of the target concentrations (Table H2). During the chronic studies, dose 

formulations were analyzed approximately every 8 weeks; all analyzed dose formulations for Swiss-Webster 

and B6C3F, mice were within 10% of the target concentrations except for two 125 ppm formulations for 

B6C3F, mice. These dose formulations were remixed. Results of the dose formulation analyses for the 

chronic studies are presented in Table H3. Results of periodic referee analyses performed by the analytical 

chemistry laboratory indicated good agreement with the results obtained by the study laboratory 

(Table H4). 
250 Oxazepam, NTP TR 443 

8 

N, 

c 

8 -

-0-

- 1 

Y 

0 

I 

FIGUREI11 

Infrared Absorption Spectrum of Oxazepam 
Nucleus: Proton 

Inslrumenl:Varlan VXR-300 FT-NMR 

Projecl No.:8412-01 

Task Designallon:BSICV-2063 

Balch No.: 01 

Lo1 No.: 86017.01 

Oxazepam 

Dale: 10/12/87 

Operalor: A. Clark 

Sample Temperature: Amblerlt 

Internal Reference:DMSO-d6 

Solvenl: DMSO-de 

9 

e 

a 

0 

a U

P 

(a) 

(b) 

(C) 

(d) 

(e)

(9

(Q) 

4.78 

6.40" 

7.21 

7.27 

7.40-7.54 

7.63 

10.84w 

Jd.I=-gHZ 

Jc-f=-2M 

1.02 

0.88 

0.99 1.oo 

4.86 

1.15 

0.80 

1 

1 

1 

1 

5 

1 

1 

"Exchangeable proton 

4 0

3 

f!

N 

3 

N

Y 
252 Oxazepam, NTP TR 443 

TABLEH1 

Preparation and Storage of Dose Formulations in the Feed Studies of Oxazepam 

14-Week Studies 

Preparation 

Dose formulations were prepared monthly. Premix was 

prepared by mixing feed and oxazepam (wh); premix and 

remaining feed were layered in a Patterson-Kelley twin-shell 

blender and mixed for 15 minutes with the intensifier bar in 

operation for the first 5 minutes. 

ChemicalLotNumber 

86017.01 

Maximum Storage Time 

3 weeks 

Storage Conditions 

In sealed, labeled, plastic buckets, storedat 5" C 

Study Laboratory 

Battelle Columbus Laboratories 

Columbus, OH 

Referee Laboratory 

Midwest Research Institute, 

Kansas City, MO 

Chronic Studies 

Same as 14-week studies exceptthat dose formulations were 

prepared every 2 weeks. 

Same as 14-week studies 

Same as 14-week studies 

Same as 14-week studies 

Same as 14-week studies 

Same as 14-week studies 
Chemical Characterization and Dose Formulations 253 

TABLE€12 

Results of Analysis of Dose Formulations Administered to Swiss-Webster and B6C3F, Mice 

in the 14-Week Feed Studies of Oxazepam 

Date Prepared Date Analyzed 

~ 

Swiss-Webster Mice 

3 May 1988 5 May 1988 

1 June 1988 2 June 1988 

6 July 1988 8July 1988 

24 August 1988 25 August 1988 

B6C3F, Mice 

3 May 1988 5 May 1988 

10 May 1988 12 May 1988 

1June 1988 2 June 1988 

Target 

Concentration 

(PPW 

625 

625 

625 

10,Ooo 

10,000 

10,000 

625 

1,250 

2,500 

5,000 

10,000 

625 

1,250 

2,500 

5,000 

10,000 

625 

1,250 

2,500 

5,000 

10,000 

625 

625 

625 

10,000 

10,000 

10,000 

625 

1,250 

2,500 

5,000 

10,000 

625 

1,250 

2,500 

5,000 

10,000 

Determined 

Concentrationa 

(PPm) 

633b 

656' 

660d 

9,784b 

10,004c 

9.999d 

617 

1,214 

2,435 

4,968 

9,975 

655 

1,242 

2,484 

4,941 

10,196 

665 

1,311 

2,519 

5,013 

10,022 

633b 

656' 

660d 

9,784b 

10,004' 

9.999d 

584 

1,230 

2,537 

5,063 

10,405 

617 

1,214 

2,435 

4,968 

9,975 

Difference 

from Target 

(94 

+1 

+5 

+6 

-2 

0 

0 

-1 

-3 

-3 

-1 

0 

+5 

-1 

-1 

-1 

+2 

+6 

+5 

+1 

0 

0 

+1 

+5 

+6 

-2 

0 

0 

-7 

-2 

+1 

+1 

+4 

-1 

-3 

-3 

-1 

0 
254 Oxazepam, NTP TR 443 

TABLEH2 

Results of Analysis of Dose Formulations Administered to Swiss-Webster and B6C3F, Mice 

in the 14-Week Feed Studies of Oxazepam (continued) 

Target

Date Prepared Date Analyzed Concentration 

(PPm) 

B6C3F, Mice (continued) 

6 July 1988 8 July 1988 625 

1,250 

2,500 

5,OoO 

10,OoO 

1 August 1988 4 August 1988 

1,250 

626 

2,500 

5,000 

10.000 

a Resultsofduplicateanalyses 

Sample selection from top right of twin-shell blender 

Sample selection from top left of twin-shell blender 

Sample selection from bottom of twin-shell blender 

Determined 

Concentration 

(PPm) 

655 

1,242 

2,484 

4,941 

10.196 

628 

1,251 

2,504 

5,014 

9.983 

Difference 

from Target 

(%) 

+5 

-1 

-1 

-1 

+2 
Chemical Characterization and Dose Formulations 255 

TABLE€13 

Results of Analysis of Dose Formulations Administered to Swiss-Webster and B6C3F, Mice 

in the Chronic Feed Studies of Oxazepam 

Determined Target Difference 

Date Prepared Date Analyzed Concentrationa Concentration from Target

(PPW (PPW (94 

Swiss-Webster Mice 

1May 1989 4 May 1989 5,m 5,086b +2 

5,000 4,916' -2 

5,000 5,125d +3 

30 June 1989 6-7 July 1989 2,500 2,541 +2 

5,Ooo 4,972 -1 

11August 1989 15-16 August 1989 2,500 2,482 -1 

5,000 4,928 -1 

6 October 1989 11-12 October 1989 2,500 2,604 +4 

5,000 5,050 +1 

1December 1989 5 December 1989 2,500 2,493 0 

5,000 5,056 +1 

26 January 1990 31 January 1990 2,500 2,432 -3 

5,000 4,984 0 

6 April 1990 11April 1990 2,500 2,530 +1 

5,000 5,080 +2 

4 June 1990 5 June 1990 2,500 2,527 +1 

5,m 5,020 0 

27 July 1990 1August 1990 2,500 2,488 0 

5,000 4,978 0 

B6C3F, Mice 

1 May 1989 3 May 1989 125 128b +2 

125 130' +4 

125 1 32d +6 

4 May 1989 5,000 5,086b +2 

5,000 4,916' -2 

5,000 5,125d +3 

19June 1989 20-21 June 1989 125 133 +6 

2,500 2,656 +6 

5,000 5,031 +1 

30 June 1989 6-7 July 1989 125 137 +10 

2,500 2,541 +2 

5,000 4,972 -1 

11 August 1989 15-16 August 1989 125 138 +10 

2,500 2,482 -1 

5,000 4.928 -1 
256 Oxazepam, NTP TR 443 

TABLE133 

Results of Analysis of Dose Formulations Administered to Swiss-Webster and B6C3Fl Mice 

in the Chronic Feed Studies of Oxazepam (continued) 

Date Prepared 

B6C3Fl Mice (continued) 

16 August 1989 

6 October 1989 

1December 1989 

6 December 1989 

26 January 1990 

6 April 1990 

4 June 1990 

27 July 1990 

28 September 1990 

16 November 1990 

11January 1991 

8 March 1991 

6 May 1991 

~~~ ~~ 

Date Analyzed 

16-17 August 1989e 

11-12 October 1989 

5 December 1989 

6 December 1989e 

31 January 1990 

11April 1990 

5 June 1990 

1August 1990 

3 October 1990 

19 November 1990 

15 January 1991 

11 March 1991 

7 May 1991 

a Resultsofduplicateanalyses 

Sample selection from bottom of twin-shell blender 

Sample selection from top rightof twin-shell blender 

Sample selection from top leftof twin-shell blender 

e Analysisresults of remix 

Target Determined Difference 

Concentration Concentration from Target 

(PP@ (PPm) (%) 

125 132 +6 

125 126 +1 

2,500 2,604 +4 

5,000 5,050 +1 

125 140 +12 

2,500 2,493 0 

5,000 5,056 +1 

125 126 +1 

125 127 +2 

2,500 2,432 -3 

5,000 4,984 0 

125 128 +2 

2,500 2,530 +1 

5,000 5,080 +2 

125 122 -2 

2,500 2,527 +1 

5,000 5,020 0 

125 135 +8 

2,500 2,488 0 

5,000 4,918 0 

125 125 0 

2,500 2,461 -1 

5,000 4,818 -2 

125 124 -1 

2,500 2,487 -1 

5,000 5,034 +1 

125 120 -4 

2,500 2,525 +1 

5,000 4,950 -1 

125 124 -1 

2,500 2,495 0 

125 126 +1 

2,500 2,386 -5 
Chemical Characterization and Dose Formulations 257 

TABLEH4 

Results of Referee Analysis of Dose Formulations Administered in the 14-Week and 

Chronic Feed Studies of Oxazepam 

Date Prepared 

14-Week Studies 

Swiss-Websler Mice 

1 June 1988 

24 August 1988 

B6C3Fl Mice 

10 May 1988 

6 July 1988 

Chronic Studies 

Swiss-Webster Mice 

30June 1989 

26 January 1990 

BKJF, Mice 

19June1989 

30 June 1989 

26 January 1990 

a Results of duplicateanalysis 

Results of triplicate analysis; mean 

Target Concentration 

(PPm) 

10,Ooo 

625 

1,250 

5.000 

2,500 

5,000 

125 

2,500 

5,Ooo 

k standard deviation 

Determined Concentration (mp/mL) 

Study Referee 

Lnborato$ Laboratoryb 

9,999 10,100 k 58 

665 628 & 18 

1,230 1,216 k 6 

4,941 4.889 k 104 

2,541 2,520 k 60 

4,984 5,070 k 10 

133 132 k 2 

2,541 2,520 k 60 

4,984 5,070 k 10 
258 

Oxazepam, NTP TR 443 
259 

APPENDIX I 

FEED AND COMPOUND CONSUMPTION 

IN THE CHRONIC FEED STUDIES 

TABLEI1 Feed and Compound Consumption by Male Swiss-Webster Mice 

in the 57-Week Feed Study of Oxazepam .................................. 260 

TABLE I2 Feed and Compound Consumption by Female Swiss-Webster Mice 

in the 57-Week Feed Study of Oxazepam .................................. 261 

TABLE13 Feed and Compound Consumption by Male B6C3F, Mice in the 2-Year Feed Study 

ofOxazepam ........................................................ 262 

TABLE I4 Feed and Compound Consumption by Female B6C3F, Mice in the 2-Year Feed Study 

ofOxazepam ....................................................... 263 
260 Oxazepam, NTP TR,443 

TABLEI1 

Feed and Compound Consumption by Male Swiss-Webster Mice in the 57-Week Feed Study of Oxazepam 

2 4.6 27.9 4.2 

~~ 

28.6 364 4.0 28.7 690 

6 5.1 31.9 4.5 32.4 351 4.7 33.4 700 

10 4.6 34.3 4.3 34.5 310 4.5 35.2 642 

13 4.7 36.0 4.1 36.0 286 4.4 36.2 605 

17 4.7 38.4 3.9 37.5 262 4.0 37.4 532 

21 4.1 40.3 3.8 38.7 246 4.0 38.2 522 

25 4.4 41.6 3.8 39.8 242 3.9 39.2 502 

29 4.4 42.2 3.9 40.8 241 4.0 39.4 506 

33 4.5 42.8 4.1 40.7 251 4.2 39.2 530 

41 4.1 42.2 3.8 40.3 238 4.2 39.2 532 

45 4.1 41.7 3.7 39.4 236 4.1 37.8 542 

49 4.4 41.5 3.9 38.6 253 4.1 37.4 549 

53 4.4 41.7 3.7 37.9 247 3.8 36.4 521 

Mean for weeks 

1-13 4.8 32.5 4.3 32.9 328 4.4 33.4 659 

14-52 4.4 41.3 3.9 39.6 246 4.1 38.5 527 

53 4.4 41.7 3.7 37.9 247 3.8 36.4 521 

a Grams of feed consumed per animal per day; study terminated at week 57 

Milligrams of oxazepam consumed per day per kilogram Lwdy weight 
Feed and Compound Consumption 261 

TABLEI2 

Feed and Compound Consumption by Female Swiss-Webster Mice in the 57-Week Feed Study 

of Oxazepam 

2 4.8 22.8 3.9 25.4 381 3.6 24.8 732 

6 5.6 26.3 5.1 29.6 428 5.1 29.3 863 

10 5.3 28.3 5.o 31.1 402 4.8 31.2 762 

13 5.0 29.4 4.5 31.8 352 4.4 31.9 695 

17 5.0 31.4 4.3 33.8 317 4.2 33.6 620 

21 4.4 32.4 4.1 34.9 291 4.2 33.9 625 

25 4.8 33.3 4.0 35.9 276 4.2 34.7 611 

29 4.9 34.5 4.1 36.3 281 4.1 34.9 591 

33 4.9 34.8 4.3 36.8 295 4.5 35.2 642 

37 4.6 35.0 4.0 37.4 267 4.3 35.5 610 

41 4.5 34.8 4.2 36.6 286 4.8 34.6 690 

45 4.8 34.3 4.1 36.4 279 4.4 34.7 632 

49 4.9 34.8 4.2 36.7 286 4.5 34.3 661 

53 5.0 35.0 4.3 36.3 299 4.3 34.4 619 

Mean for weeks 

1-13 5.1 26.7 4.6 29.5 391 4.5 29.3 763 

14-52 4.7 33.9 4.1 36.1 287 4.4 34.6 631 

53 5.0 35.0 4.3 36.3 299 4.3 34.4 619 

a Grams of feed consumed per animal per day; study terminated at week 57 

Milligrams of oxazepam consumed per day per kilogram body weight 
262 Oxazepam, NTP TR 443 

TABLE13 

Feed and Compound Consumption by Male B6C3F, Mice in the 2-Year Feed Study of Oxazepam 

0 PPm 125 ppm 2,500 ppm 5,000 ppm 

Body Feed Body Feed Dose/ Body Feed Dose/ Body Feed Dose/ 

we%ht (glday) Weight(%day) Day' Weight Day (glday) Weight Day 

Week (9) (€9 (~%k"aY) (€9 (nWkg/day) (€9 (m%k"ay) 

3.02.8

6 4.3 27.5 4.3 29.3 18 4.5 29.2 382 4.8 28.7 836 

10 4.4 32.0 4.2 32.9 16 4.5 31.7 352 4.9 31.5 780 

13 4.2 34.6 4.2 35.4 15 4.4 33.5 332 4.7 33.3 702 

17 4.1 38.5 4.0 38.2 13 4.2 35.9 290 4.3 34.9 616 

21 4.0 41.2 4.1 39.9 13 4.0 36.7 276 4.4 35.2 624 

25 4.0 43.5 3.9 41.8 12 4.0 38.1 266 4.4 36.0 605 

29 3.9 44.3 3.7 43.0 11 3.6 38.8 234 4.0 36.7 547 

2 3.3 22.8 3.4 23.7 18 23.8 313 23.4 589 

4.44.6

37 4.0 46.6 3.9 45.1 11 4.2 40.1 259 4.4 37.6 589 

41 4.0 47.1 3.9 46.1 10 4.1 40.8 250 4.6 38.0 611 

45 4.3 47.4 4.3 46.3 12 4.3 40.8 266 4.8 37.8 641 

33 4.1 46.3 4.1 44.4 12 39.4 277 37.1 625

4.4.83

4.55.2

57 4.5 48.0 4.4 47.9 11 4.8 41.4 289 5.7 36.4 788 

49 4.3 47.5 4.1 46.9 11 41.0 264 37.7 639

53 4.5 47.3 4.2 47.5 11 41.1 274 37.1 705

4.55.2 35.8 726 

65 4.6 48.7 4.4 48.3 12 4.7 40.5 288 5.7 34.7 816 

69 4.5 48.3 4.4 48.2 11 4.6 39.7 291 5.4 33.9 792 

61 4.4 48.5 4.4 48.3 11 41.3 273 

4.75.4

77 4.4 48.8 4.1 48.9 11 5.O 37.1 338 4.8 33.4 724 

73 4.6 48.8 4.5 48.4 12 38.4 303 33.6 803

4.64.3

3.94.3

89 4.6 49.4 4.2 48.9 11 5.4 34.5 388 4.7 32.6 724 

93 4.7 49.1 4.4 48.7 11 5.1 33.1 386 

97 4.8 48.3 4.1 48.2 11 5.3 33.0 401 

101 4.4 47.7 4.2 47.8 11 4.4 33.2 332 

104 4.4 48.3 4.2 47.6 11 4.4 33.5 329 

Mean for weeks 

1-13 4.1 29.2 4.0 30.3 17 4.1 29.6 345 4.3 29.2 727 

14-52 4.1 44.7 4.0 43.5 11 4.1 39.1 265 4.5 36.8 611 

53-104 4.5 48.5 4.3 48.4 11 4.7 36.7 324 5.1 34.0 742 

a Grams of feed consumed per animal per day 

Milligrams of oxazepam consumed per day per kilogram My weight 

81 4.3 48.6 4.2 48.6 11 36.1 319 33.0 648

85 4.3 49.4 4.3 49.6 11 35.4 275 32.8 659
Feed and Compound Consumption 263 

TABLEI4 

Feed and Compound Consumption by Female B6C3F, Mice in the 2-Year Feed Study of Oxazepam 

0 ppm 125 ppm 2,500 PPm 5,000 ppm 

Body Feed Body Feed Dosd Body Feed Dose/ Body Feed Dose/ 

weight (@day) Weight Dayb(&day) Weight Day (@day) Weight Day 

Week (9) (g) (mg/kg/hy) (8) (m@kg/day) (9) - (WwdaY) 

1.8 20.5 444 

10 5.8 27.3 5.7 29.2 24 5.4 28.2 482 5.1 27.2 939 

2.3 

6 5.9 23.3 5.8 25.1 29 5.1 

2 3.6 19.1 3.0 20.7 18 21.0 271 

5.4 25.1 512 24.7 1,085 

28.5 907 

31.5 732 

32.5 709 

33.6 689 

13 6.1 30.3 5.6 31.8 22 5.2 5.2 30.3 429 

17 5.6 35.5 5.1 35.9 18 4.7 4.6 33.4 348 

21 5.6 38.7 5.1 38.4 17 4.6 4.6 34.6 334 

25 5.2 41.7 4.8 40.3 15 4.6 4.6 36.6 313 

29 5.1 43.9 4.6 42.0 14 4.3 37.7 285 4.6 34.4 674 

33 5.3 45.9 5.0 43.2 14 4.9 38.4 317 5.1 35.2 728 

37 4.9 47.8 4.5 44.2 13 4.4 39.4 280 4.6 36.0 640 

41 4.9 48.7 4.5 45.3 12 4.5 4.9 40.3 282 36.8 662

45 5.1 49.9 5.0 45.3 14 4.7 5.2 39.9 297 36.2 723

49 5.0 50.8 4.8 46.2 13 4.8 5.2 40.3 36.9 706 

5.4 

6.1 

298 

4.8 

5.4 

47.0 13 4.9 

53 5.2 51.0 4.7 46.3 13 40.6 295 36.4 743

57 5.2 51.9 5.1 46.5 14 40.6 331 35.8 849

61 5.1 53.2 5.0 6.0 40.9 301 35.1 848 

65 5.2 53.4 5.1 46.6 14 5.26.3 41.0 315 34.2 926

69 5.1 53.6 4.8 46.8 13 5.2 6.3 40.7 318 33.9 934

73 5.0 54.4 4.9 47.5 13 5.6 6.2 39.3 356 33.5 920

77 5.O 54.4 4.8 47.3 13 5.7 5.8 38.5 373 33.3 870

81 5.0 55.1 4.7 47.3 12 5.5 37.8 366 4.9 33.1 745 

85 4.7 56.0 4.8 47.2 13 5.4 36.8 366 4.2 33.4 624 

89 5.0 54.6 4.8 47.1 13 5.9 36.1 411 

93 4.8 55.2 4.9 46.8 13 5.4 35.2 384 

97 4.8 54.1 4.8 46.5 13 5.6 35.6 390 

101 4.9 53.2 4.9 45.5 14 5.2 35.0 374 

Mean for weeks 

1-13 5.3 25.0 5.0 26.7 23 4.5 26.2 423 4.4 25.2 844 

14-52 5.2 44.8 4.8 42.3 14 4.6 37.8 306 4.8 34.8 696 

53-101 5.0 53.9 4.9 46.8 13 5.4 38.3 352 5.7 34.3 829 

a Grams of feed consumed per animal per day 

Milligrams of oxazepam consumed per day per kilogram body weight 
264 Oxazepam, NTP TR 443 
265 

TABLEJl 

TABLE52 

TABLE53 

TABLE54 

APPENDIX J 

INGREDIENTS, NUTRIENT COMPOSITION, 

AND CONTAMINANT LEVELS 

IN NIH-07 RAT AND MOUSE RATION 

Ingredients of NIH-07 Rat and Mouse Ration ............................... 266 

Vitamins and Minerals in NIII-07 Rat and Mouse Ration ...................... 266 

Nutrient Composition of NIII-07 Rat and Mouse Ration ....................... 267 

Contaminant Levels in NIH-07 Rat and Mouse Ration ........................ 268 
266 Oxazepam, NTP TR 443 

TABLEJ1 

Ingredients of NIH-07 Rat and Mouse Rationa 

Ingredientsb 

Ground #2 yellow shelled corn 

Ground hard winter wheat 

Soybean meal (49% protein) 

Fish meal (60% protein) 

wheat middlings 

Dried skim milk 

Alfalfa meal (dehydrated, 17% protein) 

Corn gluten meal (60% protein) 

Soy oil 

Dried brewer’s yeast 

Dry molasses 

Dicalcium phosphate 

Ground limestone 

Salt 

Premixes (vitamin and mineral) 

a NCI, 1976; NIH, 1978 

Percent by Weight 

24.50 

23.00 

12.00 

10.00 

10.00 

5.00 

4.00 

3.00 

2.50 

2.00 

1.50 

1.25 

0.50 

0.50 

0.25 

Ingredients were ground to pass through a U.S. Standard Screen No. 16 before being mixed. 

TABLE52 

Vitamins and Minerals in NIH-07 Rat and Mouse Rationa 

Vitamins 

A 

Choline 

Folic acid 

Niacin 

d-Pantothenic acid 

Riboflavin 

Thiamine 

B12 

Pyridoxine 

Biotin 

Minerals 

Iron 

Manganese 

Zinc 

Copper

Iodine 

Cobalt 

a Per ton (2,OOO Ib)offinishedproduct 

Amount 

5,500,000 IU 

4,600,Ooo IU 

2.8 g 

20,Ooo IU 

560.0 g 

2.2 g 

30.0 g 

18.0 g 

3.4 g 

10.0 g 

4,Ooo rcg

1.7 g 

140.0 mg 

120.0 g 

60.0 g 

16.0 g 

4.0 g 

1.4 g 

0.4 g 

Source 

Stabilized vitamin A palmitate or acetate 

D-activated animal sterol 

Menadione 

Choline chloride 

d-Calcium pantothenate 

Thiamine mononitrate 

Pyridoxine hydrochloride 

d-Biotin 

Iron sulfate 

Manganous oxide 

Zinc oxide 

Copper sulfate 

Calcium iodate 

Cobalt carbonate 
Feed Analyses 267 

TABLE53 

Nutrient Composition of NIH-07 Rat and Mouse Ration ~~ 

Nutrient 

Protein (8by weight) 

Crude Fat (% by weight) 

Crude Fiber (% by weight) 

Ash (% by weight) 

Amino Acids (% of total diel) 

Arginine 

Cystine 

Glycine 

Histidine 

Isoleucine 

Leucine 

Lysine 

Methionine 

Phenylalanine 

Threonine 

Tryptophan 

Tyrosine 

Valine 

Essenlial Fatty Acids (96of total diel) 

Linoleic 

Linolenic 

Vitamins 

Vitamin A (IUkg) 

Vitamin D (IUkg) 

a-Tocopherol (ppm) 

Thiamine (ppm) 

Riboflavin (ppm) 

Niacin (ppm) 

Pantothenic acid (ppm) 

Pyridoxine (ppm) 

Folic acid (ppm) 

Biotin (ppm) 

Vitamin BIZ(ppb) 

Choline (ppm) 

Minerals 

Calcium (%) 

Phosphorus (%) 

Potassium (%) 

Chloride (%) 

Sodium (%) 

Magnesium (%) 

Sulfur (%) 

Imn (PP@ 

Manganese (ppm) 

Zinc (ppm) 

Copper (PPm) 

Iodine (ppm) 

Chromium (ppm) 

Cobalt (ppm) 

Range 

Mean f Standard 

Deviation 

23.5 f 0.88 

5.2 f 0.29 

3.6 f 0.56 

6.5 f 0.20 

1.308 f 0.060 

0.306 * 0.084 

1.150 f 0.047 

0.576 f 0.024 

0.917 f 0.029 

1.946 2 0.055 

1.270 f 0.058 

0.448 f 0.128 

0.987 f 0.140 

0.877 f 0.042 

0.236 f 0.176 

0.676 2 0.105 

1.103 f 0.040 

2.393 f 0.258 

0.280 f 0.040 

7,425 f 1,737 

4,450 f 1,382 

37.95 f 9.406 

18.72 f 1.72 

7.92 * 0.87 

103.38 2 26.59 

29.54 * 3.60 

9.55 f 3.48 

2.25 f 0.73 

0.25 f 0.04 

38.45 f 22.01 

3,089 * 328 

1.21 * 0.08 

0.95 * 0.04 

0.883 * 0.078 

0,526 f 0.092 

0.313 f 0.390 

0.168 f 0.010 

0.280f 0.064 

360.54 f 100 

91.97 f 6.01 

54.72 f 5.67 

11.06 f 2.50 

3.37 f 0.92 

1.79 * 0.36 

0.681 & 0.14 

21.30 -

4.80 -

2.60 - 6.20 -

1.210 - 0.181 - 1.060 - 0.531 - 0.881 -

1.850- 1.200 - 0.306 - 0,665 - 0.824 - 0,107 -

0.564 - 1.050 -

1.830 - 0.210 -

5,060 - 3,OOO - 22.5 - 15.0 - 6.10 - 65.0 - 23.0 -

5.60 -

1.80 - 0.19 -

10.6 -

2,400 -

1.08 -

0.88 - 0.772 - 0.380-

0.258 - 0.151 - 0.208 - 255.0 -

81.70 -

46.10 -

8.09 - 1.52-

1.04 - 0.490 -

25.20 

5.80 

4.80 

6.97 

1.390 

0.400 

1.210 

0.607 

0.944 

2.040 

1.370 

0.699 

1.110 

0.940 

0.671 

0.794 

1.170 

2.570 

0.320 

12,540 

6,300 

48.90 

22.0 

9.00 

150.0 

34.0 

14.0 

3.70 

0.32 

65.0 

3,430 

1.37 

1.03 

0.971 

0.635 

0.371 

0.181 

0.420 

523.0 

99.40 

64.50 

15.39 

4.13 

2.09 

0.780 

Number OC Samples 

18 

18 

18 

18 

8 

8 

8 

8 

8 

8 

8 

8 

8 

8 

8 

8 

8 

7 

7 

18 

4 

8 

18 

8 

8 

8 

8 

8 

8 

8 

8 

18 

18 

6 

8 

8 

8 

8 

8 

8 

8 

8 

6 

8 

4 
268 Oxazepam, NTP TR 443 

TABLE54 

Contaminant Levels in NIH-07 Rat and Mouse Ration 

Mean 2 Standard 

Deviationa Range Number of Samples 

Contaminants 

Arsenic (ppm) 0.36 2 0.18 0.06 - 0.60 18 

Cadmium (ppm) co.10 18 

Lead (PPm) 0.20 f 0.07 0.10 - 0.30 18 

Mercury (PP@ co.05 18 

Selenium (ppm) 0.33 f 0.11 0.10 -0.52 18 

Aflatoxins (ppb) c5.00 18 

Nitrate nitrogen (ppm 14.48 2 4.36 5.0 - 21.0 18 

Nitrite nitrogen (ppm) b" 0.20 2 0.21 <0.10 - 1.00 18 

BHA (PPm)" 1.33 2 0.77 <1.00 - 4.00 18

BHT (PPmlC 1.55 2 1.42 <1.10 - 7.00 18

Aerobic plate count (CFLJ/g)d 121,222 f 91,920 25,Ooo - 380,Ooo 18 

Coliform (MPN/g)e 20.91 k 20.22 <3.00 - 75.00 18 

E. coli (MPN/g) 3.38 f 1.61 c3.00 - 9.00 18 

Total nitrosoamines (ppb)f 7.02 f 2.87 2.00 - 13.70 18 

N-Nitrosodimethylamine (p b)f 5.30 f 2.30 1.00 - 11.00 18 

N-Nitrosopyrrolidine (ppb) P 1.72 f 1.14 1.00- 4.30 18 

Pesticides 

a-BHCg <0.01 18 

fl-BHC <0.02 18 

T-BHC co.01 18 

6-BHC <0.01 18 

Heptachlor <0.01 18 

Aldrin co.01 18 

Heptachlor epoxide <0.01 18 

DDE co.01 18 

DDD <0.01 18 

DDT co.01 18 

HCB co.01 18 

Mirex co.01 18 

Methoxychlor co.05 18 

Dieldrin co.01 18 

Endrin <0.01 18 

Telodrin <0.01 18 

Chlordane co.05 18 

Toxaphene co.1 18 

Estimated PCBs co.2 18 

Ronnel co.01 18 

Ethion <0.02 18 

Trithion <0.05 18 

Diazinon co.1 18 

Methyl parathion <0.02 18 

Ethyl parathion co.02 18 

Malathion 0.20 +- 0.15 0.05 - 0.48 18 

Endosulfan 1 co.01 18 

Endosulfan 2 <0.01 18 

Endosulfan sulfate cO.03 18 
Feed Analyses 269 

TABLE54 

Contaminant Levels in NIII-07 Rat and Mouse Ration (continued) 

a For values less than the limit of detection, the detection limit is given for the mean. 

Sources of contamination: alfalfa, grains, and fish meal 

Sources of contamination: soy oil and fish meal 

CFU = colony forming unit 

e MPN = mostprobablenumber 

AII values were correct for % recovery. 

g BHC = hexachlorocyclohexaneor benzenehexachloride 
270 Oxazepam, NTP TR 443 
271 

APPENDIX K 

SENTINEL ANIMAL PROGRAM 

METHODS.................................................................... .. 272 
272 Oxazepam, NTP TR 443 

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 all 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. 

Swiss-Webster Mice 

For the 1Cweek study, samples were obtained from five male and five female control mice at terminal 

sacrifice. These samples were processed appropriately and were submitted to Microbiological Associates 

(Bethesda, MD) for viral titer screening. The following tests were performed: 

Method of Analvsis 

ELISA 

Ectromelia virus 

GDVII (mouse encephalomyelitis virus) 

Mouse adenoma virus 

MHV (mouse hepatitis virus) 

MVM (minute virus of mice) 

PVM (pneumonia virus of mice) 

Sendai 

Hemagglutination Inhibition 

K (papovavirus) 

Polyoma virus 

Immunofluorescence Assay 

EDIM (epizootic diarrhea of infant mice) 

LCM (lymphocytic choriomeningitis virus) 

Reovirus 3 

Time of Analvsis 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 
Sentinel Animal Program 273 

For the 57-week study, serum samples for viral screening were collected from up to five male and five 

female sentinel mice at 6, 12, and 13 months into the study. Additional samples were collected at 10, 11, 

and 12 months from mice exhibiting neurological signs; test results were negative. Blood from each 

collection was processed appropriately, shipped to Microbiological Associates, and screened for the 

following: 

Method of Analvsis 

ELISA 

Ectromelia virus 

GDVII 

EDIM 

LCM 

Mouse adenoma virus 

MHV 

PVM 

Reovirus 3 

Sendai 

Hemagglutination Inhibition 

K 

Polyoma virus 

Immunofluorescence Assay 

EDIM 

MVM 

B6C3F, Mice 

Time of Analvsis 

6, 12, and 13 months 

6, 12, and 13 months 

12 and 13 months 

6, 12, and 13 months 

6, 12, and 13 months 

6, 12, and 13 months 

6, 12, and 13 months 

6, 12, and 13 months 

6, 12, and 13 months 

6, 12, and 13months 

6, 12, and 13months 

6 months 

6, 12, and 13 months 

For the 14-week study, samples were obtained from five male and five female control mice at terminal 

sacrifice. These samples were processed appropriately and were submitted to Microbiological Associates 

for viral titer screening. The following tests were performed: 

Method of Analvsis 

ELISA 

Ectromelia virus 

GDVII 

Mouse adenoma virus 

MHV 

MVM 

PVM 

Sendai 

Hemagglutination Inhibition 

K 

Polyoma virus 

Immunofluorescence Assay 

EDIM 

LCM 

Reovirus 3 

Time of Analvsis 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 
274 Oxazepam, NTP TR 443 

For the 2-year study, serum samples for viral screening were collected from up to five male and five female 

sentinel mice at 6, 12, and 18 months into the study. Serum for the 24-month screening was obtained 

from four male and two female mice in the 2,500 ppm groups and one male and three female mice in the 

125 ppm groups. Blood from each collection was processed appropriately, shipped to Microbiological 

Associates, and screened for the following: 

Method of Analvsis 

ELISA 

Ectromelia virus 

EDIM 

GDVII 

LCM 

Mouse adenoma virus 

MHV 

Mycoplasma arthritidis 

Mycoplasma pulmonis 

PVM 

Reovirus 3 

Sendai 

Hemagglutination Inhibition 

K 

MVM 

Polyoma virus 

Immunofluorescence Assay 

EDIM 

LCM 

Mouse adenoma virus 

MHV 

MVM 

Reovirus 3 

All test results were negative. 

Time of Analvsis 

6, 12, 18, and 24 months 

12 and 18 months 

6, 12, 18, and 24 months 

6, 12, and 18 months 

6, 12, 18, and 24 months 

6, 12, 18, and 24 months 

24 months 

24 months 

6, 12, 18, and 24 months 

6, 12, 18, and 24 months 

6, 12, 18, and 24 months 

6, 12, 18, and 24 months 

18and 24 months 

6, 12, 18, and 24 months 

6 and 24 months 

24 months 

24 months 

24 months 

6 and 12 months 

18 months 
275 

APPENDIX L 

RAS PROTO-ONCOGENE ACTIVATION 

OF LIVER NEOPLASMS FROM B6C3Fl MICE 

ADMINISTERED OXAZEPAM IN FEED FOR 2 YEARS 

Theodora R. Devereux, Catherine M. White, John R. Bucher, 

Robert R. Maronpot, and Marshall W. Anderson 

National Institute of Environmental Health Sciences 

Research Triangle Park, North Carolina 

INTRODUCTION................................................................. 276 

MATERIALSAND METHODS........................................................ 276 

RESULTS..................................................................... 277 

DISCUSSION................................................................... 278 

REFERENCES .................................................................. 279 

TABLEL1 H-ras MutationsinOxazepam-InducedLiverNeoplasms 

fromB6C3FlMice ................................................... 280 
276 Oxazepam, NTP TR 443 

RAS.PROTO-ONCOGENE ACTIVATION 

OF LIVER NEOPLASMS FROM B6C3F, MICE 

ADMINISTERED OXAZEPAM IN FEED FOR 2 YEARS 

INTRODUCTION 

Liver neoplasms are commonly seen in B6C3Fl mice in 2-year studies, occurring with a typical incidence of 

30% to 40% in control males and 10% to 20% in control females. Chemical-induced liver neoplasms in 

mice have a high frequency of proto-oncogene activation, particularly by point mutations of H-, K-, or 

N-ras genes (Barbacid, 1987). The frequency of ras activation in these neoplasms is often greater than that 

detected in neoplasms occurring in control animals (Reynolds et al., 1987), and there is evidence for 

chemical specificity in the pattern of oncogene activation (Wiseman et al., 1986). The specific types of 

oncogene-activating mutations induced by a chemical carcinogen often agree with what is expected based 

on the DNA adducts formed by that agent (Wiseman et al., 1986, You et al., 1989). Even for 

"nongenotoxic" carcinogens, the patterns of ras gene mutations in neoplasms can give clues about the 

mechanism of tumorigenesis (Fox et al., 1990, Devereux et al., 1993). 

The purposeof this study was to examine liver neoplasms from male and female BBBF, mice 

administered oxazepam for the presence of activated ras genes. Oncogene activation in chemical-induced 

and "spontaneous" neoplasms was compared to identify mechanisms that may be involved in the induction 

of hepatic neoplasia in these animals. 

MATERIALSAND METHODS 

Neoplasm isolation. The liver neoplasms analyzed in this study were collected from mice in the 2-year 

Battelle Columbus (Columbus, OH) study at moribund or terminal sacrifice. Upon necropsy, sections of 

each neoplasm were fixed and processed for histological examination, and the remainder of each neoplasm 

was frozen in liquid nitrogen and stored at -70" C. Tissues were transferred to NIEHS where the 

oncogene study was performed. 

DNA isolation. DNA was isolated from small (~0.1 g) pieces of 20 or more liver neoplasms from each 

group receiving oxazepam. The DNA isolation procedure (Marmur, 1961) was scaled down and performed 

in 1.5 mL Eppendorf tubes. 

DNA amplipcation. DNA was amplified by the polymerase chain reaction (PCR) (Saiki et al., 1988), and 

details on the use of nested primers have been described previously (Smit et al., 1988, Devereux et al., 

1991).Reactions were carried out in volumesof 20 to 50 p L and consisted of 0.2 to 0.5 pg genomic DNA, 

two amplification primers (lpM), 200pM dNTPs (dATP, dCTP, dGTP, dlTP), reaction buffer [50mM KC1, 

lOmM Tris (pH 8.4 at room temperature), 1mM MgCl,], and 0.5 units of Taq polymerase (Promega, 

Madison, WI). The primers used for amplification of the different exons of the K-ras and H-ras genes are 

listed in Devereux et al. (1991)and Devereux et nl. (1993). Incubations containing DNA from normal 

tissue and no DNA controls were run with all sets of reactions. For those samples to be sequenced, 

asymmetric PCR (one primer at normal concentration and one primer diluted 1:20) was performed with 

the outer PCR reaction as the source of DNA template for this amplification reaction. Amplified DNA 

was desalted and unused primers and dNTPs were removed by spin dialysis in a Centricon 30 tube 

(Amicon, Danvers, MA). The amount of DNA was estimated by measuring optical density at a wavelength 

of 260 nm, and then the samples were evaporated and stored at -20" C until further use. 

Slot-blot oligonucleotide hybridization. Amplified DNA samples (10 to 50 ng) were denatured in 

0.4M NaOHBM NaCl and applied to Nytran nylon filters (0.45 pm mesh) using a slot-blot apparatus 

(Schleicher and Schuell, Keene, NH). The filters were hybridized to 5'-32P end labeled 19 base 
Liver Proto-oncogene Activation 277 

oligonucleotides centered on the second base of codon 61 (or other codons of interest) of the H-rus or 

K-ras genes. The 19-oligomer probes contained either the wild type sequence (CAA for codon 61) or 

mutant sequence (AAA, CGA, or CTA for codon 61). Following hybridization, the blots were washed 

according to the method of Saiki et al. (1986) and exposed to X-ray film for 2 to 24 hours. 

Direct sequencing. Direct sequencing of the amplified exon 2 of the H-rus gene was performed as described 

by Tindall and Stankowski (1989). The sequences of the primers used for the different regions of the ras 

genes are also given by these authors, and the primers were end labeled with y"P labeled ATP. Following 

the sequencing reaction, samples were electrophoresed on 8% acrylamide gels containing urea. Gels were 

dried and exposed to X-ray film overnight. 

Single stranded conformation polymorphism. Single stranded conformation polymorphism (SSCP) of 

amplified exons 1, 2, or 3 of the H-ras gene and exon 1of the K-ras gene in some liver neoplasm samples 

was performed to screen neoplasms for mutations according to the method of Orita et al. (1989). Briefly, 

DNA was amplified by PCR in 20 pL volumes (20 cycles - 94" C, 1 min.; 50" C, 30 sec.; 72" C, 30 sec.) 

with 200pM dNTPs using outer amplification primers (4 pmol each); a second amplification was performed 

with inner amplification primers (20 pmol each) (30 cycles - 94" C, 1 min; 52" C, 1 min; 72" C, 30 sec). 

One pL from the first reaction was added to 19p L to start the second set of cycles. At this point, a 

mini-agarose gel was run with 5 p L of the PCR reaction to check for successful amplification. A third 

PCR reaction with CY-~~P labeled dATP (0.2 pCi), 2pM dNTPs, and 20 pmol each inner primer was

incubated with 1pL from the second reaction as the template DNA (15 cycles - 94" C, 1min; 52" C, 

1min; 72" C, 30 sec). The crude product (2 pL) was mixed with 50 p L of a 0.1% sodium dodecyl 

sulfate-l0mM EDTA mixture, followed by 1:l dilution with a 95% formamide-20mM EDTA-0.05% 

bromophenol blue-0.05% xylene cyano1 loading solution. Diluted samples were heat denatured by boiling 

for 2 to 5 minutes and then placed on ice and loaded on a 12% non-denaturing acrylamide gel containing 

5% glycerol. Electrophoresis was carried out at 4" C with constant power at 20 watts for 16 hours on a 

Model S2 sequencing gel apparatus (BRL, Gaithersburg, MD). The gels were dried and exposed to film 

for 4 to 24 hours at room temperature. 

RESULTS 

In order to determine if the oxazepam-induced neoplasms contained an H-rus mutation profile similar to 

that observed with "spontaneous" neoplasms, similar sample groups of 20 or more adenomas and 

carcinomas from each exposure group were screened by PCR amplification of H-rus exon 2 followed by 

selective oligonucleotide hybridization with slot blots of the amplified DNA for the three common 

codon 61 mutations (Table Ll). SSCP was used as an alternative screening method for detection of 

mutations in DNA from some of the neoplasm samples. Results from the screening were confirmed by 

direct sequencing. In neoplasms from animals in the 125 ppm exposure group, 13/37 (35%) exhibited 

H-rus mutations in codon 61. Nine of these showed C to A transversions in base 1, and four had A to G 

transitions in base 2. Mutations occurred in 58% of the neoplasms taken from control mice and examined 

in the present study and in 65% of historical control B6C3Fl mouse liver neoplasms. While the frequency 

of codon 61 mutations in the 125 ppm group was only about half that observed with control liver 

neoplasms, the mutation spectrum of the H-rus genes detected was similar to that in the "spontaneous" 

neoplasms. In the liver neoplasms from the 2,500 ppm exposure group, only one H-ras codon 61 mutation 

was identified, and no mutations were detected in codon 61 in neoplasms from the 5,OOO ppm exposure 

group. 

In subsequent analyses, neoplasm DNA from the 125 and the 2,500 or 5,OOO ppm groups was analyzed for 

mutations in codons 12, 13,or 117 of the H-rus gene (Table L1) and codons 12 or 13of the K-rus gene 

(not shown), the other known hotspots for rus activation in mouse liver neoplasms. However, no 

mutations in these regions of the genes were detected in any of the neoplasms. 
278 Oxazepam, NTP TR 443 

DISCUSSION 

The formation of both "spontaneous" and chemical-induced liver neoplasms in B6C3Fl mice has often been 

associated with activation of the H-rus gene (Wiseman et al., 1986; Reynolds et ul., 1987; Fox et ul., 1990; 

Devereux et ul., 1993). In this mouse strain, activated H-rus genes have been detected in "spontaneous" 

liver neoplasms at a high frequency, which suggests that this gene is important in liver neoplasm 

formation. Despite the prevalence of H-rus mutations in liver neoplasms in this mouse strain, distinct 

specific mutation spectra have been identified in neoplasms induced by certain genotoxic agents. For 

example, specific mutation patterns in codon 61 of the H-rus gene, which are different from the profile in 

control liver neoplasms, have been detected in neoplasms induced by treatment with N-hydroxy-lacetylaminofluorene, vinyl carbamate, and l-hydroxy-2,3-dehydroestragole(Wiseman et ul., 1986). For 

many genotoxic carcinogens, the specific rus mutation pattern identified in murine neoplasms is associated 

with DNA adducts derived from the chemicals (Wiseman et ul., 1986, Belinsky et al., 1989; You et uL, 

1989). These studies indicated that rus activation is an early event in the development of these neoplasms 

and that rus gene mutation patterns in neoplasm sets may be important in understanding the mechanisms 

by which certain chemicals cause cancer. 

In addition to studies with genotoxic carcinogens, recent studies have analyzed B6C3Fl mouse liver 

neoplasms induced by "nongenotoxic" chemicals for rus activation. Recently it was reported that the 

frequency and the pattern of H-rus gene mutations identified in methylene chloride-induced liver 

neoplasms did not differ significantly from those detected in control neoplasms (Devereux et ul., 1993). 

Results from that study suggest that the activation of the H-rus gene in the chemical-induced neoplasms is 

not directly related to chemical exposure. In another study (Fox et al., 1990), detection of H-rus mutations 

in phenobarbital-, chloroform-, or ciprofibrate-induced liver neoplasms was significantly lower than that 

detected in neoplasms in control animals. Results from that study suggested that pathways other than 

H-rus activation are involved in the development of liver neoplasms induced by those compounds. 

Furthermore, those "nongenotoxic" compounds appear to give a selective growth advantage to cells that 

lack mutations in the H-rus gene. 

In the present study, codon 61 mutations in the H-rus gene were detected in 13/37 (35%) of the 

hepatocellular neoplasms from the 125 ppm group as compared to 58% of the neoplasms in control mice, 

only one neoplasm (2.5%) in the 2,500 ppm group, and none in the 5,OOO ppm group of mice. Of those 

13neoplasms from the 125 ppm group of mice with an activated H-rus gene, the mutation spectrum 

identified was similar to that detected in liver neoplasms that had an activated H-rus gene in control mice 

from both this study and others. These data indicate that oxazepam treatment fosters the formation of 

neoplasms that do not contain H-rus mutations, and thus, it is likely that the neoplasms in the 125 ppm 

group include some related to chemical exposure. These findings are significant because the neoplasm 

incidence data alone are insufficient to determine if the neoplasms that formed in the 125 ppm group were 

influenced in any way by oxazepam administration. These findings are also relevant when considering that 

the 125 ppm dietary level of oxazepam resulted in serum oxazepam concentrations very close to the 

targeted range for human therapeutic use. 
Liver Proto-oncogene Activation 279 

REFERENCES 

Barbacid, M. (1987). Ras genes. Annu.Rev. Biochem. 56, 779-829. 

Belinsky, S.A., Devereux, T.R., Maronpot, R.R., Stoner, G.D., and Anderson, M.W. (1989). Relationship 

between the formation of promutagenic adducts and the activation of the K-ras protooncogene in lung 

tumors from A/J mice treated with nitrosamines. Cancer Res. 49, 53055311. 

Devereux, T.R., Anderson, M.W., and Belinsky, S.A. (1991). Role of rus protooncogene activation in the 

formation of spontaneous and nitrosamine-induced lung tumors in the resistant C3H mouse. 

Carcinogenesis 12, 299-303. 

Devereux, T.R., Foley, J.F., Maronpot, R.R., Kari, F., and Anderson, M.W. (1993). Ras protooncogene 

activation in liver and lung tumors from B6C3Fl mice exposed chronically to methylene chloride. 

Carcinogenesis 14, 795-802. 

Fox, T.R., Schumann, A.M., Watanabe, P.G., Yano, B.L., Maher, V.M., and McCormick, J.J. (1990). 

Mutational analysis of the H-rus oncogene in spontaneous C57BL/6 x C3H/He mouse liver tumors and 

tumors induced with genotoxic and nongenotoxic hepatocarcinogens. Cancer Res. 50, 4014-4019. 

Marmur, J. (1961). A procedure for isolation of deoxyribonucleic acid from micro-organisms. J. Mol. Biol. 

3, 208-218. 

Orita, M., Youichi, S., Sekiya, T., and Hayashi, K. (1989). Rapid and sensitive detection of point 

mutations and DNA polymorphisms using the polymerase chain reaction. Genontics 5, 874-879. 

Reynolds, S.H., Stowers, S.J., Patterson, R.M., Maronpot, R.R., Aaronson, SA., and Anderson, M.W. 

(1987). Activated oncogenes in B6C3Fl mouse liver tumors: Implications for risk assessment. Science 

237, 1309-1316. 

Saiki, R.K., Bugawan, T.L., Horn, G.T., Mullis, K.B., and Erlich, H.A. (1986). Analysis of enzymatically 

amplified b-globin and HLA-DQa DNA with allele-specific oligonucleotide probes. Namre 342, 163-166. 

Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., and Erlich, HA 

(1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 

239, 487-491. 

Smit, V.T.H.B.M., Boot, A.J.M., Smits, A.M.M., Fleuren, G.J., Cornelisse, C.J., and Bos, J.L. (1988). 

KRAS codon 12 mutations occur very frequently in pancreatic adenocarcinomas. Nucleic Ac& Res. 16, 

7773-7782. 

Tindall, K.R., and Stankowski, L.F., Jr. (1989). Molecular analysis of spontaneous mutations at the 

gpt locus in Chinese hamster ovary (AS52) cells. Mutat. Res. 220,241-253. 

Wiseman, R.W., Stowers, S.J., Miller, E.C., Anderson, M.W., and Miller, J.A. (1986). Activating mutations 

of the c-Ha-ras protooncogene in chemically induced hepatomas of the male B6C3Fl mouse. Roc. Natl. 

Acad Sci. USA 83,5825-5829. 

You, M., Candrian, U., Maronpot, R.R., Stoner, G.D., and Anderson, M.W. (1989). Activation of the 

Ki-ras protooncogene in spontaneously occurring and chemically induced lung tumors of the strain A 

mouse. Roc. Natl. Acad Sci USA 86, 3070-3074. 
280 Oxazepam, NTP TR 443 

TABLEL1 

H-ras Mutations in Oxazepam-InducedLiver Neoplasms from B6C3F, Mice 

A. Data Summary 

Neoplasms with Codon 61a Codons Codon 

Treatment Mutations AAACTA CGA 12, 13 117 

Control-historicalb 102/156 (65%) 58 30 13 0 1 

Control 11/19 (58%) 6 4 1 ND ND 

ppmOxazepam-125 13/37 (35%) 9 4 0 0 0 

ppm Oxazepam-2,500 1/20 (5%) 0 0 1 0 ND 

ppm Oxazepam-5,000 on0 (0%) 0 0 0 ND 0 

B. Mutations by Sex and Neoplasm Types for Oxazepam (125 ppm) Liver Neoplasms 

Neoplasms with Codon 61 

Neoplasm Type Sex of Mice Mutations AAA CGA 

Adenomas Female 6/20 (30%) 4 2 

Male 2i9 (22%) 1 1 

Carcinomas Female 3/4 (75%) 2 1 

Male 2/4 (50%) 2 0 

a AAA, CGA, CTA=mutant sequences for codon 61; CAA=wild type sequence. 

Fox er al. (1990); Reynolds et al. (1987); and Devereux (unpublished). H-ras mutation profile not significantly different between 

liver neoplasms from male and female B6C3Fl mice. ND=not done; O=not detected. 
281 

APPENDIX M 

MEASURES OF REPLICATIVE DNA SYNTHESIS 

IN LIVER IN A 90-DAY FEED STUDY 

OF OXAZEPAM IN MALE B6C3Fl MICE 

Michael L. Cunningham, Robert R. Maronpot, and John R. Bucher 

National Institute of Environmental Health Sciences 

Research Triangle Park, North Carolina 

INTRODUCTION ................................................................. 282

MATERIAISANDMETHODS ........................................................ 282 

RESULTS ..................................................................... 283 

DISCUSSION................................................................... 284 

REFERENCES .................................................................. 286 

TABLEM1 Survival, Mean Body Weights, and Feed Consumption 

of Male B6C3Fl Mice Administered Oxazepam in Feed for 90 Days .............. 288 

TABLE M2 Serum Oxazepam Concentrations in Male B6C3Fl Mice 

Administered Oxazepam in Feed for 90 Days ............................... 289 

TABLE M3 BrdU Labeling Indices and Liver WeighVBody Weight Ratios 

of Male B6C3Fl Mice Administered Oxazepam in Feed for 90 Days .............. 289 
282 Oxazepam, NTP TR 443 

MEASURES OF REPLICATIVE DNA SYNTHESIS 

IN LIVER IN A 90-DAY FEED STUDY 

OF OXAZEPAM IN MALE B6C3Fl MICE 

INTRODUCTION 

The 14-week toxicity studies of oxazepam conducted by Battelle Columbus (Columbus, OH) in SwissWebster and B6C3Fl mice gave evidence of marked compound-related increases in liver weight and 

hepatocyte hypertrophy without significant evidence of cytotoxicity. Because studies have shown a 

"promoter" type activity of oxazepam for hepatocellular carcinogenesis (Diwan et al., 1986), and effects of 

oxazepam on the liver of rodents similar to those of chemicals such as phenobarbital and mirex (Cattley 

and Popp, 1989; Schulte-Hermann et al., 1983; Ward et al., 1988, Yarbrough ef al.,1991), the potential of 

oxazepam to stimulate cell replication in the liver was studied. 

Male B6C3Fl mice received diets containing oxazepam at the concentrations used in the 2-year Battelle 

study (125, 2,500, and 5,000 ppm) as well as a lower concentration (25 ppm) that was expected to produce 

blood levels within or below the therapeutic dose range for humans. Mice were evaluated for liver cell 

replication at 15, 30, 45, and 90 days by measuring scheduled DNA synthesis using a bromodeoxyuridine 

(BrdU) labeling method. Livers were also evaluated histopathologically for evidence of hepatocellular 

hypertrophy and cytotoxicity. Blood was taken for analysis of oxazepam concentrations, and serum was 

evaluated for clinical evidence of cytotoxicity. 

MATERIALSAND METHODS 

Oxazepam was acquired from Roussel Corporation (Englewood Cliffs, NJ), and was the same material 

used in the chronic Battelle Columbus (Columbus, OH) studies. Dosed feed was prepared biweekly, by 

mixing ground feed and oxazepam in a Patterson-Kelley blender for 15 minutes with the intensifier bar in 

operation for the first 5 minutes. Aliquots were collected and analyzed by high performance liquid 

chromatography (HPLC) using acetophenone as internal standard. Measured concentrations of oxazepam 

in feed were within 10% of target concentrations. Powdered, dosed feed was delivered to mice in 

DataMAX 50 mouse feeders (Lambert Associates, Flourtown, PA) to allow accurate estimation of feed 

consumption with limited spillage. 

Male B6C3Fl mice (Charles River Breeding Laboratories, Raleigh, NC), weighing approximately 24 g, 

received water and standard NIH-07 feed ad libitum and were maintained under alternating 12-hour 

periods of light and dark at 21" to 23" C and 40% to 60% relative humidity. The animals were acclimated 

to this environment for 2 weeks prior to beginning the experiment. Mice were randomly assigned to 

treatment groups of 10 animals each per time point. All animals were identified by tail tattoos with 

indelible ink. 

Mice were observed twice daily, and feed consumption was measured three times per week. Mice were 

sacrificed by carbon dioxide asphyxiation and exsanguination. Blood samples were collected from the 

retroorbital sinus and allowed to clot for approximately 30 minutes at room temperature, after which they 

were centrifuged at 13,600 x g for 5 minutes. Serum was collected, an aliquot frozen for oxazepam 

analysis, and the remainder immediately analyzed for several clinical pathology parameters (Appendix N). 

Serum oxazepam analysis. Frozen serum was allowed to thaw and 0.1 mL aliquots were made basic with 

10 pL of 0.1 M NaOH. Oxazepam was extracted from the aqueous layer with 1mL ethyl acetate. The 

organic layer containing the oxazepam was removed and evaporated to dryness under nitrogen. Samples 

were reconstituted in 100 pL of 60% methanol/40% phosphate buffer (0.05 M; pH 2.8). Analysis of 

oxazepam was conducted using an HPLC system (Waters Associates, Milford, MA) consisting of two 
Replicative DNA Synthesis in Liver 283 

model 510 HPLC pumps, a 712 WISP multiple sample injector, a C,, column, and a 49OE multiwavelength 

detector at 230 nm. Samples were run isocratically at 0.8 mL/minute at 40% phosphate buffer/60% 

methanol. Serum oxazepam was quantitated against an oxazepam standard curve created with spiked 

serum standards. 

DNA synthesis measurements. Seven days prior to sacrifice, osmotic minipumps (Aka Corporation, Palo 

Alto, CA, model 2002) were implanted subcutaneously on the backs of the mice. These minipumps 

delivered bromodeoxyuridine (BrdU) (Sigma Chemical Co., St. Louis, MO) at 15 pghr. Seven days later, 

the animals were killed by CO, inhalation and blood was collected for clinical chemistgy measurements and 

for analysis of oxazepam levels as described above. Livers were blotted and weighed. A mid-lobe radial 

section of the right anterior lobe was fmed in neutral buffered formalin for 24 hours. A cross section of 

small intestine was also fmed as a positive control for the proper operation of the minipump and the 

staining technique because intestinal crypt cells are constantly in S phase. Tissues were embedded in 

paraffin and serial sections mounted onto poly-1-lysine coated slides. Following deparaffination and 

rehydration, one set of slides was stained with hematoxylin and eosin for histopathological analysis and 

another set stained for BrdU incorporationby a variation on the method of Sugihara et al. (1986), as 

described previously (Cunningham and Matthews, 1991; Cunningham et al., 1991, 1992). Slides were 

treated with 2 N HCl for 30 minutes at 37" C to allow the DNA to become single stranded. The acid 

treatment was quenched with boric acid buffer (pH 7.6) for 1minute at room temperature, followed by 

digestion in 0.01% trypsin (Sigma, St. Louis, MO) and rinsed in PBT [phosphate buffer, pH 7.2, containing 

1%bovine serum albumin (Sigma, St. Louis, MO), 0.05% Tween 20 (Bio-Rad, Richmond, CA) and 

7.2% NaCl]. Nonspecific antibody binding was eliminated by blocking (20 minutes) with normal horse 

serum (1:20)(Vector Laboratories, Inc., Burlington, CA). The slides were then incubated with a 

150 dilution of rat anti-BrdU monoclonal antibody (Accurate Corp., Westbury, W )for 20 minutes at 

room temperature. Following two PBT washes, the slides were incubated with a 1:lOOdilution of a 

biotinylated rabbit anti-rat antibody (Vector, Burlington, CA) for 20 minutes at room temperature and 

visualized with the avidin biotin peroxidase complex (ABC) method using a Vectastain (peroxidase 

standard) kit (Vector, PK4004, Burlington, CA). Nuclear binding of the BBC reagent (labeled nuclei) was 

visualized by staining for 6 minutes with 3,3'-diaminobenzidine (Sigma, St. Louis, MO) to give a dark 

brown color, and nonlabeled nuclei were stained with hematoxylin to yield a blue color. Random areas of 

the slides were chosen for counting stained and unstained hepatocyte nuclei (>1,000 hepatocytes/animal). 

Statistics were performed using a Student's t-test. For the purposes of this study, measures of replicative 

DNA synthesis are assumed to represent cell replication events. This may be an overestimation because 

changes in cell ploidy that commonly occur in rodent liver have not been distinguished and considered. 

RESULTS 

While there were no deaths considered to be compound related during the study (Table Ml), animals in 

the 2,500 and 5,OOO ppm groups appeared sedated and lethargic, and during the first 2 week some exposed 

mice became trapped in the feeders and died. The mice appeared to adapt to the marked pharmacologic 

effect of oxazepam after this period, and no further deaths occurred. At 15, 30, and 45 days, feed 

consumption by 2,500 and 5,ooO ppm mice was generally significantly less than that by controls 

(Table Ml). Mice in the 2,500 and 5,000 ppm groups gained less weight than those in the control, 25, or 

125 ppm groups at 15,30, and 45 days. At 90 days, feed consumption by 2,500 ppm mice was similar to 

that by controls as was the mean body weight of this group (Table Ml). Feed consumption by the 25 ppm 

group at 90 days was slightly greater than controls and was reflected by greater body weight gain 

(Table Ml). 

Serum oxazepam concentrations increased with dose in the 25, 125, and 2,500 ppm groups, but 

concentrations observed in animals receiving 5,000 ppm were .similar to those in mice receiving 2,500 ppm 

at each time point. The highest serum oxazepam concentrations in each exposure group occurred at the 

15-day sacrifice. Serum oxazepam concentrations were also increased in all exposure groups at the 45-day 
284 Oxazepam, NTP TR 443 

evaluation. Serum oxazepam concentrations at 30 and 90 days were somewhat lower than those at 15 and 

45 days (Table M2). 

Histopathologic evaluation of hematoxylin- and eosin-stained slides revealed little evidence of cytotoxicity 

or hepatocyte degeneration and no generalized or periportal inflammation. This was in basic agreement 

with the results of the clinical pathology studies which showed mild cholestasis without hepatocellular 

necrosis (Appendix N). The major histopathological feature in mice exposed to oxazepam was 

hepatocellular hypertrophy characterized by enlarged hepatocytes with pale pink, nonvacuolated cytoplasm 

consistent with proliferation of smooth endoplasmic reticulum. In the 25 and 125 ppm groups, the 

hypertrophy was minimal, was in the centrilobular region, and encompassed hepatocytes 2 to 6 cells deep 

emanating from the central vein. At 2,500 and 5,OOO ppm, the hypertrophy became more extensive and the 

area of enlarged hepatocytes included the periportal region. Although the largest hepatocytes were usually 

found around the central vein, all hepatocytes in mice exposed to 2,500 and 5,OOO ppm were markedly 

enlarged and pale, and there was generalized occlusion of hepatic sinusoids. There was no apparent 

change in hepatocyte ploidy. 

The extent of hepatocellular replication was examined in relation to the amountof oxazepam consumed 

and the relative increase in liver growth over the course of this study. Male B6C3Fl mice exposed to 125, 

2,500, or 5,OOO ppm oxazepam exhibited a significant increase in the rate of hepatocyte replication at 

15 days (Table M3). By this point, (representing cumulative replicative synthesis during days 8 to 15), 

there were fourfold to fivefold increases in BrdU labeling indices at each exposure level compared to those 

of the controls. The mean labeling index at the 25 ppm level was almost twice the control value, but it 

was not statistically different from the control (PcO.05). Relative liver weights of 2,500 and 5,OOO ppm 

mice were significantly greater than those of the controls at the 15-day time point and similar elevated 

ratios were seen in these groups throughout the 90-day study. Relative liver weights of 25 and 125 ppm 

mice were only slightly elevated and were significantly different only when control values appeared low. 

With the exception of a variable, but significantly elevated, cell replication rate at 45 days in the 2,500 ppm 

group, there were no other significant differences in labeling index between exposed and control animals. 

DISCUSSION 

A significant increase in the rate of liver cell replication was noted after 15 days of exposure to 125, 2,500, 

and 5,OOO ppm oxazepam. This increase was dose related, although the rate in the 25 ppm group was 

similar to that of the control. The rate of cell replication returned to control levels after 30 days of 

exposure, although the control rate at 30 days was twice that seen at 15 days. Control values for 

hepatocellular labeling indices using BrdU administered to B6C3Fl mice via osmotic minipumps for 7 days 

have been reported to vary from approximately 4% to 7% (Eacho et al., 1992), which was the range 

observed for control mice in the present study. It is unlikely that this decline in DNA synthesis in 

oxazepam-treated mice could be due to the lower serum levels of oxazepam noted at 30 days because the 

levels in animals in the 2,500 and 5,000 groups still exceeded that sufficient to stimulate DNA replication 

in the 125 ppm group at 15 days. 

The labeling indices of mice treated with higher levels of oxazepam for 15 days closely approximate the 

levels observed in mice treated with 500 mg phenobarbitalb drinking water for 14 days. Klaunig et al. 

(1991)observed a labeling index of approximately 30% in male B6C3Fl mice as determined by 

3H-thymidine delivered by osmotic minipump for 7 days prior to sacrifice. Male CD-1 mice exposed to 

0.1% phenobarbital in the diet produced a labeling index of approximately 30% after 1week of exposure 

compared to about 2% in untreated controls as measured by 7-day BrdU labeling with osmotic minipumps. 

However, by 5 weeks of exposure to phenobarbital, the labeling indices were similar to those in controls 

(Smith et al., 1991). In the current study with oxazepam, by 30 days of exposure, the labeling indices in 

the livers of all exposed groups of mice were similar to that of the control. A decrease in cell replication 
Replicative DNA Synthesis in Liver 285 

has also been observed following chronic administration of phenobarbital to Wistar rats (Schulte-Hermann 

et aL, 1983). This effect is similar to that with trophic hormones and suggests that a feedback mechanism 

may exist to prevent excessive cell proliferation in the continued presence of a mitogenic stimulus. Jirtle 

and Meyer (1991) have shown that chronic administration of phenobarbital to rats results in a transient 

mitogenic effect on the liver, followed by a reduction in the ability of hepatocytes to respond to other 

mitogenic stimuli, such as epidermal growth factor. In addition, the periportal hepatocyte concentration of 

transforming growth factor p, a mitoinhibitory peptide, is increased with chronic phenobarbital 

administration. These mitoinhibitory effects apparently do not occur in preneoplastic foci, possibly 

accounting for their preferential growth during chronic phenobarbital treatment. 

In in vitro studies using lymphoma cells, it was demonstrated that the mitogenic activity of prolactin is 

potentiated by the stimulation of peripheral benzodiazepine receptors at low concentrations (lo9M) of 

agonist (Laird et af., 1989). Activation of peripheral benzodiazepine receptors have also been reported to 

induce differentiation in a variety of cell types (Clarke and Ryan, 1980; Matthew et aL, 1981; Wang et aL, 

1984a), and benzodiazepines have been shown to inhibit proliferation of mouse thymoma cells in culture, 

with a potency that correlates with their affinity to bind to the peripheral benzodiazepine receptor (Wang 

et af., 1984b). Thus, there are several possible mechanisms by which oxazepam may influence 

hepatocellular replication. The GABAergic neurons in the central nervous system have binding sites for 

both the benzodiazepines and phenobarbital (Olsen et af., 1986), however, there are no reports thatthe 

peripheral benzodiazepine receptor may also bind barbiturates. 

In summary, oxazepam appears to stimulate replicative DNA synthesis in the mouse liver, producing an 

approximately 5-fold increase in cell replication within 15 days without associated cytotoxicity. This effect 

is dose dependent and is not sustained with chronic exposure. 
286 Oxazepam, NTP TR 443 

REFERENCES 

Cattley, R.C., and Popp, J.A. (1989). Differences between the promoting activities of the peroxisome 

proliferator WY-14,643 and phenobarbital in rat liver. Cancer Res. 49, 3246-3251. 

Clarke, G.D., and Ryan, P.J. (1980). Tranquillizers can block mitogenesis in 3T3 cells and induce 

differentiation in Friend cells. Nature 287, 160-161. 

Cunningham, M.L., and Matthews, H.B. (1991). Relationship of hepatocarcinogenicity and hepatocellular 

proliferation induced by mutagenic noncarcinogens vs carcinogens. 11. 1-vs2-Nitropropane. ToxicoZ.AppZ. 

Pharmacol. 110, 505-513. 

Cunningham, M.L., Foley, J., Maronpot, R.R., and Matthews, H.B. (1991). Correlation of hepatocellular 

proliferation with hepatocarcinogenicity induced by the mutagenic noncarcinogen:carcinogen pair -

2,6- and 2,4-diaminotoluene. Toxicol. Appl. Pharmacol. 107, 562-567. 

Cunningham, M.L., Elwell, M.R., and Matthews, H.B. (1992). Site-specific cell proliferation in renal 

tubular cells by the renal tubular carcinogen tris (2,3-dibromopropyl)phosphate. Environ. Health Perspecr. 

Diwan, B.A., Rice, J.M., and Ward, J.M. (1986). Tumor-promoting activity of benzodiazepine 

tranquilizers, diazepam and oxazepam in mouse liver. Carcinogenesis 7, 789-794. 

Eacho, P.I., Lanier, T.L., Foxworthy, P.S., and Hoover, D.M. (1992). Effect of the peroxisome proliferator 

LY171883 on hepatocellular replication in female B6C3Fl mice. Fundam. Appl. Toxicol. 18, 632-634. 

Jirtle, R.L., and Meyer, SA. (1991). Liver tumor promotion: Effect of phenobarbital on EGF and protein 

kinase C signal transduction and transforming growth factor41 expression. Dig. Dis. Sci 36, 659-668. 

Klaunig, J.E., Siglin, J.C., Schafer, L.D., Hartnett, J.A., Weghorst, C.M., Olsen, M.J., and Hampton, J.A. 

(1991). Correlation between species and tissue sensitivity to chemical carcinogenesis in rodents and the 

induction of DNA synthesis. In Chemically Induced Cell Proliferation: Implications for Risk Assessment 

(B.E. Butterworth, T.J. Slaga, W. Farland, and M. McClain, Eds.), pp. 185-194. Wiley-Liss, Inc., New 

York. 

Laird, H.E. 11, Gerrish, ICE., Duerson, K.C., Putnam, C.W., and Russell, D.H. (1989). Peripheral 

benzodiazepine binding sites in Nb 2 node lymphoma cells: Effects on prolactin-stimulated proliferation 

and ornithine decarboxylase activity. Eur. J. Pharmacol. 171, 25-35. 

Matthew, E., Laskin, J.D., Zimmerman, E.A., Weinstein, I.B., Hsu, K.C., and Engelhardt, D.L. (1981). 

Benzodiazepines have high-affinity binding sites and induce melanogenesis in B16/C3 melanoma cells. 

Proc. Natl. Acad Sci USA 78, 3935-3939. 

Olsen, R.W., Yang, J., King, R.G., Dilber, A., Stauber, G.B., and Ransom, R.W. (1986). Benzodiazepine 

receptor function. V. Barbiturate and benzodiazepine modulation of GABA receptor binding and 

function. Life Sci 39, 1969-1976. 

Schulte-Hermann, R., Schuppler, J., Timmermann-Trosiener, I., Ohde, G., Bursch, W., and Berger, H. 

(1983). The role of growth of normal and preneoplastic cell populations for tumor promotion in rat liver. 

Environ. Health Perspect. 50, 185-194. 
Replicative DNA Synthesis in Liver 287 

Smith, P.F., O’Brien, KA., and Keenan, K.P. (1991). Evaluation of bromodeoxyuridine labeling in 

hepatomegaly produced by peroxisomal proliferation or P-450 induction in rodents. In Chemically Induced 

Cell Proliferation: Implications for Risk Assessment (B.E. Buttenvorth, T.J. Slaga, W. Farland, and 

M. McClain, Eds.), pp. 285-289. Wiley-Lis, New York. 

Sugihara, H., Hattori, T., and Fukuda, M. (1986). Immunohistochemical detection of bromodeoxyuridine 

in formalin-fixed tissues. Histochemistry 85, 193-195. 

Wang, J.K.T., Morgan, J.I., and Spector, S. (1984a). Differentiation of Friend erythroleukemia cells 

induced by benzodiazepines. Roc.Natl. Acad Sci USA 81, 3770-3772. 

Wang, J.KT., Morgan, J.I., and Spector, S. (1984b). Benzodiazepines that bind at peripheral sites inhibit 

cell proliferation. Proc. Natl. Acad Sci 81, 753-756. 

Ward, J.M., Hagiwara, A, Anderson, L.M., Lindsey, IC, and Diwan, B.A. (1988). The chronic hepatic or 

renal toxicity of di(2-ethylhexyl)phthalate, acetaminophen, sodium barbital, and phenobarbital in male 

B6C3F1 mice: Autoradiographic, immunohistochemical, and biochemical evidence for levels of DNA 

synthesis not associated with carcinogenesis or tumor promotion. Toxicol. Appl. Pharmcacol. 96, 494-506. 

Yarbrough, J., Cunningham, M., Yamanaka, H., Thurman, R., and Badr, M. (1991). Carbohydrate and 

oxygen metabolism during hepatocellular proliferation: A study in perfused livers from mirex-treated rats. 

Hepatology 13, 1229-1234. 
288 Oxazepam, NTP TR 443 

TABLEM1 

Survival, Mean Body Weights, and Feed Consumption of Male B6C3F, Mice Administered Oxazepam 

in Feed for 90 Days 

Final Weight 

Mean Body Weightb (e) Relative Feed 

Concentration Survivala Initial Change Final to Controls Consumption' 

(PPm) (%) 

15 Days 

0 lono 23.7 f 1.5 28.5 f 1.9 4.8 f0.8 3.07 f 0.55 

25 lono 23.5 f0.8 29.3 f 1.5 5.8 f 1.3 102.8 2.77 f 0.17 

125 9/10 24.1 f 1.6 29.8 f 2.6 5.7 f 1.1 104.6 2.92 f 0.77 

2,500 10/10 23.5 f 1.8 25.5 f 1.8.. 2.0 f 1.3.. 89.5 1.77 f 1.09*' 

5,OOo lono 22.9 f 1.4 23.9 f 1.8.. 1.0 f l.5*' 83.9 1.55 f 0.91'' 

30 Days 

0 10/10 22.9 f 0.8 29.4 f 2.0 6.5 f 2.3 3.27 f 0.68 

25 10/10 25.5 f 1.9" 32.2 f 2.2. 6.7 f 2.2 109.5 3.45 f 0.68 

125 9/10 24.7 f 1.6 30.0 f 1.8 5.5 f 1.2 102.0 2.98 f 0.77 

2,500 8/10 24.4 f 1.4 27.9 f 3.0 3.6 f 2.4. 94.9 2.23 f 1.23' 

5,OOo 6/10 23.8 f 2.3 27.6 f 3.4 3.3 f 1.3' 93.9 2.30 f 1.16 

45 Days 

0 lono 23.6 f 1.2 31.6 f 2.1 8.0 f 1.6 3.36 f 0.72 

25 8/10 24.7 f 1.7 32.9 f 2.0 8.0 f 1.2 104.1 3.48 f 0.70 

125 9/10 24.4 f 1.1 31.9 f 1.6 7.5 f 0.5 100.9 3.22 f 1.14 

2,500 9/10 24.2 f 1.2 30.0 f 1.3 5.8 f OB** 94.9 2.29 f 1.00* 

5,0oo 5/10 24.3 f 1.2 27.8 f 2.4** 3.3 f 1.7.. 88.0 2.22 f 1.03* 

90 Days 

0 lono 23.6 f 1.3 33.4 f 2.4 9.8 f 1.4 3.03 f 0.99 

25 6/10 23.6 f 0.7 35.1 f 1.3 11.7 f 0.8' 105.1 3.39 f 1.08 

125 loll0 24.1 f 1.0 34.3 f 1.6 10.2 f 1.0 102.7 3.06 f O.% 

2,500 9/10 25.1 f 1.2 34.2 f 1.5 9.1 f 1.4 102.4 2.82 f 0.95 

5,OOo 8/10 24.1 f 1.2 32.5 f 1.4 8.2 f 1.3. 97.3 2.69 f 0.97 

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

.* PSO.01 

a Number of animals survivinglnumber initially in group 

Weights and weight changes are given as mean f standard deviation 

Feed consumption is given as grams consumed per animal per day 
Replicative DNA Synthesis in Liver 289 

TABLEM2 

Serum Oxazepam Concentrations in Male B6C3F, Mice Administered Oxazepam in Feed for 90 Daysa 

Dose 15 30 45 90 

(PPm) Days Days Days Days 

25 0.6 f 0.06 0.4 f 0.1 0.5 f 0.1 0.6 f 0.1 

125 5.9 f 0.6 3.9 f 0.4 4.9 f 0.7 2.1 f 0.3 

2,500 27.8 f 5.1 13.0 f 7.7 23.9 f 8.8 12.4 f 3.3 

5,Ooo 27.9 f 5.7 7.7 f 4.5 21.0 f6.8 15.4 f 3.9 

~~~ ~~~~ ~ ~ ~ ~~ ~ ~ ~ ~ ~~ 

a Values are expressed in pg/mL serum f standard deviation; 6 to 10 animals per group were evaluated 

TABLEM3 

BrdU Labeling Indices and Liver Weight/Body Weight Ratios of Male B6C3F, Mice 

Administered Oxazepam in Feed for 90 Daysa 

Concentration Number Fold Labeling Indexb Increase Weight/ Liver

(PPm)ControlCroupper Over of Mice Body Weight (%) 

15 Days 

0 10 3.63 f 3.57 5.6 f 0.7 

25 10 6.65 f 6.19 1.83 5.6 f 0.3 

125 9 12.97 f 5.32.' 3.57 5.6 2 0.4 

2,500 10 16.31 2 5.18" 4.49 7.9 2 0.5.. 

5,Ooo 10 17.50 f 4.13.. 4.82 9.1 f 0.6.' 

30 Days 

0 10 7.92 f 4.15 4.4 f 0.4 

25 10 10.10 f 3.48 1.28 5.1 f 0.7" 

125 9 6.37 f 3.30 0.80 5.3 f0.2.. 

2,500 8 13.16 f 8.65 1.66 8.0 f 0.3;. 

5,OOO 6 6.17 f 1.95 0.78 9.5 f 0.5.. 

45 Days 

0 10 8.68 k 2.64 5.1 2 0.3 

25 8 9.23 f 3.07 1.M 5.1 f 0.6 

125 9 8.37 f 7.50 0.96 6.0 f 0.2.' 

2,500 9 21.46 f 15.72. 2.47 8.4 & 0.6.. 

5.Ooo 5 6.28 f 2.16 0.72 9.8 f 0.5.. 

90 Days 

0 10 3.41 f 2.57 4.1 f 0.4 

25 6 3.13 2 1.84 0.92 5.3 f 0.5.. 

125 10 3.73 -e 1.87 1.09 5.1 2 0.5** 

2,500 9 2.57 A 2.06 0.75 9.0 f 0.2.. 

5,OOO 8 3.56 k 3.13 1.04 10.8 f 0.7'. 

. Significantly different from the control (PsO.05) by Dunnett's test 

I. PSO.01 

a Dataarepresented as means f standard deviation 

Number of hepatocytes with labeled nuclei/1,000 hepatocytes scored 
290 Oxazepam, NTP TR 443 
291 

APPENDIX N 

CLINICAL PATHOLOGY ANALYSES 

IN MALE B6C3Fl MICE 

IN A 90-DAY FEED STUDY OF 0XAZEPA.M 

Morrow Thompson and Michael L. Cunningham 

National Institute of Environmental Health Sciences 

Research Triangle Park, North Carolina 

INTRODUCTION ................................................................. 292

MATERIALSANDMETHODS ........................................................ 292 

RESULTS ..................................................................... 292 

DISCUSSION................................................................... 293 

REFERENCE ................................................................... 293 

TABLEN1 ClinicalChemistryData for Male B6C3F, MiceAdministeredOxazepam 

in Feed for 90 Days .................................................. 294 
292 Oxazepam, NTP TR 443 

CLINICAL PATHOLOGY ANALYSES 

IN MALE B6C3F, MICE 

IN A 90-DAY FEED STUDY OF OXAZEPAM 

INTRODUCTION 

The 14-week toxicity studies of oxazepam conducted by Battelle Columbus (Columbus, OH) in SwissWebster and B6C3Fl mice gave evidence of marked compound-related increases in liver weight and 

hepatocyte hypertrophy without significant necrosis. To further evaluate the extent of necrotic damage, a 

clinical pathology study was performed on the same animals used in the study of liver cell replication 

(Appendix M). Male B6C3Fl mice received oxazepam in the diet at concentrations of 25, 125, 2,500, or 

5,OOO ppm for up to 90 days. Clinical pathology analyses were made on serum taken from animals killed 

after 15, 30, 45, and 90 days of treatment. 

MATERIALS AND METHODS 

Oxazepam was acquired from Roussel Corporation (Englewood Cliffs, NJ), and was the same material 

used in the 2-year Battelle studies. Dosed feed was prepared biweekly by adding ground feed and 

oxazepam and mixing in a Patterson-Kelley blender for 15 minutes with the intensifier bar in operation for 

the first 5 minutes. Aliquots were collected for analysis by high performance liquid chromatography 

(HPLC) using acetophenone as internal standard. Measured concentrations of oxazepam in feed were 

within 10% of target concentrations. Powdered, dosed feed was delivered to mice in DataMAX 50 mouse 

feeders (Lambert Associates, Flourtown, PA) to allow accurate estimation of feed consumption with 

limited spillage. 

Male B6C3Fl mice (Charles River Breeding Laboratories, Raleigh, NC) weighing approximately 24 g, 

received water and standard NIH-07 feed ad libitum and were maintained under alternating 12-hour 

periods of light and dark at 21" to 23" C, and 40% to 60% relative humidity. The animals were acclimated 

to this environment for 2 weeks prior to beginning the experiment. Mice were randomly assigned to 

treatment groups of 10 animals each per time point. All animals were identified by tail tattoos with 

indelible ink. 

Mice were observed twice daily, and feed consumption was measured three times per week. At 15, 30, 45, 

and 90 days, mice were anesthetized with CO, and bled from the retroorbital sinus using heparinized 

microcapillary tubes. Blood was collected in plastic tubes containing a serum separator gel (Microtainer, 

Becton Dickinson, Rutherford, NJ). Samples were allowed to clot at room temperature and were 

centrifuged at 13,600 X g for 5 minutes. Biochemical analyses were performed using an automated 

analyzer (Monarch 2000, Instrumentation Laboratory Inc., Lexington, MA). Activities or concentrations of 

the following were determined: alkaline phosphatase (AP),alanine aminotransferase (ALT), creatine 

kinase (CK), sorbitol dehydrogenase (SDH), S'nucleotidase (5'N), bile acids, albumin, total protein, 

creatinine, and cholesterol. Reagents and applications were from Instrumentation Laboratory except those 

for bile acids, SDH, and 5'N. For these assays, reagent kits were obtained from Sigma Chemical Company 

(St. Louis, MO) and adapted for the analyzer. 

RESULTS 

At each evaluation there were moderate increases in serum activities of AP and 5'N in the 2,500 and 

5,OOO ppm groups (Table Nl). The increases were statistically significant with the exception of the AP 

activities measured at 15 days and 5'N at 90 days in the 2,500 ppm group. Mild, significant increases in 

activities of SDH occurred in 2,500 ppm mice at 15 and 90 days and in 5,000 ppm mice at 30, 45, and 
Clinical Pathology Measures 293 

90days. Other significant changes included increases in cholesterol concentration in 2,500 and 5,000 ppm 

mice at 15 and 45 days, increases in bile acids in 2,500 and 5,000 ppm mice at 15 and 45 days and in the 

5,OOO ppm group at 90 days, and increases in total protein in 5,000 ppm mice at 30, 45, and 90 days. 

Changes in bile acids were inconsistent, and the magnitude of the changes overall was considered minimal. 

DISCUSSION 

The increased activities of A P and S'N and concentrations of cholesterol and bile acids are compatible 

with mild cholestasis. Whether this represents a cellular or a physical mechanism of cholestasis cannot be 

determined based on these biochemical data. An example of a direct cellular effect that results in 

cholestasis is a decrease in hepatocellular membrane fluidity produced by treatment with some steroids 

(e.g., ethynyl estradiol) (Zimmerman and Lewis, 1987). A physical mechanism that can result in 

cholestasis is mild compression of canaliculi and bile ducts produced by increased numbers of parenchymal 

cells. The physical mechanism would be consistent with the histologic appearanceof the livers, and the 

effects on liver weight (Appendix M). The minimal to mild increases in activities of SDH (without 

changes in ALT)are suggestive of leakage of the enzyme from intact rather than necrotic cells. As with 

indicators of cholestasis, this may have resulted from compression of cells related to increased rates of 

replication. The increases in total protein appear to be produced by globulins. A distinction was not 

made between immunoglobulins and globulins of hepatic origin. Overall, changes in clinical pathology 

parameters indicated mild cholestasis consistent with compression of bile canaliculi by the hypertrophied 

hepatocytes resulting in obstructed bile flow. There was little evidence of impaired liver function or 

significant hepatocyte injury. 

REFERENCE 

Zimmerman, H.J., and Lewis, J.H. (1987). Drug-inducedcholestasis. Med. Toxicol. 2, 112-160. 
TABLEN1 

Clinical Chemistry Data for Male B6C3Fl Mice Administered Oxazepam in Feed for 90 Days" 

Concentration AP ALT CK SDH 5'N Bile Acids Albumin Protein Creatinine Cholesterol 

(PPd IU/L IUlL IUlL IUlL IUlL pmoUL gldL gldL mgldL mgldL 

15 Days 

0 (n= 10) 60 f 15 29 f 7 307 f 155 28 f 4 17.7 f 4.5 14 f 3 3.3 f0.3 5.9 f 0.3 0.63 f0.1 136 f 6 

25 (n= 10) 51 f 10 40 f 17 212f 111 40 f 10* 19.2 f3.8 11 f 5 3.2 f0.4 5.8 f 0.3 0.69 f0.1 129 f 17 

125 (n=9) 47 f 10 36 f 11 361 f 231 31 f2 19.4 f2.7 11 f 3 2.9 f 0.3' 5.9 f0.3 0.64 f 0.1 122 & 14 

2,500 (n= 10) 81 f 19 42 f 17 207 f 101 54 f 15** 68 f 13** 22 f 4** 3.6 f0.2 6.2 f 0.4 0.75 f0.1** 157 f 15** 

5,000 (n= 10) 98 f29** 29 f 7 255 f 118 38 f6 97 f 32** 19 f 3** 3.5 f 0.3 6.1 f 0.4 0.64 f0.1 153 f 14* 

30 Days 

0 (n= 10) 57 f9 34 f 17 239 f 158 31 f 9 20 f 6 16.1 f4.5 3.1 f 0.2 5.9 f 0.3 0.53 f 0.1 135 f 6 

25 (n= 10) 32 f 5 33 f 12 253 f 153 32 f 8 16 f 6 14.0 f 5.8 2.6 f0.1** 5.9 f0.2 0.54 f 0.1 112 f 15 

125 (n =9) 37 f 10 28 f 8 300 f 167 32 f3 23 f 4 12.9 f 1.3 2.8 f 0.2 6.0 f 0.2 0.53 f0.1 120 f 12 

2,500 (n=8) 81 f23* 32 f 8 270 f 115 33 f6 69 f 28** 16.9 f 4.2 3.0 f 0.4 5.9 f0.3 0.54 f 0.1 149 & 41 

5 ,ooo (n =6) 133 f 39** 30 f 7 207 f 80 44 f lo** 126 f34+* 16.6 f 3.6 3.2 f 0.5 6.3 f OS* 0.52 f0.1 145 & 15 

45 Days 

0 (n=lO) 46 f 6 30 f 8 514 f 272 27.1 f 4.2 17.1 f 1.6 20.0 f 2.4 3.1 f 0.2 5.7 f 0.3 0.62 f 0.1 114 f 11 

25 (n=8) 37 & 9 32 f 9 325 f 225 30.0 f 4.0 16.5 f 1.6 17.6 f 2.2 2.7 f0.2** 5.6 f 0.2 0.65 f 0.1 113 f 8 

125 (n =9) 32 f 5 28 f3 377 f273 27.4 f 2.7 20.0 f 4.7 17.9 f 1.8 2.7 f 0.2" 5.6 & 0.4 0.60 f0.1 108 & 13 

2,500 (n=9) 71 f 25** 25 f 5 247 f 278 32.0 f2.8 41.1 f 9.4** 16.8 f 1.0** 3.0 f 0.3 5.6 f0.3 0.58 f 0.1 133 f IS** 

5,000 (n =5) 138 & lo** 28 f 4 282 f 122 42.0 f 7.1'" 110 f 34** 16.7 +- 2.3* 3.1 f 0.3 6.2 f 0.2** 0.60 f0.1 149 f 5** 

90 Days 

0 (n= 10) 42 f 5 29 f 4 330 f241 28.8 f 4.7 19.4 f 2.3 14.7 f 1.3 3.0 f 0.1 5.5 & 0.2 0.68 f0.1 120 f 5 

25 (n=6) 41 f9 26 f 8 148 & 105 29.7 f 3.8 17.0 f 2.4 14.6 & 3.1 2.8 f 0.3 5.8 f0.2 0.60 f 0.1 109 f 4 

125 (n-10) 37 f 11 32 f 9 312 f 256 30.7 f 5.2 19.1 f 2.1 15.7 f 3.1 2.8 f 0.3 5.6 -+ 0.3 0.60 f 0.0 121 k 9 C

E

2$500 (n =9) 93 f 12** 47 f33 247 f 85 45.7 f25* 39.8 f 5.9** 17.5 f 6.0 2.9 f 0.2 5.8 & 0.4 0.60 f 0.1 129 f 18 N 

5,000 (n= 8) 137 f 19** 37 f 9 287 f 106 57.1 f lo** 97.3 f 39** 19.2 f 2.2** 2.8 f 0.1 6.2 f 0.3** 0.66 f0.1 126 f 10 f -

3 

* 5 Significantly differentfrom the control (PSO.05) by Dunnett's test 

** P50.01 -3 

1 

a X Data ace presented as means f standard deviations: AP=alkaline phosphatase; ALT=alanine aminotnnsferase; CK=creatine kinase; SDH-sorbitol dehydrogenase; 5'N=5'nucleotidase P

P 

Gd 
295 

APPENDIX 0 

TOXICOKINETICS OF OXAZEPAM 

IN B6C3Fl AND SWISS-WEBSTER MICE 

Jinhua Yuan, Thomas J. Goehl, Lily Hong, and James Clark 

National Institute of Environmental Health Sciences 

Research Triangle Park, North Carolina 

INTRODUCTION ................................................................... 296

MATERIALsANDMEr'HODS .......................................................... 296 

RESULTS ........................................................................ 297 

DISCUSSION................................................................... 298 

REFERENCES .................................................................. 299 

FIGURE01 Plasma Concentrations of Oxazepam after IV Administration 

of 20 mgkg to B6C3Fl and Swiss-Webster Mice .. . . . .. . . . . . . . . .... . . . . .... . . 300 

FIGURE02 PlasmaConcentrations of Oxazepam 

after Gavage Administration to B6C3Fl Mice . . . . . . . . .. . . . . . . . .. . . . . . . ... . . . 301 

FIGURE03 PlasmaConcentrations of Oxazepam 

after Gavage Administration to Swiss-Webster Mice , , , .. . . . . . . . . . . . . . . . .. . . . . 302 

FIGURE04 PlasmaConcentrationsinMale B6C3FI 

Mice Administered Oxazepam in Feed for 3 Weeks ... . . . . . . . . . .. . . . . . . . . . . .. . 303 

TABLE01 Toxicokinetic Parameters Obtained from B6C3Fl and Swiss-Webster Mice 

after Intravenous Administration of Oxazepam .. . . . . . . . . . . . . ... . . . . . . . . ..... 304 

TABLE 02 Toxicokinetic Parameters Obtained from B6C3Fl and Swiss-Webster Mice 

after Oral Gavage Administration of Oxazepam .. . . . . . . .. . . . . . . ... . . . .. . . . . . 304 

TABLE03 Estimated Average Daily Feed Consumption for Male B6C3FI Mice 

Administered Oxazepam in Feed for 3 Weeks , ., . . . . . .... . . . . . . ... . . . . .. . . . . 305 
296 Oxazepam, NTP TR 443 

TOXICOKINETICS OF OXAZEPAM 

IN B6C3Fl AND SWISS-mBSTER MICE 

INTRODUCTION 

In the 14-week Battelle Columbus (Columbus, OH) feed studies, B6C3Fl and Swiss-Webster mice received 

diets containing oxazepam at concentrations of 0, 625, 1,250, 2,500, 5,OOO, or 10,OOO ppm, and blood 

samples were collected at weeks 2 and 12. Serum oxazepam concentrations ranged from 6 to 37 pg/mL at 

week 2 and 6 to 22 pg/mL at week 12. In the 2-year Battelle studies, mice received oxazepam in feed at 0, 

125 (B6C3Fl only), 2,500, or 5,OOO ppm. Blood samples were collected at 66 weeks from B6C3Fl mice and 

at 57 weeks from Swiss-Webster mice. The serum oxazepam concentrations ranged from about 1pg/mL in 

125 ppm mice to 6 to 10pg/mL in 2,500 and 5,OOO ppm mice, and were significantly lower than those 

observed in the 14-week studies. The lower concentrations were likely due to typically lower feed 

consumption by older animals, and to an increased capability of the mice to metabolize oxazepam. 

Toxicokinetic studies of oxazepam were conducted in B6C3Fl and Swiss-Webster mice to aid interpretation 

of the results of the mouse carcinogenicity studies and to improve the use of the studies for risk 

assessment. The studies were designed to define: 1) the elimination profiles of oxazepam after 

intravenous (IV) administration, 2) the equivalent single gavage doses that would produce peak plasma 

concentrations similar to those observed in the dosed feed studies, 3) the linear absorption and elimination 

range of oxazepam after oral gavage doses, and 4) the bioavailability of oxazepam from dosed feed. 

Because the ultimate goal is to describe the kinetics of serum oxazepam in the dosed feed studies, the 

pharmacokinetics were simulated using a previously developed model (Yuan, 1993), and the simulation 

results were verified by measurements of actual serum oxazepam concentrations during a short dosed feed 

study. 

MATERIALSAND METHODS 

Oxazepam was procured from Roussel Corporation (Englewood Cliffs, NJ). Identity and purity were 

confirmed by independent analyses. The oxazepam IV formulations were prepared by dissolving oxazepam 

in 80% aqueous dimethyl acetamide at 10 mg/mL. In order to reduce the potential hemolytic side effects 

of the IV vehicle, a small injection volume (2 mL/kg) was used. The gavage formulations (5, 20, and 

40 mg/mL) were prepared by suspending oxazepam in 0.5% aqueous methylcellulose. The dosing volume 

was 10 mL/kg. The homogeneity and stability of the gavage formulations were confirmed prior to use. 

Oxazepam dosed feed formulations (125 and 2,500 ppm) were prepared by directly mixing oxazepam with 

powdered rodent feed (NIH-07). The concentrations of all dose formulations were independently 

confirmed using high performance liquid chromatography (HPLC). 

For the IV studies, male and female B6C3Fl and Swiss-Webster mice (11 weeks old) were obtained from 

Charles River Breeding Laboratories (Raleigh, NC) and were quarantined and acclimated to laboratory 

conditions for one week prior to study start. For each strain, 30 male and 30 female mice were 

administered 20 mgkg oxazepam intravenously via the tail vein. Blood samples were collected from 

anesthetized (70% CO,, 30% 0,)mice at 2, 15, and 30 minutes, and 1, 2, 3, 4, 6, 8, and 10 hours. Each 

mouse was sampled only once. 

For the gavage studies, groups of 24 mice were administered a single oxazepam dose of 50, 200 or 

400 mg/kg. Blood samples were collected from the orbital sinus of three animals per time point at the 

same time intervals as in the IV study. Plasma was separated from blood and stored at -20" C until 

analysis. 
Toxicokinetics 297 

For dosed feed studies, male B6C3F, mice (6 weeks old) were obtained from Charles River Breeding 

Laboratories (Raleigh, NC) and were quarantined and acclimated to laboratory conditions for 2 weeks 

prior to study start. After randomization, mice were housed five per cage on hardwood chip bedding in 

polystyrene cages covered with polyester filter sheets. The animal room was maintained at 20" to 24" C, 

and 40% to 60% relative humidity, with a 12-hour light/dark cycle. Mice received oxazepam dosed feed 

formulations (125 and 2,500 ppm) and water ad Zibinrm. Feed consumption data were collected during the 

study. Blood samples were collected at various time points from one mouse per time point. Each mouse 

was sampled only once. Plasma was separated for oxazepam analysis. 

Oxazepam anaEysis. Plasma oxazepam concentrations were determined by an HPLC method. Briefly, an 

aliquot of 500 pL of acetonitrile containing an internal standard (acetophenone, - 23 pg/mL) was added 

to 200 pL of plasma for extraction. The mixture was then vortexed for 20 seconds. The content of each 

vial was decanted into separate 1mL disposable syringes equipped with 13mm filters (0.2 pm). The 

solutions were filtered directly into 500 pL polypropylene HPLC autosampler vials. The 100 pL 

acetonitrile extract was injected directly into a Waters 510 HPLC which was equipped with an autosampler 

and a Waters 486 UV detector operated at 238 nm. A Phenomenex Ultracarb 7 ODS 30 (250 X 4.6 mm 

ID) with a Whatman Pellicular ODS guard column (20 x 2 mm ID) was used. The mobile phase was 

45% acetonitrile:S5% water containing 0.5% (vb) glacial acetic acid at flow rate of 1mL/min. The 

method was validated to be linear over a range of 0.2 to 50 pg/mL. The recovery of oxazepam from 

plasma was complete. The limit of detection for the method was 0.04 pg/mL, which was three times the 

standard deviation obtained from triplicate determinations of the lowest concentration sample. The limit 

of quantitation, defined as the concentration at which the standard deviation and relative error are less 

than lo%, was 0.24 pg/mL. Stability studies of spiked plasma indicated that oxazepam was stable in 

plasma for at least 4 weeks. 

Data anaEysis. Oxazepam plasma concentration-versus-time data sets obtained after IV administration 

were evaluated for estimation of toxicokinetic parameters using the program NONLIN@ (Metzler et aL, 

1974). The data were fitted to a two-compartment model: 

C(t)=A exp(-A&)+ B exp(-A,t) 

Where: C(t) stands for the plasma concentration at time t, A, and A, are the rate constants for the first 

and second phases of the decline, respectively, and A and B are the corresponding zero-time intercepts. 

These four parameters were estimated by nonlinear regression using a least-squares method and a 

weighting factor equal to the square of the reciprocal of the concentration calculated by the model. 

Clearance (Cl) was computed by dividing dose by area under the curve (AUC). Apparent volume of the 

central compartment (V,) and volumes of distribution at steady state (Vss)were calculated by Dose/(A+B) 

and (NA: + B/A:)/AUC, respectively. Half-life values for t,, and t, were calculated as (ln2)/A, and 

(ln2)/A2, respectively. 

The initial values to be used in the NONLIN@ program were estimated by a manual curve stripping 

method. Standard errors of the toxicokinetic parameters were obtained from the NONLIN@ program 

output. Non-compartmental analysis methods were used to evaluate the gavage data. AUC values and 

their standard deviations after gavage dosing were estimated using the trapezoidal rule. Student's t-test 

was used whenever appropriate. 

RESULTS 

Intravenous snrdies. After IV injection of 20 mg/kg, the estimated half-life of distribution of oxazepam in 

B6C3Fl and Swiss-Webster mice ranged from 12 to 19 minutes (Table 01). The terminal elimination 

half-life of oxazepam in Swiss-Webster mice was about 6 hours for males and 7 hours for females, while in 

B6C3Fl mice, the estimated terminal elimination half-life was 7 hours for males and 5 hours for females. 
298 Oxazepam, NTP TR 443 

A two-compartment model was found to be adequate to fit the IV data for both B6C3Fl and 

Swiss-Webster mice (Figure 01). The estimated apparent volume of distribution at steady state (V,) in 

mice was larger than the vascular or the body water space, which indicates tissue distribution of oxazepam, 

At most time points, and in both strains, female mice had a higher plasma oxazepam concentration than 

males. This difference was also reflected by the smaller V, value for the females. 

Gavage studies. Plasma concentrations of oxazepam after gavage administration were fit to a onecompartment model using a least-squares fit procedure. After gavage administration of 50, 200, and 

400 mgkg, peak plasma oxazepam concentrations (C,J increased nonlinearly with dose and were less than 

or equal to 31 pg/mL (Table 02). Hypothermia and sedation were observed in mice after dosing with 

oxazepam at 200 and 400 mgkg. Elimination profiles of oxazepam at all gavage doses were determined 

(Figures 02 and 03). The peak serum concentration achieved after a single gavage dose of 400 mgkg was 

close to the maximum serum oxazepam concentration observed in the 14-week Battelle studies. The time 

to reach the maximum plasma oxazepam concentrations (T,d in most mice was 2 to 3.5 hours. 

Swiss-Webster mice tended to have a later peak time, which may be due to a delay in stomach emptying. 

The calculated bioavailabilities of oxazepam in mice at most doses were less than 50% (Table 02). Again, 

female mice had higher plasma oxazepam concentrations than males. 

Dosedfeed study. The results of analyses of oxezapam concentration in the plasma of male B6C3Fl mice 

exposed to 125 or 2,500 ppm are shown in Figure 04. Possibly because of the bitter taste and sedative 

effect of oxazepam, feed consumption by exposed groups was initially less than controls, but gradually 

increased (Table 03). As expected, plasma oxazepam concentrations in exposed mice showed individual 

variations and changed with the feeding circadian rhythm. A quasi-steady-state was achieved after 4 days 

ad libitum access to the dosed feed. 

Based on mouse feeding habits (Duffy er af., 1991), daily feed consumption (Table 03), and kinetic 

parameters generated with the gavage study at 50 mag, the plasma oxazepam concentrations during the 

dosed feed study were predicted using a previously developed simulation model (Yuan, 1993). The 

predicted results are shown in Figure 04. 

Simulation with the computer model further suggested that absorption of oxazepam from dosed feed 

between 125 and 2,500 ppm was proportionate and bioavailability (or that fraction of the total dose that 

appeared in the systemic circulation as parent drug) was about 43% for both levels. The simulated plasma 

concentrations for the second and third weeks of the study at 2,500 ppm were higher than the actual values 

(Figure 04). This may be due to an overestimate of the daily feed consumption (Table 03), and/or to 

increased metabolism or elimination of oxazepam. 

DISCUSSION 

The bioavailability of oxazepam in humans following a small oral dose (approximately 0.2 mg/kg) was 

reported to be about 93% and the peak serum concentration was reached in 1.7 to 2.8 hours (Sonne et al., 

1988). Metabolism studies conducted in mice indicated that 30% of a 22 mgkg gavage dose of 

14C-oxazepam was recovered in urine during a 5-day collection period. The majority of the radioactivity 

(57.8% of dose) was recovered in the feces, but was not identified (Sisenwine er af.,1987). If most of the 

radioactivity represented unabsorbed oxazepam, this would agree with current findings that the 

bioavailability of oxazepam in rodents after a gavage dose of 50 mgkg is between 43% and 56%, due 

primarily to incomplete absorption. At higher doses, bioavailability tends to be even lower, which is also 

consistent with incomplete absorption. However, findings from the comparative metabolism studies 

(Appendix P) and those reported by Sisenwine and Tio (1986) for rats suggest that enterohepatic 

circulation of oxazepam, involving the cleavage of glucuronides in the gut, may be significant. This would 

mean that the amount of unchanged drug found in feces would be an overestimate of the unabsorbed 
Toxicokinetics 299 

fraction of a given dose. Thus, until the extent of enterohepatic cycling is determined for these strains of 

mice, the determined numbers for bioavailability should be considered as apparent bioavailability. 

Because of the smaller apparent volume of distribution of oxazepam in female mice, plasma oxazepam 

concentrations in females at most time points (including Cm3 were significantly higher than in males. 

Therefore, females might be more vulnerable to adverse pharmacologic and possibly carcinogenic or toxic 

effects of the parent compound than males. 

The Tmaxin Swiss-Webster mice occurred later than in B6C3Fl mice, suggesting slower absorption of 

oxazepam. This difference in the rate of absorption may be caused by a strain difference in the 

pharmacological response to oxazepam. It is possible that the Swiss-Webster mouse may be more sensitive 

to the sedative effects of oxazepam, which would delay stomach emptying. 

In conclusion, the toxicokinetics of oxazepam in B6C3Fl and Swiss-Webster mice were similar. Differences 

in the elimination profiles of males and females after IV administration were observed in both strains of 

mice, with females having higher plasma concentrations. The terminal elimination half-life ranged from 

5 to 7 hours in each strain and sex. Plasma oxazepam concentrations during prolonged administration via 

dosed feed could be reasonably simulated using pharmacokinetic parameters derived from single dose 

gavage and IV studies. The bioavailability of oxazepam from dosed feed in male B6C3Fl mice was about 

43% and the absorptionof oxazepam was first order. Deviations from the predicted plasma concentrations 

at higher doses with long-term administration may be due to enhanced elimination. 

REFERENCES 

Duffy, P.H., Feuers, R.J., Leakey, J.E.A.,and Hart, R.W. (1991). Chronic caloric restriction in old female 

mice: Changes in the circadian rhythms of physiological and behavioral variables. In Biological Effects of 

Dietary Resniction (L.Fishbein, Ed.), pp. 245-263. Springer-Verlag, New York. 

Metzler, C.M., Elfring, G.K.,and McEwen, AJ. (1974). A package of computer programs for 

pharmacokinetic modeling. Bionlenics 30,562-563. 

Sisenwine, S.F., and Tio, C.O. (1986). The metabolic disposition of oxazepam in rats. Drug Metab. Dispos. 

14, 41-45. 

Sisenwine, S.F., Tio, C.O., Liu, A.L., and Politowski, J.F. (1987). The metabolic fate of oxazepam in mice. 

Drug Metnb. Dispos. 15, 579-580. 

Sonne, J., Loft, S.,Dosing, M., Vollmer-Larsen, A., Olesen, K.L., Victor, M., Andreasen, F., and 

Andreasen, P.B. (1988). Bioavailability and pharmacokinetics of oxazepam. Eur. J. Cfin.Pharmacol. 35, 

385-389. 

Yuan, J.H. (1993). Modeling blood/plasma concentrations in dosed feed and dosed drinking water 

toxicology studies. Toxicol. Appl. Pharmacof. 119, 131-141. 
10 

Oxazepam, NTP TR 443 

B6C3F1 

1 

30 

I 

0 

I 

2 

I 

4 

I I 

6 

I I I 

a 

I 

10 

I I 

12 

1 

Swiss Webster 

10 

0 

I 

2 

I 1 

4 

I 

6 

I I 

a 10 

I 

12 

I 

Time (hr) 

FIGURE01 

Plasma Concentrations of Oxazepam after IV Administration 

of 20 mg/kg to B6C3F, and Swiss-Webster Mice. Each data 

point [(0)male, (A) female] represents the mean & standard 

error for three mice. Solid (male) and dashed (female) lines 

are the two-compartment model results. 
Toxicokinetics 301 

10 

h 

E 

\

0 1 

W 

1 

0 4 8 12 28 16 20 24

B 

0 4 8 12 16 20 24 28 

Time (hr) 

FIGURE02 

Plasma Concentrations of Oxazepam after Gavage Administration 

to B6C3F, Mice. Each data point [(O) 50 mgkg, (A) 200 mgkg, 

(0)400 mgkg] represents the mean It standard error for three mice. 

Solid (50 mgkg), dashed (200 mgkg), and dotted (400 mgkg) lines 

are the one-compartment model results. Figure (A), males; 

Figure (B), females. 
302 Oxazepam, NTP TR 443 

-. 

10 -- ---

& r- 4

1 

0 124 8 16 20 24 28 

B 

40 

10 

12 8 0

FIGURE03 

Plasma Concentrations of Oxazepam after Gavage Administration 

to Swiss-Webster Mice. Each data point [(O) 50 mag, (A) 200 mgkg, 

(0)400 mgkg] represents the mean 2 standard error for three mice. 

Solid (50 mgkg), dashed (200 mgkg), and dotted (400 mgkg) lines 

are the one-compartment model results. Figure (A), males; 

Figure (B), females. 

4 16 20 24 28 

Time (hr) 
Toxicokinetics 303 

100 

Day

1 "1""' 

h

\ 

E 

1

0 

Y 

c

.-0

10 

* 

2 

c 

a 

c 

0 

c 

0 

0 1 

E2- .1 

Q 

.Ol,-,-I 

0 1 2 

' , 

3 . , 4 ' l5 ' 6 l ' 7 , ' 8 l9 ~ l10 ~ 11 l ' 12 r ' l13 14 ' 15 l ~ 16 l17 ~ l18 ~ 19 l : ' l ~ l ~ l ' 

Day of Exposure 

FIGURE04 

Plasma Concentrations of Oxazepam in Male B6C3F, Mice Administered 

OxazepaminFeedfor 3 Weeks. Each data point [(O) 125 ppm, 

(A) 2,500 ppm] represents an individual mouse. Lines (bottom, 125 ppm; 

top, 2,500 ppm) are the prediction based on the developed dosed feed 

computer model. 
304 Oxazepam, NTP TR 443 

TABLE01 

Toxicokinetic Parameters Obtained from B6C3Fl and Swiss-Webster Mice after Intravenous 

Administration of Oxazepam 

B6C3Fl 

M 0.28 20 f 0.04 1.1f 0.170.30 2.61 f 0.12 6.9 f 1.9 71 f 10

F 20 0.29 f 0.02 1.24 & 0.170.26 1.86 f 0.19 4.6 f 0.6 69f 4

Swiss-Webster 

M 20 0.30f 0.02 0.97 f 0.13 2.50 0.19 f 0.06 6.1 f 0.9 67 f 5 

F 20 0.24 f 0.02 1.4 f 0.17 2.23 0.32 f 0.20 6.8 f 1.1 85 f 8 

a V,=apparent volume of the central compartment 

V,=volume of distrubution at steady-state 

tnA,=half-life at rate of decline during first phase 

tnA2=half-lifeat rate of decline during second phase 

TABLE02 

Toxicokinetic Parameters Obtained from B6C3Fl and Swiss-Webster Mice 

after Oral Gavage Administration of Oxazepama 

B6C3Fl 

Male 50 4.9 f 0.64 1.o 76f 4 43 f 6 

200 9.4f 1.4 2.0 181 f 14 25f4 

400 18 f 6 2.0 304 f 25 21 & 3 

Female 50 8.4 f 1.1 2.0 101 f 11 51 f 7 

200 14.8 f 2 2.0 249 f 22 3624 

400 20 f 1.2 3.5 287 f 26 21 f 2 

Swiss-Webster 

Male 50 6.1 & 0.8 2 113 f 5 51 f 15 

200 13.6 f 1.0 3.5 268 f 24 40 f 5 

400 17.4 f 3 3.5 535 f 40 40 f 4 

Female 50 9.2f 0.4 3.5 118 f 6 56 f 6 

200 22 f 3 3.5 335 f 21 40 & 4 

400 31 f 7 6 505 f 44 30f4 

a All parameters listed were obtained using noncompartmental analysis method. Trapezoidal rule and endpoint correction were used 

to estimate AUC value. 

C,,=peak serum oxazepam concentration 

T,,=time at which peak plasma oxazepam concentration was reached 

AUC=area under the curve 
Toxicokinetics 305 

TABLE03 

Estimated Average Daily Feed Consumption for Male B6C3F, Mice Administered Oxazepam 

in Feed for 3 Weeksa 

Days 125 ppm 2,500 ppm 

0 to 2 0.5 0.2 

2 to 4 2.3 0.7 

4 to 7 4.5 3.0 

7 to 9 4.2 2.6 

9 to 11 3.9 4.4 

11 to 14 4.9 4.6 

14 to 16 4.2 4.0 

16 to 18 5.0 3.2 

a Feed consumption is expressed asgrams per animal per day. 
306 Oxazepam, NTP TR 443 
307 

APPENDIX P 

COMPARATIVE METABOLISM STUDIES 

IN B6C3Fl AND SWISS-VVEBSTER MICE 

ADMINISTERED OXAZEPAM BY ORAL GAVAGE 

Robert J. Griffin and Leo T.Burka 

National Institute of Environmental Health Sciences 

Research Triangle Park, North Carolina 

INTRODUCTION ................................................................. 308

MATERIALSANDMEI'HODS ........................................................ 308 

RESULTS ..................................................................... 309 

DISCUSSION ................................................................... 310 

REFERENCES .................................................................. 311 

FIGUREP1 MetabolitesinUrine and Feces fromMaleSwiss-WebsterMice 

Administered 500 mgkg Oxazepam . . . . . . . . . . . . . .. . . . . . . . . . . . . ..... . . . . . . . 312 

FIGUREP2 Oxazepam Metabolites in Man ... . . . ... . . ..... . . . . . . . . . . . . . . . .. . . ... . . . . 313 

FIGURE P3 Oxazepam Metabolites in Male B6C3F, Mice (500 mgkg) ... . . . . . . . . . .. . . . .... . 314 

TABLEP1 RecoveryofRadiolabelfromMiceAdministeredOxazepam by Oral Gavage ... . . . . . 315 

TABLEP2 Major Urinary Metabolitesin Mice 

Administered Oxazepam by Oral Gavage . . . ... . .. . .. . . . . . . . .... . . . . . . . . . .. . 315 

TABLEP3 Total Major Urinary and Fecal MetabolitesinMice 

Administered Oxazepam by Oral Gavage . . . . . . . . . . . . .. . . . . . .... . . . ..... . . .. 316 

TABLEP4 CovalentBinding to Hepatic Protein Following Oxazepam Treatment in Mice ... . .. . 316 
308 Oxazepam, NTP TR 443 

COMPARATIVE METABOLISM STUDIES 

IN B6C3Fl AND SWISS-WEBSTER MICE 

ADMINISTERED OXAZEPAM BY ORAL GAVAGE 

INTRODUCTION 

The manner in which oxazepam is metabolized by humans has been determined by metabolite 

identification in urine (Sisenwine et al., 1972). The metabolic fate of oxazepam in the male Charles River 

COBS CD-1 mouse has also been evaluated, and a brief comparison of the differences in the metabolism 

of oxazepam in several species has been reported (Sisenwine et al., 1987). No comparisons of oxazepam 

metabolism in different strains of mice have appeared in the literature, and no studies using the 

Swiss-Webster mouse strain were located. Because of the observation of liver neoplasms in B6C3Fl and 

Swiss-Webster mice in 2-year studies, and their very early occurrence in the latter strain, studies were 

carried out to identify the major pathways for metabolism of oxazepam in these two strains. In addition, 

elimination of the various metabolites by the two strains was compared, and the possibility that reactive 

intermediates might be formed as a consequence of oxidative metabolism in the mouse was explored. 

MATERIALSAND METHODS 

Oxazepam labeled with 14C in the 3 position of the diazepine ring, was obtained from Amersham (UK). 

Standards used to establish the identities of metabolites were provided by Wyeth-Ayerst 

(Philadelphia, PA). These included 3'-hydroxyoxazepam, 4'-hydroxy-3'methoxyoxazepam, 

6-chloro-4-phenyl2(1H)-quinazoline(CPQ), and R- and S-oxazepam glucuronides. In addition, 

4'-hydroxyoxazepam was synthesized according to the procedure described by Sisenwine and Cesario (1986) 

for the synthesis of 3'-hydroxyoxazepam, by substituting p-methoxybenzycyanide for 

rn-methoxybenzylcyanide in the first step. 6-Chloro-4-phenyl-2(lH)-quinazolinecarboxylic acid (CPQ-acid) 

was prepared according to Sternbach et al. (1964). 

Male and female B6C3Fl and Swiss-Webster mice (18 to 22 g) were obtained from Frederick Cancer 

Research Facility (Frederick, MD) and Charles River Breeding Laboratories (Portage, MI), respectively, 

and quarantined for 6 days. Animals were dosed orally (5 mL/kg) with 14C-labeled oxazepam 

(5 microcuries) in carrier drug in a vehicle of water/emulphor/ethanol (8:l:l) at 25, 250, and 500 m@g. 

Some animals received 2,500 ppm oxazepam in dosed feed for 2 weeks prior to dosing with labeled 

material. Dosed feed was removed 16 hours prior to study to allow clearance of the major portion of the 

drug. Immediately after oral gavage dosing, animals were placed in glass metabolism cages for 72 hours 

for collection of urine, feces, and expired carbon dioxide and then killed. Carbon dioxide was trapped in 

ethanolamine/ethylene glycol monomethyl ether (3:7). 

Collected urine was centrifuged at 1,000 x g to sediment particulates, and directly injected into a Waters 

Associates (Milford, MA) HPLC. Separation was achieved on a C18, 5 micron, Rainin Microsorb column 

(4.6 X 250 mm). The mobile phase was a linear gradient of 85% dibutylamine phosphate (pH 6.5, 

10mM)/15% methanol, to 100% methanol, programmed over 23 minutes, with a flow rate of 1.2 mL/min. 

Detection was by UV absorption (Beckman model 163 Variable Wavelength Detector, Beckman Inst., 

Palo Alto, CA) and radiochemistry (Radiomatic Flo-One, Tampa, FL). 

Feces were homogenized in sodium acetate buffer (pH 6.8, 100mM), precipitated in acetonitrile (1:3), 

vortexed, allowed to extract at 4"C overnight, and then centrifuged. The supernatant was analyzed by the 

high performance liquid chromatography (HPLC) method outlined above. Feces were dried and 

combusted for determination of total radioactivity. The extraction process generally removed more than 
Comparative Metabolism 309 

85% of the total radioactivity. Activity remaining in the carcass was determined following hydrolysis in 

3 mL of tetraethylammonium hydroxide per gram. 

Some urinary and fecal metabolites were identified by co-chromatography with known standards. 

Conjugates were identified after treatment with Qpe IX &glucuronidase or Qpe VI1 sulfatase and 

saccharolactone (all from Sigma Chemical Company, St. Louis, MO). Final identification of isolated peaks 

was confirmed by 1H-nuclear magnetic resonance (NMR) spectroscopy (General Electric GN-500 NMR 

Spectrometer, Fremont, CA), or by mass spectrometry (ZAB4F, VG Analytical, Manchester, UK) with 

glycerol or dithiothreitol as a matrix. 

Residual radioactivity assumed to represent covalently bound oxazepam metabolites was assayed in the 

livers of the animals killed 72 hours after receiving the radiolabel. Livers were removed and homogenized 

(100mM sodium acetate, pH 6.8, 4 mL/g tissue) and aliquots were exhaustively extracted three times with 

80% methanol, followed by trichloroacetic acid (0.4M), and a 3:l mixture of ether and ethanol until no 

further radioactivity could be removed. The protein pellets were solubilized in 0.1 N NaOH, and 

radioactivity/mg protein was determined. 

RESULTS 

The recovery of administered radiolabel in the feces, urine, expired air, and the amount remaining in the 

carcass 72 hours after the oral dose is shown in Table P1. The values given are percent of administered 

radioactivity & the standard error from four animals per dose group. Information is given for animals 

receiving a single oral gavage dose of 25, 250 or 500 mg/kg, and for animals pretreated for 14 days with 

feed containing 2,500 ppm oxazepam and then given 500 mg/kg by oral gavage. There did not appear to 

be major dose-, sex-, or strain-dependent differences in the proportion of materials excreted by the various 

routes, although females tended to excrete somewhat more label in expired breath than did males. 

Pretreatment with oxazepam did result in a consistent increase in the amount of label recovered in the 

urine and decreases in the amount in the feces and the amount remaining in the carcass. 

Shown in Figure P1 are the separationsof oxazepam metabolites in the urine and feces from male 

Swiss-Webster mice given a dose of 500 mgkg. The major peaks in both the urine and feces are oxazepam 

glucuronide; CPQ-acid, a nonenzymatic degradation product of oxazepam; and unchanged oxazepam. The 

bulk of the material excreted in the feces is unchanged drug, possibly representing unabsorbed oxazepam. 

Additional peaks found in urine appear to comprise metabolites that have undergone oxidative 

metabolism, and include 4'-hydroxyoxazepam and its glucuronide, a single peak containing 

3'-hydroxyoxazepam and 4'-hydroxy-3'-methoxyoxazepam, and peaks that are as yet unidentified. 

4'-Hydroxyoxazepam was also identified in the feces, and a unique fecal metabolite remains unidentified. 

The five main urinary metabolites, expressed as percent of total urinary radioactivity, are shown in 

Table P2. A number of strain-dependent differences were noted. B6C3F, mice excreted more oxazepam 

as the glucuronide and less as hydroxylated and other metabolites or unchanged drug than did 

Swiss-Webster mice. Females eliminated less unchanged oxazepam, and more CPQ-acid, 

4'-hydroxyoxazepam and an unknown metabolite than did males. Pretreatment appeared to cause 

increased excretion of glucuronidated metabolites, more unknown metabolite, and less unchanged drug and 

CPQ-acid. 

These five major metabolites were also determined in feces and the total urinary and fecal excretion 

patterns are given in Table P3. Overall, the manner in which oxazepam is metabolized appears similar in 

the two strains of mice. Pretreatment appeared to result in an increase in total glucuronidated metabolites 

in urine and a reduction in elimination of unchanged drug in the feces. 
310 Oxazepam, NTP TR 443 

An evaluation of residual radioactivity in the livers of mice showed a dose-dependent accumulation that 

was quite similar for the two strains of mice (Table P4). Pretreatment for 14 days resulted in lower levels 

of residual radioactivity. 

DISCUSSION 

The data from numerous studies of oxazepam metabolism in humans indicate that a larger portion of an 

administered dose is excreted via the urine than was found in the current mouse studies (Knowles and 

Ruelius, 1972; Alvan et aZ., 1977; Greenblatt, 1981; Sonne et af.,1988). This could be due in part to the 

proportionately higher doses used in mice than are typically given to humans, and it could reflect a larger 

unabsorbed fraction. An alternative explanation for the large amount of unchanged drug in the feces of 

mice is that some of it may represent oxazepam glucuronides that entered via the bile and were cleaved by 

bacterial action. This is supported by the observation that about 50% of the radioactivity extracted from 

the liver of animals treated similarly to those described earlier was present as oxazepam glucuronide, a 

much higher fraction than appears inthe urine. 

Humans appear to excrete from 95% to 99% of urinary metabolites as the glucuronide (Figure P2), along 

with small amounts of the other indicated metabolites. However, three of these, CPQ, 

4'-hydroxyoxazepam glucuronide, and 4'-hydroxy-3'-methoxyoxazepam glucuronide are common to those 

identified in the current mouse studies (Figure P3). The identification of the 3'- and 4'-hydroxylated 

forms indicates the involvement of oxidative metabolism in both humans and mice, and suggests the 

potential for formation of a reactive epoxide. 

Residual radioactivity in the liver presumably represents protein bound metabolites, likely the result of 

oxidative metabolism. Binding increased nearly linearly with oral dose over the range 25 to 500 mgkg, but 

was in the pmol/mg protein range. For comparison, following a 750 mgkg dose of acetaminophen, binding 

to liver protein was reported to be approximately 20 nmol/mg (Jollow et al., 1973). Thus, oxazepam 

should not be considered a particularly potent protein binding agent in mouse liver. 

Prior and presumed chronic administration of oxazepam to mice would likely increase the relative fraction 

of the drug metabolized to the glucuronide and the total amount of metabolites excreted in the urine, thus 

increasing the resemblance of the metabolic pattern to that of humans. The origin of the diazepine ring 

condensation products CPQ and CPQ-acid is not clear, although along with the open ring forms found in 

the human studies, they are possible nonenzymatic reaction products that may be present in the tissue or 

that may form during the extraction and analysis procedures. 

Overall, there was no marked difference in the metabolic profile or in residual hepatic protein binding of 

oxazepam between the B6C3Fl and Swiss-Webster mice that would suggest a possible cause for the very 

early onset of hepatic neoplasia in the Swiss-Webster strain. 
Comparative Metabolism 311 

REFERENCES 

Alvan, G., Siwers, B., and Vessman, J. (1977). Pharmacokinetics of oxazepam in healthy volunteers. Acta 

Pharmacol. Toxicol. 40, 40-51. 

Greenblatt, D.J. (1981). Clinical pharmacokinetics of oxazepam and lorazepam. Cfin. Pharmacokinet. 6, 

89-105. 

Jollow, D.J., Mitchell, J.R., Potter, W.Z., Davis, D.C., Gillette, J.R., and Brodie, B.B. (1973). 

Acetaminophen-induced hepatic necrosis. 11. Role of covalent binding in vivo. J. Pharmacol. Exp. Ther. 

187, 195-202. 

Knowles, J.A., and Ruelius, H.W. (1972). Absorption and excretion of 7-chloro-1,3-dihydro-3-hydroxy5-phenyl-2H-l,4-benzodiazepine-2-one(oxazepam) in humans. Anneimirtelforschung 22, 687-692. 

Sisenwine, S.F., and Cesario, O.T.(1986). The metabolic disposition of oxazepam in rats. Drug Metab. 

Disp~~. 14, 41-45. 

Sisenwine, S.F., Tio, C.O., Schrader, S.R., and Ruelius, H.W. (1972). The biotransformation of oxazepam 

(7-chloro-l,3-dihydro-3-hydroxy-5-phenyl-2H-1,4-benzodiazepine-2-one) in man, miniature swine and rat. 

Anneimittelforschung 22, 682-687. 

Sisenwine, S.F., Tio, C.O.,Liu, A.L., and Politowski, J.F. (1987). The metabolic fate of oxazepam in mice. 

Drug Metab. Dkpos. 15, 579-580. 

Sonne, J., Loft, S., Dossing, M., Vollmer-Larsen, A., Olesen, K.L., Victor, M., Andreasen, E, and 

Andreasen, P.B. (1988). Bioavailability and pharmacokinetics of oxazepam. Eur. J. Clin.Pharmacol. 35, 

385-389. 

Sternbach, L.H., Reeder, E., Stempel, A, and Rachlin, A.I. (1964). Quinazolines and 1,4-benzodiazepines. 

XVIII. The acetylation of chlordiazepoxide and its transformation into 6-chloro-4-phenyl2-quinazolinecarboxyaldehyde.J. 0-g. Chem. 29, 332-336. 
312 Oxazepam, NTP TR 443 

200 

I50 

100 

50 

0 

Feces 

150 -

100 -

50 

0 5 10 I5 20 

RETENTION TIME (nlin) KEY 

A 4-hydroxyoxazepam-glucuronide 

B unknown RT-16.2 

C unknown RT-17.2 

D oxazepam-glucuronide 

E 4-hydroxy+xazepam 

F peak 19.2 

G CPQ-acid 

H unknown RT-20.8 

J 

i 

unknown RT-12.6 

oxazepam 

FIGUREP1 

Metabolites in Urine and Feces from Male Swiss-Webster Mice 

Administered 500 mdkg Oxazepam 
Comparative Metabolism 313 

OXAZEPAMMETABOLITES IN MAN 

FROM SlSENWlNE et al. 1972. 

H 

0 

2-AMINO-5-CHLOROBENZOPHENONE 

0 

CPO K 

I O 

OXAZEPAM - &%tH _3 4-OH-OXAZEPAM 

GLUCURONIDE GLUCURONIDE CI N (> 95 Yo) + 4-OH-3-OMe-OXAZEPAM 

GLUCURONIDE 

J OXAZEPAM 

H 0 OH H 0 OH I II I I II I N-C-CH 

CI 

CI 

2'-OENZOYL-4'-CHLORO- 2'-BENZOYL-4'-CHLORO- 2,2-DIHYDROXYACETANILlDE 2-HYDROXY-2-UREIDOACElANlLlDE 

2'-BENZOYL-4'CHLORO- 2'-BENZOYL-4'-CHLORO- 2,2-DIHYDROXYACETANILlDE 2-HYDROXY-2-UREIDOACETANILIDE GLUCURONIDE GLUCURONIDE 

FIGUREP2 

Oxazepam Metabolites in Man 
314 Oxazepam, NTP TR 443 

OXAZEPAMMETABOLITES IN THE MALE B6C3F1 MOUSE (500mglkg) 

q - O+ co2 

CI 

CPO(1.6%) CPO-ACID (11.1%) z 

H 

/

OXAZEPAM 

- -

UNKYOWN 12.6 (0.7%) GLUCURONIDE UNKNOWN 16.2 (1.1%) (15.0%) UNKNOWN 17.2 (0.4%) 

UNKNOWN 20.8 (0.4%) 

OH 

CI 4-OH-OXAZEPAM (2.6%) 

@OH 

@O Me '3-OH-OXAZEPAM (e 0.3%)

4-OH-OXAZEPAM I 

OH GLUCURONIDE 

(1=I%) 4-OH-3-OMe-OXAZEPAM (< 0.3%) 

FIGUREP3 

Oxazepam Metabolites in Male B6C3F, Mice (500 mg/kg) 
Comparative Metabolism 315 

TABLEP1 

Recovery of Radiolabel from Mice Administered Oxazepam by Oral Gavagea 

Gavage 

Carcass Expiration Feces StrainUrine DOSe Sex Days Total 

(wdw Pretreated 

B6C3Fl 

25 0 M 62.1 f 7.0 23.6 f 3.3 1.8 f 0.1 2.3 f 0.2 90.4 f 4.6 

250 0 M 45.7 f 0.6 37.2 f 4.5 1.5 f 0.1 1.5 f 0.4 87.1 f 4.6 

500 0 M 55.2 f 4.2 22.8 f 2.3 1.4 2 0.3 2.0 f 0.2 81.5 f 2.7 

500 14 M 34.9 f 3.8 29.1 f 5.3 1.9 f 0.1 1.1 f 0.1 67.5 f 2.4 

500 0 F 40.4 f 4.5 29.5 f 1.9 2.3 f 0.1 1.6 f 0.1 75.7 f 6.0 

500 14 F 36.6 f 1.9 41.8 f 3.8 2.5 2 0.1 0.9 f 0.1 82.5 .c 2.7 

Swiss-Webster 

25 0 M 63.7 f 1.9 25.2 f 3.7 2.3 f 0.2 3.2 f 0.8 95.6 f 1.2 

250 0 M 47.8 f 0.9 27.1 f 2.4 1.9 f 0.1 1.7 f 0.3 79.3 f 2.2 

500 0 M 66.1 f 9.4 24.1 f 6.8 1.6 f 0.4 3.3 f 1.2 91.4 f 4.2 

500 14 M 25.0 f 1.1 40.4 k 2.9 1.9 f 0.1 1.2 f 0.3 68.9 f 1.8 

500 0 F 52.6 f 6.1 18.5 f 3.8 3.2 f 0.7 3.4 f 1.7 79.4 f 4.4 

500 14 F 43.1 f 2.9 42.6 f 4.9 2.9 f 0.1 1.2 f 0.1 90.3 f 2.2 

a Data are presented as percent of administered radiolabel f standard error; n=4 

TABLEP2 

Major Urinary Metabolites in Mice Administered Oxazepam by Oral Gavagea 

Gavage Metabolite 

Sex Days Dose 

(mg/kg) Pretreated Oxazepam CPQ-Acid 40H-OX OXAZ-GLUC 40H-OX-GLUC UNK-16.2 

B6C3Fl 

25 0 M 15.3 f 2.5 18.8 f 0.3 3.4 f 0.3 47.7 f 1.0 3.2 f 0.5 6.7 f 0.5 

250 0 M 10.1 f 2.0 11.2 f 1.1 1.6 f 0.5 67.7 f 2.9 2.3b 3.7 f 0.4 

500 0 M 19.7 f 2.7 12.9 f 1.0 3.3 f 1.1 55.0 f 4.8 1.7b 4.0 f 0.5 

500 14 M 1.3 f 0.3 10.5 2 0.7 2.9 f 0.2 69.7 f 1.3 2.3 .c 0.5 7.4 f 0.3 

500 0 F 4.2 f 1.0 17.7 f 0.7 1.2 f 0.2 62.4 f 1.9 3.7 f 0.3 5.6 f 0.5 

500 14 F 1.0 f 0.2 10.5 f 0.5 2.0 f 0.4 71.6 f 0.6 3.0 f 0.2 7.4 f0.3 

Swiss-Webster 

25 0 M 36.1 & 4.8 23.1 f 1.8 5.8 f 0.8 16.9 f 1.3 5.0 f 1.0 8.1 2 1.1 

250 0 M 22.2 f 5.5 20.3 f 1.5 2.5 f 1.1 37.5 f 6.4 6.1 f 1.3 8.0 f 0.7 

500 0 M 23.5 & 4.6 20.7 f 5.3 4.3 f 1.1 37.4 f 12.2 4.5 2 1.7 7.5 f 1.7 

500 14 M 4.5 f 1.2 13.5 f 0.8 ND~ 72.1 f 1.5 2.6 f 0.4 7.3 f 0.5 

500 0 F 14.9 f 3.2 30.6 f 6.3 2.2 f 0.8 32.1 f 12.0 8.2 f 2.5 6.7 f 1.4 

500 14 F 2.2 f 0.4 22.3 f 4.8 2.2 f 0.6 60.0 f 5.9 3.5 f 0.4 8.9 f 1.1 

a Data are presented as percent of total urinary radioactivity f standard error; n=4 

n=3; no standard error available 

ND=not detected 
316 Oxazepam, NTP TR 443 

TABLEP3 

Total Major Urinary and Fecal Metabolites in Mice Administered Oxazepam by Oral Gavagea 

Gavage Metabolite 

D eSex Days 

(mgntg) Pretreated Oxazepam CPQ-Acid 40H-OX OW-GLUC UNK-16.2 40H-OX-GLUC

B6C3Fl 

25 0 M 46.8 f 11.15.2 f 2.0 13f 0.1 13.0 f 1.5 1.1 f 0.6 1.6 f 0.3

250 0 M 31.2 f8.73.3 f 1.21.2 f 0.1 29.7 f 4.2 l.Ob 1.4 f 0.2 

500 0 M 45.0 f 3.4 5.8 f 0.9 1.6 f 0.4 14.7 f 1.7 0.4b 0.9 f 0.0 

500 14 M 19.9 f 1.6 7.3 f 0.2 2.6 f 0.3 23.7 f 3.0 0.6 f 0.1 2.1 f 0.4 

500 0 F 19.2 f 1.4 13.4 f 1.4 1.1 f 0.1 23.0 f 2.5 1.1 ? 0.1 1.6 ? 0.2 

500 14 F 19.2 f 1.5 8.7 f 0.6 2.1 f 0.1 35.8 f 3.4 1.4 f 0.2 3.1 f 0.3 

Swiss-Webster 

25 0 M 51.2 f 1.5 13.6 f 1.9 3.2f 0.6 4.5 f 0.2 1.3 f 0.4 2.1 f 0.5 

250 0 M 34.7 f 3.4 11.6 f 0.5 2.2 f 0.8 11.9 f 2.4 1.6 f 0.2 2.3 f 0.2 

500 0 M 39.2 f 7.2 11.1 f 0.5 2.6 f 0.4 15.8 f 5.6 1.1 f 0.3 1.7 f 0.3 

500 14 M 15.4 f 1.4 8.5 f 0.7 1.1 f 0.3 31.0 f 2.5 1.0 f 0.1 2.9 f 0.3 

500 0 F 29.6 f 3.8 13.1 f 2.8 2.2 f 0.7 12.8 f 2.8 1.8 f 0.5 1.2 f 0.3 

500 14 F 21.9 f 2.8 12.9 f 2.6 2.5 f 0.6 32.2f 4.5 1.6 f 0.1 3.6 f 0.2 

a Data arepresented as percent of administered radiolabel f standard error; n=4 

n=3; no standard error available 

TABLEP4 

Covalent Binding to Hepatic Protein Following Oxazepam Treatment in Micea 

Gavage 

Dose B6C3Fl Males B6C3F, Females Swiss-Webster Males Swiss-Webster Females 

(mgntg) 

25 7.7 f 1.2 ND~ 8.8 f 1.2 ND 

250 63.9 f 7.8 ND 68.4 f 13.2 ND 

500 129.4 f 13.4 133.2 f 19.6 124.4 f 29.7 322.9 f 105.8 

500C 75.8 f 4.6 93.7 f 8.0 63.4 f 5.1 107.7 f 7.8 

a Values are the mean of four samples f the standard error of the mean; units are presented in (pmol boundhg of protein) 

ND=not determined 

These groups were pretreated with oxazepam-dosed feed (2,500ppm) for 2 weeks prior to receiving 500 m&g by oral gavage. 
NATIONAL TOXICOLOGY PROGRAM TECHNICAL REPORTS 

PRINTED AS OF AUGUST 1993 

TR No. CHEMICAL TR No. CHEMICAL 

201 2,3,7,8-Tetrachlorodibenzo-p-dioxin(Dermal) 

206 1,2-Dibromo-3-chloropropane 

207Cytembena 

208 F'D & C Yellow No. 6 

209 2,3,7,8-Tetrachlorodibenzog-dioxin(Gavage) 

210 1,ZDibromoethane 

211C.I.AcidOrange 10 

212 Di(2-ethylhql)adipate 

213 Butyl Benzyl Phthalate 

214Caprolactam 

215Bisphenol A 

21611-AminoundecanoicAcid 

217 Di(2-Ethylhq1)phthalate 

219 2,6-Dichloro-p-phenylenediamine 

220C.I.AcidRed 14 

221LocustBeanGum 

222C.I.DisperseYellow 3 

223Eugenol 

224TaraGum 

225 D & C Red No. 9 

226C.I.SolventYellow 14 

227GumArabic 

228VinylideneChloride 

229GuarGum 

230 Agar 

231StannousChloride 

232Pentachloroethane 

2332-BiphenylamineHydrochloride 

234AllylIsothiocyanate 

235Zearalenone 

236D-Mannitol 

237 1,1,1,2-Tetrachloroethane 

238Ziram 

239 Bis(2-chloro-1-Methylethy1)ether 

240PropylGallate 

242Diallyl Phthalate (Mice) 

243Trichlorethylene (Rats and Mice) 

244PolybrominatedBiphenylMixture 

245Melamine 

246ChrysotileAsbestos(Hamsters) 

247L-AscorbicAcid 

2484,4'-MethylenedianilineDihydrochloride 

249AmositeAsbestos(Hamsters) 

250BenzylAcetate 

2512,4- & 2,6-Toluene Diisayanate 

252GeranylAcetate 

253 Allyl Isovalerate 

254Dichloromethane(MethyleneChloride) 

2551,2-Dichlorobenzene 

257DiglycidylResorcinol Ether 

259EthylActylate 

261Chlorobenzene 

2631,2-Dichloropropane 

266Monuron 

2671,2-PropyleneOxide 

269Telone 11s (1,3-Dichloropropene) 

271HCBlue No. 1 

272Propylene 

273 Trichloroethylene (Four Rat Strains) 

274 Tris(2-ethylhexy1)phosphate

275 2-Chloroethanol 

276 8-Hydroxyquinoline

277 Tremolite 

278 2,6-Xylidine

279 Amosite Asbestos 

280 Crocidolite Asbestos 

281 HC Red No. 3 

282 Chlorodibromomethane 

284 Diallylphthalate (Rats)

285 C.I. Basic Red 9 Monohydrochloride 

287 Dimethyl Hydrogen Phosphite 

288 1,3-Butadiene 

289 Benzene 

291 Isophorone

293 HC Blue No. 2 

294 Chlorinated Trisodium Phosphate 

295 Chrysotile Asbestos (Rats) 

296 Tetrakis(hydroxymethy1) phosphonium Sulfate& 

Tetrakis(hydroxymethy1) phosponium Chloride 

298Dimethyl Morpholinophosphoramidate 

299C.I.DisperseBlue 1 

300 3-Chloro-2-methylpropene 

301o-Phenylphenol 

3034-Vinylcyclohexene 

304ChlorendicAcid 

305ChlorinatedParaffins (G,43%chlorine) 

306Dichloromethane(MethyleneChloride) 

307EphedrineSulfate 

308ChlorinatedPariffins(C,,, 60% chlorine) 

309DecabromodiphenylOxide 

310MarineDieselFueland JP-5 Navy Fuel 

311Tetrachloroethylene(Inhalation) 

312n-ButylChloride 

313Mirex 

314MethylMethacrylate 

315OxytetracyclineHydrochloride 

316 1-Chloro-2-methylpropene 

317ChlorpheniramineMaleate 

318Ampicillin Trihydrate 

319,4-Dichlorobenzene 

320Rotenone 

321Bromodichloromethane 

322PhenylephrineHydrochloride 

323DimethylMethylphosphonate 

324 Boric Acid 

325Pentachloronitrobenzene 

326EthyleneOxide 

327Xylenes(Mixed) 

328 Methyl Carbamate 

3291,2-Epoxybutane 

3304-Hexylresorcinol 

331Malonaldehyde,SodiumSalt 

3322-Mercaptobenzothiazole 

333 N-Phenyl-2-naphthylamine 

3342-Amino-5-nitrophenol 

335C.1.AcidOrange 3 
NATIONAL TOXICOLOGY PROGRAM TECHNICAL REPORTS 

PRINTED AS OF AUGUST 1993 (CONT.) 

TR No. CHEMICAL TR No. CHEMICAL 

336 Penicillin VK 376 Allyl Glycidyl Ether 

337 Nitrofurazone 377 o-Chlorobenzalmalononitrile 

338 Erythromycin Stearate 378 Benzaldehyde 

339 2-Amino-4-nitrophenol 379 2-Chloroacetophenone 

340 Iodinated Glycerol 380 Epinephrine Hydrochloride 

341 Nitrofurantoin 381 d-Carvone 

342 Dichlorvos 382 Furfural 

343 Benzyl Alcohol 385 Methyl Bromide 

344 Tetracycline Hydrochloride 386 Tetranitromethane 

345 Roxalsone 387 Amphetamine Sulfate 

346 Chloroethane 388 Ethylene Thiourea 

347 D-Limonene 389 Sodium Azide 

348 a-Methyldopa Sesquihydrate 390 3,3'-Dimethylbenzidine Dihydrochloride 

349 Pentachlorophenol 391 Tris(2-chloroethyl) Phosphate 

350 Tribromomethane 392 Chlorinated Water and Chloraminated Water 

351 p-Chloroaniline Hydrochloride 393 Sodium Fluoride 

352 N-Methylolacrylamide 394 Acetaminophen 

353 2,4-Dichlorophenol 395 Probenecid 

354 Dimethoxane 396 Monochloroacetic Acid 

355 Diphenhydramine Hydrochloride 397 C.I. Direct Blue 15 

356 Furosemide 399 Titanocene Dichloride 

357 Hydrochlorothiazide 401 2,4-Diaminophenol Dihydrochloride 

358 Ochratoxin A 402 Furan 

359 8-Methoxypsoralen 403 Resorcinol 

360 N,N-Dimethylaniline 405 C.I. Acid Red 114 

361 Hexachloroethane 406 y-Butyrolactone 

362 4-Vinyl-1-Cyclohexene Diepoxide 407 C.I. Pigment Red 3 

363 Bromoethane (Ethyl Bromide) 408 Mercuric Chloride 

364 Rhodamine 6G (C.I. Basic Red1) 409 Quercetin 

365 Pentaelythritol Tetranitrate 410 Naphthalene 

366 Hydroquinone 411 C.I. Pigment Red 23 

367 Phenylbutazone 412 4,4-Diamino-2,2-StilbenedisulfonicAcid 

368 Nalidixic Acid 413 Ethylene Glycol 

369 Alpha-Methylbenzyl Alcohol 414 Pentachloroanisole 

370 Benzofuran 415 Polysorbate 80 

371 Toluene 416 o-Nitroanisole 

372 3,3-Dimethaxybenzidine Dihydrochloride 417 p-Nitrophenol 

373 Succinic Anhydride 418 p-Nitroaniline 

374 Glycidol 419 HC Hellow 4 

375 Vinyl Toluene 434 1,3-Butadiene 

These NTP Technical Reports are available for sale from the National Technical Information Service,U.S. Department of Commerce, 

5285 Port Royal Road, Springfield, VA 22161 (703-4874650). Single copies of this Technical Report are available without charge 

(and while supplies last) from the NTP Central Data Management, NIEHS, P.O. Box 12233, MD AO-01, Research Triangle Park, NC 

27709. 
DEPARTMENT OF 

HEALTH & HUMAN SERVICES SPECIAL FOURTH-CLASS RATE 

POSTAGE AND FEES PAID Public Health Service DHHS/NIH National Toxicology Program Permit No. G-763 Central Data Management

P.O. BOX12233, MD A041 

ResearchTrianglePark, NC 27709 

. 

NIH Publication No. 93-3359 

August 1993