No. 453 

Technical Report Series 

NATIONAL TOXICOLOGY PROGRAM 

STUDIES OF NICKEL SUBSULFIDE 

TOXICOLOGY AND CARCINOGENESIS 

(CAS NO. 12035-72-2) 

IN F344/N RATS AND B6C3Fl MICE 

(INHALATION STUDIES) 

National Institutes of Health 

Public Health Service 

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES 
FOREWORD 

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primary prevention of disease. 

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TOXICOLOGY AND CARCINOGENESIS 

ON THE 

NTP TECHNICAL REPORT 

STUDIES OF NICKEL SUBSULFIDE 

(CAS NO. 12035-72-2) 

IN F344/N RATS AND B6C3Fl MICE 

(INHALATION STUDIES) 

July 1996 

NTP TR 453 

NIH Publication No. 96-3369 

National Institutes of Health 

Public Health Service 

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES 
2 

CONTRIBUTORS 

Nickel Subsulfide, NTP TR 453 

National Toxicology Program 

Evaluated and interpreted results and reported findings 

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

D.B. Walters, Ph.D. 

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

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

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

J.K. Haseman, Ph.D. 

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

T.J. Goehl, Ph.D. 

M.R. Elwell, D.V.M., Ph.D. 

M.P. Dieter, Ph.D. 

R.E. Chapin, Ph.D. 

J.R. Bucher, Ph.D. 

D.A. Bridge, B.S. 

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

Experimental Pathology Laboratories, Inc. 

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

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

Provided pathology qualily assurance 

Dynarnac Corporation 

S. Brecher, Ph.D., Principal Investigator 

Prepared qualiry assurance audits 

Research Institute 

Lovelace Inhalation Toxicology 

Conducted 16-day studies, evaluated pathology findings 

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

F.F. Hahn, D.V.M., Ph.D. 

R.L. Carpenter, Ph.D. 

J.M. Benson, Ph.D., Study Director 

C.H. Hobbs, D.V.M., Principal Investigator 

R.O. McClellan, D .V .M., Director 

Conducted 13-week studies, evaluated pathology findings 

G.M. Shopp, Jr., Ph.D. 

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

F.F. Hahn, D.V.M., Ph.D. 

Y.S. Cheng, Ph.D. 

D.G. Burt, D.V.M. 

D.E. Bice, Ph.D. 

J.M. Benson, Ph.D., Study Director 

C.H. Hobbs, D.V.M., Principal Investigator 

R.O. McClellan, D.V.M., Director 

Conducted 2-year studies, evaluated pathology findings 

K.R. Maples, Ph.D. 

P.J. Haley, D.V.M., Ph.D. 

F.F. Hahn, D.V.M., Ph.D. 

G.L. Finch, Ph.D. 

Y.S. Cheng, Ph.D. 

D.G. Burt, D.V.M. 

E.B. Barr, M.S. 

J.M. Benson, Ph.D., Study Director 

C.H. Hobbs, D.V.M., Principal Investigator 
Nickel Subsulfide, NTP TR 453 3 

NTP Pathology Working Group 

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

National Toxicology Program 

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

NiPERA, Inc. 

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

Experimental Pathology Laboratories, Inc. 

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

Pathology Associates, Inc. 

M.P. Jokinen, D.V.M., Chair 

(21 July 1994) 

Evaluated slides, prepared pathology report on rats 

National Toxicology Program 

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

National Toxicology Program 

National Toxicology Program 

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

Chemical Industry Institute of Toxicology 

K.T. Morgan, D.V.M., Ph.D. 

Duke University 

J. McDonald, M.D. 

Analytical Sciences, Inc. 

Provided statistical analyses 

S. Rosenblum, M.S. 

N.G. Mintz, B.S. 

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

(26 January 1993) 

Evaluated slides, prepared pathology report on mice 

National Toxicology Program 

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

National Toxicology Program 

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

Chemical Industry Institute of Toxicology 

J. Everitt, D.V.M. 

National Toxicology Program 

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

Experimental Pathology Laboratories, Inc. 

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

North Carolina State University 

S. Anderson, D.V.M., Observer 

Pathology Associates, Inc. 

M.P. Jokinen, D.V.M., Chair 

Biotechnical Services, Inc. 

Prepared Technical Report 

S.M. Swift, B.S. 

K.L. Shaw, B.A. 

M.J. Nicholls, B.S. 

L.M. Harper, B.S. 

S.R. Gunnels, M.S. 

D . D . Lambright, Ph . D . , Principal Investigator 
4 

CONTENTS 

REFERENCES 

DISCUSSION AND CONCLUSIONS 

RESULTS 

MATERIALS AND METHODS 

INTRODUCTION 

SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS 

TECHNICAL REPORTS REVIEW SUBCOMMITTEE 

EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY 

ABSTRACT ......................................................... 

107 

89 

49 

37 

15 

13 

12 

11 

5 

........... 

............................. 

......... 

..................................................... 

............................................ 

.......................................................... 

......................................... 

....................................................... 

APPENDIX A 

APPENDIX B 

APPENDIX C 

APPENDIX D 

APPENDIX E 

APPENDIX I 

APPENDIX H 

APPENDIX G 

APPENDIX F 

of Nickel Subsulfide 

Summary of Lesions in Male Rats in the 2-Year Inhalation Study 

......................................... 123 

of Nickel Subsulfide 

Summary of Lesions in Female Rats in the 2-Year Inhalation Study 

......................................... 163 

of Nickel Subsulfide 

Summary of Lesions in Male Mice in the 2-Year Inhalation Study 

......................................... 201 

of Nickel Subsulfide 

Summary of Lesions in Female Mice in the 2-Year Inhalation Study 

......................................... 243 

Genetic Toxicology .......................................... 293 

Organ Weights and Organ-Weight-to-Body-Weight Ratios ................ 301 

Hematology Results ......................................... 311 

Tissue Burden in Rats ........................................ 319 

Tissue Burden in Mice ....................................... 325 

APPENDIX J Reproductive Tissue Evaluations 

and Estrous Cycle Characterization ............................... 

335 

331 

APPENDIX K Chemical Characterization and Generation of Chamber Concentrations ....... 

APPENDIX L Ingredients. Nutrient Composition. and Contaminant Levels 

in NIH-07 Rat and Mouse Ration ................................. 353 

APPENDIX M Sentinel Animal Program ...................................... 357 
5 

ABSTRACT 

Ni,S, 

NICKEL SUBSULFIDE 

Synonyms: Heazlewoodite, nickel subsulphide, nickel sulfide (3:2), a-nickel sulfide (3:2) crystalline, nickel sulphide, nickel tritadisulphide, 

Chemical Formula: Ni,S, Molecular Weight: 240.25 

CAS NO. 12035-72-2 

trinickel disulfide 

normochromatic erythrocytes. 

were analyzed for frequency of micronucleated 

typhimurium, and mouse peripheral blood samples 

toxicology studies were conducted in Salmonella 

week, for 16 days, 13 weeks, or 2 years. Genetic 

to 2.2 pm) by inhalation 6 hours per day, 5 days per 

eter at each exppsure concentration ranged from 2.0 

mean value for the mass median aerodynamic diamexposed to nickel subsulfide (at least 97% pure; the 

Male and female F344/N rats and B6C3Fl mice were 

toxic and carcinogenic effects of inhalation exposure. 

pounds, for which there was little information on the 

nominated as part of a class study of nickel comrefining of certain nickel ores. Nickel subsulfide was 

lithium batteries and is a major component in the 

Nickel subsulfide is used in the manufacture of 

subsulfide/m3 in the core study died on day 14; all 

burden studies. One male exposed to 10 mg nickel 

exposed to 0, 0.6, 2.5, or 10 mg/m3 for tissue 

groups of three male and three female rats were 

12 exposure days during a 16-day period. Additional 

6 hours per day, 5 days per week for a total of 

0, 0.44, 0.88, 1.83, 3.65, and 7.33 mg nickel/m3) 

2.5, 5, or 10 mg nickel subsulfide/m3 (equivalent to 

were exposed to atmospheres containing 0, 0.6, 1.2, 

Groups of five male and five female F344/N rats 

16-DAY STUDY IN RATS 

67 pg nickel/g lung; females, 9 to 77 pg/g lung). 

increased with exposure concentration (males, 7 to 

of nickel in the lungs of exposed groups of rats 

occurred in all exposed groups. The concentrations 

lung and atrophy of the nasal olfactory epithelium 

weight of 1.2 mg/m3 males. Inflammation of the 

than those .of the controls, as was the absolute lung 

exposed groups of females were significantly greater 

weights of 2.5, 5, and 10 mg/m3 males and all 

10 mg/m3 females. Absolute and relative lung 

5 and 10 mg/m3 females and dehydration in 5 and 

included labored respiration in 10 mg/m3 males and 

Clinical findings of toxicity on day 5 of the study 

significantly lower than those of the controls. 

and females exposed to 2.5, 5, or 10 mg/m3 were 

males exposed to 5 or 10 mg nickel subsulfide/m3 

mean body weights and mean body weight gains of 

other rats survived until the end of the study. Final 

 DAY STUDY m MICE 

and female mice exposed to 10 mg nickel 

2.5, or 10 mg/m3 for tissue burden studies. All male 

male and three female mice were exposed to 0, 0.6, 

during a 16-day period. Additional groups of three 

day, 5 days per week for a total of 12 exposure days 

2.5, 5, or 10 mg nickel subsulfide/m3 for 6 hours per 

were exposed to atmospheres containing 0, 0.6, 1.2, 

Groups of five male and five female B6C3Fl mice 
6 Nickel Subsulfide, NTP TR 453 

20 pglg lung). 

(males, 10 to 20 pg nickel/g lung; females, 8 to 

female mice were greater than those of the controls 

concentrations in the lung of exposed male and 

2.5, 5, and 10 mg/m3 males and females. Nickel 

of the nasal olfactory epithelium occurred in 1.2, 

hyperplasia of the bronchial lymph nodes and atrophy 

occurred 5 mg/m3 males and females, and lymphoid 

10 mg/m3 male and female mice, fibrosis of the lung 

Inflammation of the lung occurred in 2.5, 5, and 

significantly greater than those of the controls. 

relative lung weight of 10 mg/m3 females were 

10 mg/m3 males and 5 mg/m3 females, and the 

males, the absolute and relative lung weights of 

and females. The absolute lung weight of 5 mg/m3 

day 5 included labored respiration in 10 mg/m3 males 

lower than those of the controls. Clinical findings at 

weight gains of 5 mg/m3 males were significantly 

study. Final mean body weights and mean body 

1.2 mg/m3 male also died before the end of the 

One control male, one control female, and one 

the study; the death of one female was accidental. 

subsulfide/m3 in the core study died before the end of 

significantly greater than those of the controls. 

and relative lung weights of all exposed groups were 

were minimally increased in exposed rats. Absolute 

hematocrit values, and hemoglobin concentrations 

7. In general, neutrophil and erythrocyte counts, 

2.5 mg/m3 males and females during weeks 2 through 

clinical findings included labored respiration in 

similar to those of the controls. Chemical-related 

body weights of all other exposure groups were 

cantly lower than those of the controls; final mean 

body weight gains of 2.5 mg/m3 males were signifiend of the study. Final mean body weights and mean 

burden studies. All core study rats survived until the 

were exposed to 0,O. 15,0.6, or 2.5 mg/m3 for tissue 

Additional groups of 18 male and 18 female rats 

6 hours per day, 5 days per week for 13 weeks. 

0.11, 0.22, 0.44, 0.88, and 1.83 mg nickel/m3) 

1.2, or 2.5 mg nickel subsulfide/m3 (equivalent to 0, 

exposed to atmospheres containing 0, 0.15, 0.3, 0.6, 

Groups of 10 male and 10 female F344/N rats were 

13-WEEK STUDY IN RATS 

interstitial infiltrates, or incidences of chronic inflamIncreases in the number of alveolar macrophages, 

increasing exposure concentration. 

epithelium, and the severity generally increased with 

males and females had atrophy of the nasal olfactory 

0.3 mg/m3 or greater. Most 0.6, 1.2, and 2.5 mg/m3 

nal lymph nodes was observed in rats exposed to 

Lymphoid hyperplasia of the bronchial and mediastiwere observed in 0.15 mg/m3 males and females. 

Increases in the number of alveolar macrophages 

creased with increasing exposure concentration. 

greater; the severity of these lesions generally innickel subsulfide concentrations of 0.3 mg/m3 or 

mation of the lung occurred in all groups exposed to 

lung). 

5 to 18 pg nickel/g lung; females, 5 to 17 pg/g 

in the controls in rats exposed for 13 weeks (males, 

exposure concentration and were greater than those 

Nickel concentrations in the lung increased with 

1.2 or 2.5 mg/m3. 

lymph nodes was observed in groups exposed to 

2.5 mg/m3. Lymphoid hyperplasia of the bronchial 

cytes were observed in mice exposed to 0.6, 1.2, or 

males and females. Interstitial infiltrates of lymphowere observed in the lung of 1.2 and 2.5 mg/m3 

exposure groups. Chronic inflammation and fibrosis 

present in mice from the 0.3 mg/m3 and higher 

controls. An increase in alveolar macrophages was 

females were significantly greater than those of the 

relative lung weights of 1 .2 and 2.5 mg/m3 males and 

greater than those of the controls. Absolute and 

0.6, 1.2, and 2.5 mg/m3 females were minimally 

globin concentrations and erythrocyte counts in 0.3, 

minimally greater than that of the controls. HemoLymphocyte counts in 1.2 and 2.5 mg/m3 males were 

chemical-related clinical findings were observed. 

groups were similar to those of the controls. No 

studies. Final mean body weights of all exposure 

to 0, 0.15, 0.6, or 2.5 mg/m’ for tissue burden 

groups of six male and six female mice were exposed 

day, 5 days per week for 13 weeks. Additional 

1.2, or 2.5 mg nickel subsulfide/m3 for 6 hours per 

exposed to atmospheres containing 0, 0.15, 0.3, 0.6, 

Groups of 10 male and 10 female B6C3Fl mice were 

13-WEEK STUDY IN MICE 
Nickel Subsulfide, NTP TR 453 7 

increasing exposure concentration. 

incidences and severity generally increased with 

0.6, 1.2, and 2.5 mg/m3 males and females, and 

Atrophy of the nasal olfactory epithelium occurred in 

with increasing ‘exposure concentration. 

23 pg/g lung), and these concentrations increased 

(males, 3 to 17 pg nickel/g lung; females, 6 to 

exposed mice were greater than those of the controls 

At 13 weeks, nickel concentrations in the lungs of 

%YEAR STUDY IN RATS 

and Hematology 

Survival, Body Weights, Clinical Findings, 

greater than those in the controls. 

erythrocyte count in 1 mg/m3 males were mildly 

trations in 1 mg/m3 males and females and the 

periods. Hematocrit values and hemoglobin concenrapid and shallow breathing following exposure 

the study. Chemical-related clinical findings included 

those of the controls throughout the second year of 

weights of rats exposed to 1 mg/m3 were lower than 

were similar to those of the controls. Mean body 

weights of males and females exposed to 0.15 mg/m3 

rats was similar to that of the controls. Mean body 

104 weeks. Survival of exposed males and female 

inhalation for 6 hours per day, 5 days per week for 

(equivalent to 0, 0.11, or 0.73 mg nickel/m3) by 

exposed to 0, 0.15, or 1 mg nickel subsulfide/m3 

Groups of 63 male and 63 female F344/N rats were 

lymphoid hyperplasia; and interstitial inflammation. 

macrophage hyperplasia, and proteinosis; bronchial 

inflammation; focal alveolar epithelial hyperplasia, 

males and females included fibrosis; chronic active 

neoplastic lung lesions generally observed in exposed 

bined) in males and females at 2 years. Nonalveolar/bronchiolar adenoma or carcinoma (combronchiolar carcinoma in males and females, and 

alveolar/bronchiolar adenoma in males, alveolar/ 

were exposure-related increases in the incidences of 

than those of the controls at 7 and 15 months. There 

exposed males and females were significantly greater 

In general, the absolute and relative lung weights of 

Pathology Findings 

cytoma, malignant pheochromocytoma, and benign or

increases in the incidences of benign pheochromoAt 2 years, there were significant exposure-related 

controls. 

females was significantly greater than that of the 

incidence of adrenal medulla hyperplasia in 1 mg/m3 

and of benign pheochromocytoma in females. The 

malignant pheochromocytoma (combined) in males 

were significantly greater than those of the controls. 

tory epithelial atrophy in 1 mg/m3 males and females 

mation of the nose in 1 mg/m3 females and of olfacAt 2 years, the incidences of chronic active inflamwere greater than those of the controls. 

15 months and exposed males and females at 2 years 

the bronchial lymph node of exposed males at 

controls. Incidences of macrophage hyperplasia in 

2 years were significantly greater than those of the 

15 months and in exposed males and females at 

bronchial lymph node in exposed males at 7 and 

The incidences of lymphoid hyperplasia of the 

females, 4 to 7 pg/g lung). 

lung) and 15 months (males, 4 to 3 pg nickel/g lung; 

(males, 6 to 9 pg nickellg lung; females, 6 to 9 pg/g 

were greater than those of the controls at 7 months 

Nickel concentrations in the lung of exposed rats 

Tissue Burden Analyses 

females were greater than those in the controls. 

lymphocyte, and total leukocyte counts in 1.2 mg/m3 

crit value and the segmented neutrophil, monocyte, 

respiration following exposure periods. The hematofindings in male and female mice included labored 

second year of the study. Chemical-related clinical 

were less than those of the controls throughout the 

weights of 0.6 and 1.2 mg/m3 males and females 

mice was similar to that of the controls. Mean body 

105 weeks. Survival of exposed male and female 

inhalation for 6 hours per day, 5 days per week for 

(equivalent to 0, 0.44, or 0.88 mg nickel/m3) by 

exposed to 0, 0.6, or 1.2 mg nickel subsulfide/m3 

Groups of 80 male and 80 female B6C3F, mice were 

and Hematology 

Survival, Body Weights, Clinical Findings, 

&YEAR STUDY IN MICE 
8 Nickel Subsulfide, NTP TR 453 

controls at 7 and 15 months and at 2 years. 

of males and females were greater than those of the 

interstitial infiltration; and fibrosis in exposed groups 

plasia), macrophage hyperplasia and proteinosis; 

mation; bronchialization (alveolar epithelial hypergeneral, the incidences of chronic active inflamwere significantly less than those of the controls. In 

0.6 mg/m3 males and 0.6 and 1.2 mg/m3 females 

bronchiolar adenoma or carcinoma (combined) in 

0.6 mg/m3 females and the incidences of alveolar/ 

incidence ofalveolar/bronchiolar carcinoma in 

those of the controls at 7 and 15 months. The 

and females were generally significantly greater than 

Absolute and relative lung weights of exposed males 

Pathology Findings 

significantly less than that of the controls. 

of olfactory epithelium in exposed females were 

controls. At 2 years, the incidences of degeneration 

mice were also generally greater than those of the 

epithelium and inflammation of the nose in exposed 

The incidences of atrophy of the nasal olfactory 

significantly greater than those of the controls. 

0.6 and 1.2 mg/m3 males and females at 2 years were 

in 0.6 and 1.2 mg/m3 females at 15 months, and in 

hyperplasia in 1.2 mg/m3 males at 7 and 15 months, 

those of the controls. The incidences of macrophage 

females at 2 years were significantly greater than 

15 months, and in 0.6 and 1.2 mg/m3 males and 

15 months, in 0.6 and 1.2 mg/m3 females at

bronchial lymph node in 1.2 mg/m3 males at

The incidences of lymphoid hyperplasia of the 

nickel/g lung; females, 15 to 26 pg/g lung). 

14 pg/g lung) and 15 months (males, 12 to 20 pg 

(males, 10 to 11 pg nickel/g lung; females, 10 to 

were greater than those of the controls at 7 months 

Nickel concentrations in the lungs of exposed mice 

llssue Burden Analyses 

GENETIC TOXICOLOGY 

tion for 13 weeks. 

female mice exposed to nickel subsulfide by inhalaobserved in peripheral blood samples from male or 

the frequency of micronucleated erythrocytes was 

combinations gave negative results. No increase in 

bolic activation enzymes; all other strairdactivation 

obtained in strain TAlOO with and without S9 metaic weakly positive and equivocal responses were 

the Salmonella gene mutation assay overall. SporadNickel subsulfide was considered to be equivocal in 

CONCLUSIONS 

1.2 mg/m3. 

in male or female B6C3F, mice exposed to 0.6 or 

evidence of carcinogenic activity of nickel subsulfide 

chromocytoma of the adrenal medulla. There was no 

bined) and an increased incidence of benign pheoalveolar/bronchiolar adenoma or carcinoma (comincidences of alveolar/bronchiolar carcinoma and 

subsulfide in female F344/N rats based on increased 

was clear evidence of carcinogenic activity of nickel 

pheochromocytoma of the adrenal medulla. There 

malignant, and benign or malignant (combined) 

(combined) and on increased incidences of benign, 

adenoma, carcinoma, and adenoma or carcinoma 

based on increased incidences of alveolar/bronchiolar 

activity* of nickel subsulfide in male F344/N rats 

studies, there was clear evidence of carcinogenic 

Under the conditions of these 2-year inhalation 

the olfactory epithelium in the nose. 

fibrosis in the lung and inflammation and atrophy of 

inflammation, bronchialization, hyperplasia, and 

subsulfide by inhalation for 2 years resulted in 

Exposure of male and female mice tonickel 

and hyperplasia in the adrenal medulla (females). 

and atrophy of the olfactory epithelium in the nose; 

hyperplasia, and fibrosis in the lung; inflammation 

by inhalation for 2 years resulted in inflammation, 

Exposure of male and female rats to nickel subsulfide 

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

comments and the public discussion on this report appears on page 13. 
-- 

Nickel Subsulfide, NTP TR 453 

Summary of the 2-Year Carcinogenesis and Genetic Toxicology Studies of Nickel Subsulfil 

9 

de 

Male Female Male Female 

F344/N Rats F344/N Rats B6C3Fl Mice B6C3Fl Mice 

Exposure 0, 0.15, or 1 mg 

0.73 mg nickellm’) 

(0, 0.11, or concentrations nickel subsulfide/m3 

than controls 

Body weights 1 mg/m3 group lower 

rates 

2-Year survival 13/53, 21/53, 18/53 

53/53, 51/53); focal 

effects inflammation (9/53, 

Nonneoplastic Lung: chronic active 

9/52) 

atrophy (2153, 1/53, 

olfactory epithelial 

10/53, 18/52); 

inflammation (12/53, 

Nose: chronic active 

(2/53, 48/53, 40/53) 

48/53, 52/53); fibrosis 

hyperplasia (9/53, 

macrophage 

6/53, 11/53); 

hyperplasia (2/53, 

alveolar epithelial 

- 

(14/53, 30/52, 42/53) 

pheochromocytoma 

benign or malignant 

(0/53, 2/52, 10153); 

pheochromocytoma 

malignant 

(13/53, 30/52, 38/53); 

pheochromocytoma 

benign 

Adrenal medulla: 

(0153, 6/53, 11/53) 

adenoma or carcinoma 

alveolar/bronchiolar 

(0/53, 3/53, 7/53); 

bronchiolar carcinoma 

6/53); alveolar/ 

adenoma (0153, 3/53, 

bronchiolar 

Neoplastic effects u: alveolar/ 

0.73 mg nickel/m3) 

(0, 0.11, or 

nickel subsulfide/m3 

0, 0.15. or 1 mg 

than controls 

1 mg/m3 group lower 

25/53, 25/53, 28/53 

16/52) 

atrophy (0/53, 0/53, 

olfactoly epithelial 

9/53, 20/52); 

inflammation (6/53, 

Nose: chronic active 

11/53, 16/53) 

hyperplasia (5153, 

Adrenal medulla: 

(0153, 50/53, 44/53) 

51/53, 52/53); fibrosis 

hyperplasia (8/53, 

macrophage 

10153, 11/53); 

hyperplasia (2153, 

alveolar epithelial 

51/53, 51/53); focal 

inflammation (7/53, 

Lung: chronic active 

- 

carcinoma (2/53, 6/53, 

or squamous cell 

adenoma or carcinoma 

alveolar/bronchiolar 

(0/53, 0153, 4/53); 

bronchiolar carcinoma 

m: alveolar/ 

(2/53, 7/53, 36/53) 

pheochromocytoma 

benign 

Adrenal medulla: 

9/53); 

0.88 mg nickel/m3) 

(0, 0.44. or 

nickel subsulfide/m’ 

0. 0.6, or 1.2 mg 

controls 

groups lower than 

0.6 and 1.2 mglm’ 

26/61, 25/60, 26/60 

(1/61, 27/59, 55/59) 

epithelial atrophy 

0/59, 3/59); olfactory 

inflammation (0/61, 

Nose: acute 

(0161, 3/59, 16/58) 

57/59, 58/58); fibrosis 

hyperplasia (6/61, 

macrophage 

53/59, 54/58); 

bronchialization (3/61, 

52/59, 53/58); 

inflammation (1/61, 

m: chronic active 

- 

None 

0.88 mg nickellm’) 

(0, 0.44, or 

nickel subsulfide/m3 

0, 0.6, or 1.2 mg 

controls 

groups lower than 

0.6 and 1.2 mg/m3 

36/58, 34/60, 38/60 

4 1 /60) 

atrophy (1/58, 11/59, 

olfactory epithelial 

11/59, 14/60); 

inflammation (0/58, 

Nose: acute 

(0158, 7/59, 17/60) 

57/59, 60160); fibrosis 

hyperplasia (5/58, 

macrophage 

53/59, 58\60); 

bronchialization (3158, 

46/59, 58/60); 

inflammation (1158, 

Lung: chronic active 

- 

None 
10 Nickel Subsulfide, NTP TR 453 

Summary of the 2-Year Carcinogenesis and Genetic Toxicology Studies of Nickel $ubsulfide (continued) 

Male Female Male Female 

F344lN Rats F344lN Rats B6C3Fl Mice B6C3F, Mice 

activity 

of carcinogenic 

Level of evidence Clear evidence 

Genetic toxicology 

Clear evidence No evidence No evidence 

Mouse peripheral blood in vivo: 

Micronucleated erythrocytes 

Salmonella typhimurium gene mutations: 

Negative in male and female mice 

strains TA91, TA98, TA102, and TA1535 

Equivocal with and without S9 in strain TA100, negative with and without S9 in 
Nickel Subsulfide, NTP TR 453 11 

EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

increases in malignant or benign neoplasms. 

of neoplasms that may be chemical related. 

required for clear evidence. 

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

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

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

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

between two adjacent levels. These considerations should include: 

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

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

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

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

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

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

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

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

l metastases; 

l multiplicity in site-specific neoplasia; 

l latency in tumor induction: 

tissue; 

l presence or absence of dose relationships; l statistical significance of the observed tumor increase; l concurrent control tumor incidence as well as the historical control rate and variability for a specific neoplasm; * survival-adjusted analyses and false positive or false negative concerns; l structure-activity correlations; and l in some cases, genetic toxicology. 

lesion in another sex or species); 
12 Nickel Subsulfide, NTP TR 453 

TECHNICAL REPORTS REVIEW SUBCOMMITTEE 

NATIONAL TOXICOLOGY PROGRAM BOARD OF SCIENTIFIC COUNSELORS 

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

November 29, 1994, are listed below. Subcommittee members serve as independent scientists, not as representatives of any institution, 

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

l to ascertain that all relevant literature data have been adequately cited and interpreted, l to determine if the design and conditions of the NTP studies were appropriate, 

l to ensure that the Technical Report presents the experimental results and conclusions fully and clearly, l to judge the significance of the experimental results by scientific criteria, and l to assess the evaluation of the evidence of carcinogenic activity and other observed toxic responses. 

Arnold L. Brown, M.D., Chair 

Madison, WI 

University of Wisconsin Medical School 

Thomas L. Goldsworthy, Ph.D. 

Research Triangle Park, NC 

Chemical Industry Institute of Toxicology 

Department of Experimental Pathology and Toxicology 

Pittsburgh, PA . 

University of Pittsburgh 

Department of Environmental Occupational Health 

Meryl H. Karol, Ph.D.* 

Kansas City, KS 

University of Kansas Medical Center 

Department of Pharmacology and Toxicology 

Curtis D. Klaassen, Ph.D., Principal Reviewer 

Claudia S. Miller, M.D. 

San Antonio, TX 

University of Texas Health Sciences Center 

Philadelphia, PA 

Fox Chase Cancer Center 

Irma Russo, M.D. 

Louise Ryan, Ph.D., Principal Reviewer 

Boston. MA 

Dana-Farber Cancer Institute 

Harvard School of Public Health 

Division of Biostatistics 

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

Washington, DC 

Howard University College of Medicine 

Department of Pharmacology 

Mary Jo Vodicnik, Ph.D.* 

Belgium 

Lilly MSG Development Centel 

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

Frederick, MD 

National Cancer Institute 

Janardan K. Reddy, M.D., Principal Reviewer* 

Chicago, IL 

Northwestern University Medical School 

Department of Pathology 

* Did not attend 
Nickel Subsulfide, NTP TR 453 13 

SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS 

mental Health Sciences, Research Triangle Park, NC. 

meeting‘was held at the National Institute of Environors’ Technical Reports Subcommittee. The review 

Toxicology Program’s Board of Scientific Counselsubsulfide received public review by the National 

on the toxicology and carcinogenesis studies of nickel 

On November 29, 1994, the draft Technical Report 

B6C3F, mice. 

evidence of carcinogenic activity in male or female 

activity in male and female F344/N rats, and no 

conclusions were clear evidence of carcinogenic 

nonneoplastic lesions in rats and mice. The proposed 

compound-related neoplastic lesions in rats and 

survival and body weight effects, and commenting on 

describing the xperimental design, reporting on 

and carcinogenesis studies of nickel subsulfide by 

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

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

meeting but had submitted his review, which 

proposed conclusions. He was unable to attend the 

Dr. Reddy, a principal reviewer, agreed with the 

nickel studies of an association between higher lung 

mented that there was a consistent trend in the three 

lesions in mice. Dr. J.K. Haseman, NIEHS, comand carcinomas in rats while it decreases these 

exposure increases alveolarlbronchiolar adenomas 

discussion of the observation that nickel subsulfide 

not. Dr. Klaassen said there needed to be more 

compared to a low exposure concentration that does 

concentration that affects pulmonary clearance 

tion would have been to choose a high exposure 

trations that might have yielded mechanistic informamore appropriate way to have set exposure concenhexahydrate studies. Dr. Goldsworthy suggested a 

tions selected for the nickel oxide and nickel sulfate 

that were present at the highest exposure concentrahistopathologic lung changes in the 13-week study 

the presence of a similar spectrum and severity of 

lower exposure concentration was selected based on 

selected to repeat that used in a previous study. The 

explained that the highest exposure concentration was 

appropriate but needed to be discussed. Dr. Dunnick 

the wide range of exposure concentrations in rats was 

with the proposed conclusions. He commented that 

Dr. Klaassen, the second principal reviewer, agreed 

ation was biologically significant. 

in rats. He did not know whether or not this associlung neoplasm incidences at 2 years in mice but not 

weights at the 15-month interim evaluation and lower 

sure groups. 

the nickel subsulfide study was limited to two expothe number of animals, exposure groups, and costs, 

results of a previously published study. To reduce 

studies it was considered a positive control based on 

nickel subsulfide was included for this series of 

the usual three. Dr. Dunnick replied that when 

why only two exposure groups were used instead of 

route of exposure in the workplace. Dr. Ryan asked 

Dr. Dunnick noted that inhalation is the predominant 

food islikely to be much higher in humans. 

chosen as the route of exposure since intake from 

useful to state more clearly why inhalation was 

the proposed conclusions. She thought it would be 

Dr. Ryan, the third principal reviewer, agreed with 

workers exposed to low nickel levels. 

increases in the incidence of nonneoplastic lesions in 

examinations shows no evidence of nickel-related 

and she said that the information obtained from these 

posed to nickel compounds that have been examined, 

Ms. Sivulka discussed the cohorts of workers extive of workers exposed to nickel compounds. 

could be made that current regulations are not protecreport were based on existing TLVs, an implication 

Ms. Sivulka said that because conclusions in the 

relative to existing threshold limit values (TLVs). 

the presentation of the significance of findings 

for nickel toxicity and carcinogenesis in humans and 

(NIPERA), commented on the discussion of evidence 

Producers Environmental Research Association, Inc. 

Ms. D. Sivulka, executive director of the Nickel 

votes. 

motion, which was accepted unanimously with six 

of carcinogenic activity. Dr. Ryan seconded the 

activity, and for male and female mice, no evidence 

male and female rats, clear evidence of carcinogenic 

discussed and with the conclusions as written for 

nickel subsulfide be accepted with the revisions 

Dr. Klaassen moved that the Technical Report on 
14 Nickel Subsulfide, NTP TR 453 
15 

INTRODUCTION 

Ni3S, 

NICKEL SUBSULFIDE 

Synonyms: Heazlewoodite, nickel subsulphide, nickel sulfide (3:2), &-nickel sulfide (3:2) crystalline, nickel sulphide, nickel tritadisulphide, 

Chemical Formula: Ni,S, Molecular Weight: 240.25 

CAS NO. 12035-72-2 

trinickel disulfide 

CHEMICAL AND PHYSICAL PROPERTIES 

from 2.0 to 2.2 pm. 

nickel subsulfide used in these 2-year studies ranged 

dynamic diameter at each exposure concentration of 

1986). The mean value for the mass median aeroinsoluble in water but soluble in acid (USEPA, 

point of 790" C and a density of 5.82 g/cm3. It is 

Nickel subsulfide is a black powder with a melting 

(900,000 metric tons) (U.S. Bureau of Mines, 1991). 

production of nickel is estimated at 1,000,000 tons 

is extracted from oxide ore. The total annual world 

used is extracted from sulfide ore, and the remainder 

Cuba. Approximately 55% of the nickel currently 

Canada, Australia, New Caledonia, Indonesia, and 

nickel producing countries were the Soviet Union, 

of Mines, 1984, 1985a). In 1991, the six largest 

landite) (NIOSH, 1977; Warner, 1984; U.S. Bureau 

marily as an oxide (laterite) or sulfide ore (pentNickel was first isolated in 1751 and is found priAND HUMAN EXPOSURE 

PRODUCTION, USE, 

(U.S. Bureau of Mines, 1991). The United States 

mately 200,000 tons (180,000 metric tons) annually 

The United States consumption of nickel is approxiexposure not reported) (NIOSH, 1991). 

to nickel oxide (information on nickel subsulfide 

are potentially exposed to nickel sulfate and 18,165 

Survey reported that 56,843 United States workers 

1984, 1985b). The National Occupational Exposure 

and other forms (2.1 %) (U.S. Bureau of Mines, 

(17.3%), nickel oxide (11.4%), nickel salts (1.2%), 

consumes unwrought nickel (68 %), ferronickel 

(U.S. Bureau of Mines, 1984). 

household appliances, machinery, and ships and boats 

and parts, construction, fabricated metal products, 

refining, electrical equipment and supplies, aircraft 

chemicals and allied products and in petroleum 

resistance and strength leads to its wide use in 

8% nickel. The ability of nickel to impart corrosion 

stainless steel (Warner, 1984), which contains up to 

Half of the nickel sold per year is used to make 

water include hydrated nickels (ATSDR, 1992). The 

nickel sulfate, and the most probable species found in 

sphere include complex nickel, nickel oxide, and 

most probable nickel species present in the atmoair has been found to range from 1 to 86 ng/m3. The 

than 10 ppb. Nickel concentration in United States 

tration of nickel in finished drinking water is less 

food, and household objects. The average concenLow levels of nickel are found in air, soil, water, 

Nickel constitutes about 0.008% of the earth's crust. 
16 Nickel Subsulfide, NTP TR 453 

(Fed. Regist., 1987). 

chemical in waste deposit sites in the United States 

1992). Nickel is listed as a frequently occurring 

drinking water is estimated at 2 pg per day (ATSDR, 

day (excluding cigarette smoke), and intake from 

from inhalation is estimated at 0.1 to 1 pg nickel per 

from foods is estimated at 170 pg per day, intake 

et al., 1985). Dietary intake of nickel per person 

fractions of cigarette smoke is 79 ngkigarette (Bache 

average amount of nickel in mainstream particulate 

day, 40 hours per week (NIOSH, 1977). 

averaged over a work shift of up to 10 hours per 

limit for nickel be reduced to 0.015 mg nickel/m3 

Health recommended that the permissible exposure 

The National Institute for Occupational Safety and 

water-insoluble nickel compounds (ACGIH, 1993). 

change to 0.05 mg nickel/m3 for water-soluble and 

but the ACGIH published notice of an intended 

insoluble salts and 0.1 mg/m3 for water-soluble salts, 

(ACGIH) are 1 mg/m3 for nickel metal and waterConference of Governmental Industrial Hygienists 

The threshold limit values adopted by the American 

rence of individual nickel species (ATSDR, 1992). 

trial exposure report total nickel and not the occurpresent, and most studies of environmental or industotal nickel without discerning the forms of nickel 

in the workplace. This method of analysis measures 

method for quantifying nickel in the environment and 

Atomic absorption spectroscopy is a widely used 

ABSORPTION, DISTRIBUTION, 

exposure (water-soluble forms of nickel). 

exposure (water-soluble forms of nickel), and dermal 

soluble and water-insoluble forms of nickel), oral 

excretion of nickel after inhalation exposure (waterinformation on the absorption, distribution, and 

Animal model systems have been used to obtain 

Experimental Animals 

AND EXCRETION 

temperature nickel oxide calcined at 250" C) in 

labeled nickel chloride or nickel oxide (low 

study after intratracheal administration of [63Ni]- 

et al. (1981) reported on a comparative toxicokinetic 

fate of specific forms of nickel in the lung. English 

one method used by several investigators to study the 

Intratracheal administration of nickel compounds was 

within 3 days. 

greater than 50% of the nickel cleared from the lungs 

chloride was rapidly excreted from the lung with 

sisted in the lung for more than 90 days, while nickel 

excreted in the feces and urine. Nickel oxide pernickel oxide administration, nickel was equally 

tion, was excreted primarily in the urine. After 

Wistar rats. Nickel, after nickel chloride administrain the urine (Carvalho and Ziemer, 1982). 

0.1 % on day 21 ; 96% of the chemical was excreted 

of their initial burden at day 1, and this decreased to 

eliminated from the lungs. The lungs retained 29% 

urine. By day 3, 90% of the instilled chemical was 

to Sprague-Dawley rats was excreted primarily in the 

Nickel chloride administered as an intratracheal dose 

et al., 1987). 

phase and 119 hours for the second phase (Finch 

with a clearance half-life of 2 hours for the first 

administered intratracheally to mice was also biphasic 

the calculated clearance times of nickel subsulfide 

(Valentine and Fisher, 1984). In another experiment, 

after 3 days with 33% appearing in the urine 

feces; 57% of the administered dose was excreted 

ured as 63Ni) was 60% in the urine and 40% in the 

respectively. The excretion of the chemical (meashalf-lives corresponding to 1.2 and 12.4 days, 

distinct components with initial and final biological 

istered nickel subsulfide (Ni3S2) in mice has two 

The pulmonary clearance of intratracheally admintissues (including blood, kidney, and intestine), and 

3 days, nickel was present in extrarespiratory tract 

administered to F344/N rats intratracheally was 1 to 

feces. The half-life of [63Ni]-labeled nickel sulfate 

kidney, and nickel was excreted in the urine and the 

extrarespiratory tract tissue including blood and 

nickel subsulfide exposure, nickel was detected in 

first few days after exposure. In contrast, after 

material was only excreted in the feces during the 

distributed to the extrarespiratory tract tissue, and the 

Following nickel oxide exposure, nickel was not 

5 days for nickel subsulfide (Benson et al., 1994). 

was estimated at 120 days for nickel oxide and 

subsulfide by pernasal inhalation, the lung half-life 

oxide (high temperature, green oxide) or nickel 

In F344/N rats administered [63Ni]-labeled nickel 
Introduction 17 

(Medinsky et al., 1987). 

urine was the major route for excretion of nickel 

1 year. 

to nickel oxide, a steady state would be reached after 

et al. (1980) estimated that with continuous exposure 

of 36 days (Hochrainer et al., 1980). Hochrainer 

half-life of 1 day and an alveolar clearance half-life 

750 pg/m3 to Wistar rats had a bronchial clearance 

deviation [oJ of 1.5) given as an aerosol of 

diameter ["AD] of 0.15 pm, geometric standard 

oxide (formed at 550" C; mass median aerodynamic 

relatively long half-life in the rodent lung. Nickel 

Other studies also indicated that nickel oxide has a 

tively (Tanaka et al., 1985, 1988). 

particulate MMADs of 0.6, 1.2, and 4.0 pm, respecbe 8, 11, and 21 months for nickel oxide with 

in Wistar rats exposed for 1 month were estimated to 

lives for nickel oxide (high temperature, green oxide) 

blood (Kodama et al., 1985). Lung clearance halfof nickel in the liver, kidney, spleen, heart, brain, or 

100 pg per year. There was no apparent deposition 

2 months, the lung clearance was estimated to be 

1.2 pm, og of 2.5) for 6 to 7 hours per day for 1 to 

oxide/m3 (high temperature, green oxide; "AD of 

In Wistar rats after exposure to 0.6 or 8.0 mg nickel 

nickel oxide. 

sulfate or nickel subsulfide, but not after exposure to 

extrarespiratory tract tissue after exposure to nickel 

100 days for nickel oxide. Nickel was detected in 

sulfate, 5 days for nickel subsulfide, and greater than 

exposure, the lung half-life was 1 to 3 days for nickel 

nickel administered intratracheally or by inhalation 

In summary, in absorption and distribution studies for 

(Benson et al., 1987; Dunnick et al., 1989). 

subsulfide, and the longest half-life for nickel oxide 

sulfate hexahydrate, a longer half-life for nickel 

show a relatively short half-life in the lung for nickel 

and 15 months in the 2-year studies. These data 

of rats and mice after 16 days, 13 weeks, and at 7 

subsulfide, and nickel oxide in the lungs and tissues 

deposition of nickel sulfate hexahydrate, nickel 

The present studies also report findings on the 

absorbed after oral administration of nickel sulfate 

mice, and dogs indicate that 1 % to 10% was 

Studies of other routes of nickel exposure in rats, 

et al., 1993). 

guinea pigs within 24 hours (ATSDR, 1992; Nielsen 

nickel chloride was absorbed through the skin of 

hexahydrate or nickel chloride, and less than 1 % of 

and Andersen, 1979; Boysen et al., 1984). 

workers were exposed was not identified (Torjussen 

although the specific form of nickel to which these 

retired workers years after cessation of employment, 

plasma, and nasal mucosa biopsies obtained from 

elevated nickel concentrations in specimens of urine, 

of time (Morgan and Rouge, 1983). There were 

depots in the body that retain nickel for long periods 

after facility closing, indicating that there are storage 

of refinery workers for periods of up to 6 months 

Oskarsson, 1991). Nickel was excreted in the urine 

(Bennet, 1984; Grandjean, 1984; Sunderman and 

absorbed into the blood from the respiratory tract 

1992); it is estimated that 35% of inhaled nickel is 

exposure in humans is by inhalation (Sunderman, 

In the industrial setting, a major route of nickel 

Humans 

for levels of total nickel. 

is monitored by analyzing urine, hair, or fingernails 

cancer. In the workplace setting, exposure to nickel 

lung at autopsy as those who died of other types of 

cancer had the same concentrations of nickel in the 

though workers who were diagnosed as having lung 

nickel in the lungs of nickel refinery workers, alAndersen and Svenes (1989) found elevated levels of 

sulfate to 3 % for nickel chloride (ATSDR, 1992). 

occluded skin varied from 55 % to 77% for nickel 

reported absorption of radioactive nickel applied to 

(Sunderman, 1989, 1992). In studies in humans, 

inated in either the feces or urine within 4 days 

28 hours; 100% of the absorbed nickel was elimelimination half-life for absorbed nickel averaged 

0.7 % was absorbed when administered in food. The 

absorbed when given in drinking water, while only 

human volunteers, 27 % of the administered dose was 

When nickel sulfate was administered to fasting 

TOXICITY 

1990; NIOSH, 1977; Brown and Sunderman, 1985; 

reviews (NAS, 1975; IARC, 1976, 1984, 1987, 

industrial exposure have been summarized in various 

Studies of nickel toxicity after experimental or 
18 Nickel Subsulfide, NTP TR 453 

occurred in the respiratory system. 

primary toxic response to nickel after inhalation 

1992). In experimental animals and in humans, the 

WHO, 1991; ATSDR, 1992; Nieboer and Nriagu, 

USEPA, 1986; European Chemical Industry, 1989; 

fluids. 

parallels the solubility of the compounds in biological 

hexahydrate, nickel subsulfide, and nickel oxide 

1983). The comparative toxicity of nickel sulfate 

human serum than nickel subsulfide (Kasprzak et al., 

hydroxide) have been shown to be more soluble in 

oxide in biological fluids. Soluble nickel salts (nickel 

water, nickel subsulfide is more soluble than nickel 

and nickel oxide are both relatively insoluble- in 

(Sunderman et al., 1987). While nickel subsulfide 

and greater than 11 years for nickel oxide 

dissolution half-life is 23 days for nickel subsulfide 

11 years, respectively. In rat serum, the estimated 

and nickel oxide in water are greater than 7 or 

lives (based on in vitro studies) for nickel subsulfide 

have been reported. The calculated dissolution halfsubsulfide and nickel oxide in water and rat serum 

Information on the dissolution half-lives for nickel 

place. 

or nickel subsulfide is more common in the workExposure to nickel oxide, nickel sulfate hexahydrate, 

manufacturing processes is limited (NAS, 1975). 

but the use or formation of this nickel compound in 

[Ni(CO),] is the most acutely toxic form of nickel, 

pounds are summarized in Table 1. Nickel carbonyl 

The acute toxicity values for selected nickel comExperimental Animals 

rats continuously exposed to nickel oxide at 200, 

increased erythrocyte count, were observed in Wistar 

count, increase in urine alkaline phosphatase, and 

related reduction in body weight, reduced leukocyte 

Changes in a variety of parameters, including dosetoxic response is seen in the respiratory system. 

and water-insoluble nickel compounds, the primary 

In animals, after inhalation exposure to water-soluble 

1980). 

cleaning and feeding periods) (Weischer et al., 

400, or 800 pglm3 for 120 days (except for daily 

et al., 1982; Murthy and Niklowitz, 1983). 

accumulation of phospholipid material (Migally 

membranous and lamellar structures consistent with 

contained osmophilic nickel oxide particles as well as 

cytoplasm of alveolar macrophages, phagosomes 

were increased in number and enlarged. In the 

controls, alveolar macrophages from exposed animals 

examined by electron microscopy. Compared to 

intratracheal injection (10 mg nickel oxide/mL) were 

12 hours per day, 6 days per week, for 28 days or by 

rats exposed to nickel oxide at 120 pg/m3 for 

Alveolar macrophages from lung lavage fluid from 

least toxic (Benson et al., 1989). 

hydrate was the most toxic, and nickel oxide was the 

histologic changes in the lung. Nickel sulfate hexachanges in lung lavage fluid paralleled the severity of 

The severity of lung toxicity as measured by the 

and mice exposed to the three nickel compounds. 

mation was observed histologically in the lung of rats 

differences in the magnitude of the response. Inflamnickel compounds, although there were quantitative 

fluid after exposure of rats and mice to all three 

and macrophage number were observed in the lavage 

in P-glucuronidase, total protein, neutrophil number, 

in lung lavage fluid (Benson et al., 1989). Increases 

looking for treatment-related markers of lung toxicity 

hexahydrate, or nickel subsulfide for 13 weeks and 

ing rats or mice to nickel oxide, nickel sulfate 

sialic acid. This evaluation was continued by exposglutathione reductase, glutathione peroxidase, and 

dehydrogenase, P-glucuronidase, total protein, 

the following parameters in lung lavage fluid: lactate 

hydrate, and nickel chloride, there were increases in 

exposure to nickel subsulfide, nickel sulfate hexawere seen after exposure to nickel oxide. After 

1986). No significant changes in lung lavage fluid 

related changes in lung lavage fluid (Benson et al., 

instillation was evaluated by examining treatmentride, nickel sulfate, or nickel oxide by intratracheal 

single dose of either nickel subsulfide, nickel chloRespiratory toxicity to F344/Crl rats administered a 
Introduction 19 

and Nickel Subsulfidea 

Toxicity Values for Nickel Carbonyl, Nickel Oxide, Nickel Sulfate Hexahydrate, Nickel Sulfate, 

TABLE 1 

Nickel Compound Species Route Toxicity Valueb 

Nickel carbonyl Rat 

Intraperitoneal 

Intravenous 

Subcutaneous 

Inhalation 

Mouse Inhalation 67 mg/m3 (LC,,]) 

Dog Inhalation 360 ppm (LCLo) 

Rabbit 

Cat 

Inhalation 

Inhalation 

73 g/m’ (LCLo) 

1.890 mglm’ (LC,,,) 

Nickel oxide Rat 

Intratracheal 

Intramuscular 

Subcutaneous 

Mouse 

Intraperitoneal 

Subcutaneous 

Nickel sulfate hexahydrate 

Intravenous 

Subcutaneous 

72 mglkg (LDLo) 

500 mglkg (LDLo) 

89 mg/kg (LDLo) 

500 mglkg (LDLo) 

Cat 

Intravenous 

Subcutaneous 

Rabbit 

Intravenous 

Subcutaneous 

36 mg/kg (LDLo) 

500 mglkg (LDLo) 

Guinea pig Subcutaneous 62 mg/kg (LDLo) 

Nickel sulfate Rat Intraperitoneal 500 mg/kg (LD,,) 

Mouse 

Intravenous 

Intraperitoneal 21 mglkg (LD,,) 

38 mglkg (LDLo) 

38 mglkg (LDLo) 

7 mglkg (LDLo) 

Intravenous 

Subcutaneous 

Rabbit 

Cat 

Intravenous 

Subcutaneous 

Subcutaneous 

33 mg/kg (LDLo) 

33 mglkg (LDLo) 

24 mglkg (LDLo) 

(continued) 
20 Nickel 'Subsulfide, NTP TH 453 

and Nickel Subsulfide (continued) 

Toxicity Values for Nickel Carbonyl, Nickel Oxide, Nickel Sulfate Hexahydrate, Nickel Sulfate, 

TABLE 1 

Nickel Compound Species Route Toxicity Value 

Nickel subsulfide Rat 

Intramuscular 

Intravenous 

Subcutaneous 

Inhalation 

20 mg/kg (TDLo) 

10 mg/kg (TDLo) 

125 mg/kg (TDLo) 

1 mg/kg (TCLo) 

Mouse Intramuscular 200 mg/kg (TDLo) 

a From RTECS (1987) 

TCLo = lowest toxic concentration; TDLo = lowest toxic dose. 

LC,, = median lethal concentration; LCLo = lowest lethal concentration; LD,,, = median lethal dose; LDLo = lowest lethal dose; 

ments (Table 2). 

been studied under various conditions and experilocal and systemic immunity, and this toxicity has 

water-insoluble nickel salts may cause an alteration of 

Treatment of rats and mice with water-soluble and 

1.8 mg nickel/m3 nickel subsulfide. Only mice 

exposed to nickel oxide and in mice exposed to 

forming cells were observed in the spleens of mice 

those in the controls. Low numbers of antibody1.8 mg nickel/m3 nickel subsulfide were greater than 

2.0 and 7.9 mg nickel/m3 nickel oxide and 

in lung-associated lymph nodes of mice exposed to 

nickel subsulfide. Numbers of antibody-forming cells 

and nickel oxide and to 0.45 and 1.8 mg nickel/m3 

highest concentrations of nickel sulfate hexahydrate 

lavage samples was increased in mice exposed to the 

compounds. The number of alveolar macrophages in 

nodes were increased in size after exposure to all 

than those of the controls. Lung-associated lymph 

1.8 mg nickel/m3 of nickel subsulfide were lower 

hexahydrate. Thymic weights in mice exposed to 

0.027, 0.11, and 0.45 mg nickel/m3 for nickel sulfate 

0.47, 2.0, and 7.9 mg nickel/m3 for nickel oxide; and 

0.45, and 1.8 mg nickel/m3 for nickel subsulfide; 

for 13 weeks. Exposure concentrations were 0.11, 

hexahydrate for 6 hours per day and 5 days per week 

nickel subsulfide, nickel oxide, or nickel sulfate 

sured in B6C3F, mice after inhalation exposure to 

Toxic responses to the immune system were mea- peritoqeal macrophages. 

nickel compounds affected the phagocytic activity of 

1.8 mg nickel/m3 nickel subsulfide. None of the 

macrophage phagocytic activity, as did 0.45 and 

of nickel oxide resulted in low levels of alveolar 

low mixed lymphocyte response. All concentrations 

exposed to 1.8 mg nickel/m3 nickel subsulfide had a 

trations (Haley et al., 1990). 

observed at occupationally relevant exposure concenof the nickel compound, and these effects were 

system, depending on dose and physicochemical form 

nickel can have varying effects on the immune 

Results indicate that inhalation exposure of mice to 

depressed natural killer cell activity in the spleen. 

Only 1.8 mg nickel/m3 nickel subsulfide caused a 

row and spleen of B6C3F, mice (Dieter et al., 1988). 

a depressed proliferating response in the bone marto deliver 1 16 to 396 mg/kg body weight) resulted in 

for 180 days (1 to 10 g/L drinking water, estimated 

Administration of nickel sulfate in the drinking water 

and the carcinogenic effects of nickel. 

relationship between effects on the immune system 

Further, there are no studies to examine if there is a 

not been determined (Nicklin and Nielsen, 1992). 

clinical significance of these studies in humans has 

potential of nickel to affect the immune system, the 

While xperimental studies in animals how the 
Introduction 21 

Studies on the Immunologic Effects of Nickel Compounds 

TABLE 2

Nickel Compound SpeciedRoute Treatment Response Reference 

Cell-Mediated Immunity 

Nickel chloride CBA/J mice/ Single injection, Reduced T-lymphocyte 

intramuscular 18 mg/kg proliferation 

Nickel sulfate 

Nickel subsulfide 

Nickel chloride 

Humoral Immunity 

( 1984) 

Smialowicz et al. 

Guinea pig 

B6C3F, mice Up to4.000 mg/kg/day Depressed spleen lymphoproliferative Dieter et al. (1988) 

on spleen cells Lange (1980) 

In vitro study Inhibited macrophage miration Hennighausen and 

Listeria challenge) 

in spleen cells; resistance to 

activity; PFC assay; mitogen response 

(female)/oral for 23 weeks response to LPS (no effect on NK 

monkey 

Cynomolgus 

intramuscular 

CBA/J mice/ 

intramuscular 

Swiss albino mice/ 

spleen cells 

C57BL16J mouse 

inhalation 

Swiss mice/ 

Nickel acetate 

ratdintraperitoneal 

Sprague-Dawley 

Nickel oxide 

inhalation 

Wistar rats/ 

0.06 pmol/g lung 

instillation 

Intratracheal 

cells (in lung) 

No effect on antibody-forming Haley et al. (1987) 

Single injection, Reduced antibody response to T-cell Smialowicz et al. 

18 mg/kg dependent sheep red blood cells ( 1984) 

to nickel chloride 

In vitro exposure Decreased response Lawrence (1981) 

sheep red blood cells 

immunization with 

weight followed by 

3-12 pg Nilkg body Depressed antibody formation Graham et al. (1975a) 

exposure at 250 pglm3 to sheep red blood cells 

2-hour inhalation Depressed antibody response Graham et al. (1978) 

bacteriophage 

immunized with E. coli antibody response 

11 mg/kg body weight Depressed circulating 

(1975) 

Figoni and Treagan 

25-800 pg/m3 for Decreased ability to form spleen Spiegelberg et al. 

4 weeks to 4 months antibodies to sheep red blood cells (1984) 

(continued) 
22 Nickel Subsulfide, NTP TR 453 

TABLE 2 

[Nickel Compound SpeciedRoute Treatment 

Studies on the Immunologic Effects of Nickel Compounds (continued) 

I 

Response Reference 

intramuscular 

Nickel chloride CBA/J mice/ 

Macrophage Function 

18 mglkg 

Single injection, 

Rabbits 

Nickel oxide and Wistar rats/ 

nickel chloride inhalation 

Nickel oxide 

inhalation 

Wistar rats/ 

monkey 

Nickel subsulfide Cynomolgus 

Natural Killer Cell Activity 

intramuscular 

C57BL/6J mice/ 

Nickel chloride CBA/J and 

in vitro exposure 

Alveolar macrophage 

2 weeks at 0.1 mglm’ 

6 days/week for 

12 houdday, 

800 pglm’ for 2 weeks 

0.06 pmollg lung 

instillation 

Intratracheal 

18 rnglkg 

Single injection, 

Reduced viability of macrophages Graham el al. (1975b) 

of peritoneal macrophages 

No effect on phagocytic capacity Smialowicz et al. 

( 1984) 

no change after nickel chloride 

macrophages after nickel oxide; 

Increased number of alveolar Bingham et al. (1972) 

phagocytic ability 

Decrease in alveolar macrophage Spiegelberg et al. 

( 1984) 

Lung macrophage activity decreased Haley et al. (1987) 

Depressed NK activity (against Smialowicz et al. 

Yac-1 murine lymphoma cells) (1984, 1985, 1986) 

Host Resistance 

inhalation 

nickel oxide Sprague-Dawley rats/ 

Nickel chloride and CD mice and 0.5 mg/m3 for 2 hours Enhanced respiratory infection to Adkins et al. (1979) 

Streptococcus 

sole causative agent of toxicity (ATSDR, 1992). 

always been possible to conclude that‘nickel is the 

toxic metals and/or cigarette smoke, and it has not 

rial setting, these workers are often exposed to other 

observed in workers exposed to nickel in the indust- 

(WHO, 1991). While respiratory toxicity has been 

pulmonary fibrosis in workers inhaling nickel dust 

rare (Nemery, 1990). There were also reports of 

sensitive allergic asthmatic reactions to nickel are 

sinusitis, and asal-septal perforations. Hypersures were occasionally associated with rhinitis, 

tory system. In the industrial setting, nickel exporefineries where the primary toxicity is to the respiracompounds came from studies of workers in nickel 

Most of the toxicity information on nickel and nickel 

Humans 

the exposed workers. 

inflammatory or fibrogenic responses in the lungs of 

sinter workers and found no evidence of significant 

Muir et al. (1993) reviewed X-rays of 745 former 

reticulocytes, or muscular pain in workers exposed to 

gastrointestinal effects, transient increases in blood 

vascular effects in a child ingesting nickel sulfate and 

well characterized. These reactions included cardioisolated cases where exposures to nickel were not 

reactions to nickel were reported in humans in 

of nickel toxicity (ATSDR, 1992). Other toxic 

containing jewelry and/or prosthesis is another form 

general population, contact sensitivity to nickelgeneral population and in exposed workers. In the 

Nickel contact hypersensitivity has been seen in the 
Introduction 23 

diseases has not been observed (Doll et al., 1990). 

from non-malignant respiratory effects or other 

between nickel exposure and cancer, excess mortality 

epidemiologic studies that have shown an association 

nickel-contaminated water (ATSDR, 1992). In 

CARCINOGENICITY 

Table 3. 

nogenic potential of nickel compounds is presented in 

and tabulation of over 100 experiments on the carciinjection site. A portion of the IARC (1990) listing 

been associated with a carcinogenic response at the 

nickel salts such as nickel sulfate have generally not 

neoplasms at the injection site, while the soluble 

subsulfide, nickel carbonyl, and nickel powder cause 

intramuscular injection). Nickel oxide, nickel 

pounds after local injection (e.g., subcutaneous or 

studies on the carcinogenic potential of nickel com- 

(IARC , 1990) summarized the results of experimental 

.The International 'Agency for Research on Cancer 

Experimental Animals . 

compared with a survival of 31 % in control groups. 

than 5% of the animals in exposed groups were alive 

At the end of the 108-week exposure period, fewer 

compared to less than 0.5 % of 21 5 control animals. 

12% to 14% of the 208 animals had lung tumors 

5 daydweek for 108 weeks). In the exposed groups, 

exposed to 1 mg/m3 by inhalation (6 hourdday and 

increased incidence in lung tumors in E344/N rats 

particles were between 1 and 1.5 pm) caused an 

particles were smaller than 1 pm in diameter; 25 % of 

et al. (1975) reported that nickel subsulfide (70% of 

hydrate by inhalation exposure is limited. Ottolenghi 

oxide, nickel subsulfide, and nickel sulfate hexaInformation on the carcinogenic potential of nickel 

resulting in a misleadingly low carcinoma incidence, 

that toxicity might decrease a carcinogenic response 

transplants in female F344 rats. These authors noted 

subsulfide/kg caused tumors in heterotrophic tracheal 

a single intratracheal dose of 1 or 3 mg nickel 

mucosa. Yarita and Nettesheim (1978) reported that 

potential of nickel subsulfide for the respiratory tract 

Other experimental studies indicated carcinogenic 

dence of toxic lesions). 

the 1 mg/kg group (generally with only a low inciplastic change) versus a 10% carcinoma incidence in 

mas (there was a high incidence of tracheal hyper- 

(3 mg/kg) caused only a 1.5 % incidence of carcinobased on the finding that the more toxic dose 

1971). 

carcinogenic effects of nickel (Furst and Schlauder, 

hamster may be 'less sensitive than the rat to the 

incidence of lung tumors (Wehner et al., 1975). The 

2.2) for 2 years did not have an increase in the 

diameter of 0.3 pm; geometric standard deviation of 

Hamsters exposed to 53 mg nickel oxide/m3 (median 

Donnelly , 1965). 

exposed to nickel carbonyl (Sunderman and 

showed a low incidence of lung tumors in rats 

3 timeslweek for 1 year). Follow-up studies also 

nickel carbonyl (0.03 to 0.25 mg/m3 for 30 minutes 

lung tumors in groups of Wistar rats exposed to 

Sunderman et al. (1959) found a low incidence of 

ment-related lesions were observed. 

100, 1,000, 2,500 ppm) for 2 years, and no treathexahydrate in the diet of Wistar rats or dogs (0, 

Ambrose et a2. (1976) administered nickel sulfate 

1975); the maximum tolerated dose was not reached. 

(Schroeder et al., 1964; Schroeder and Mitchener, 

tions (5 ppm) induced no lung lesions in Swiss mice 

Lifetime exposure to nickel acetate at low concentraafter oral administration is limited (IARC, 1990). 

Information on the carcinogenic potential of nickel 

(IARC, 1987). 

to different concentrations of nickel compound(s) 

compounds or the risk for cancer based on exposure 

to evaluate the carcinogenic risk for individual nickel 

although there was insufficient information available 

nickel compounds as human carcinogens (Group I), 

nickel refinery workers, IARC classified nickel and 

on the finding of lung and/or nasal sinus tumors in 

(IARC, 1976, 1987, 1990; Doll et al., 1990). Based 

ated with an increase in lung and nasal sinus tumors 

Exposure to nickel in the workplace has been associHumans 
24 Nickel Subsulfide, NTP TR 453 

in Experimental Animalsa 

Summary of Studies Used to Evaluate the Carcinogenicity of Nickel Compounds 

TABLE 3

Nickel Compound SpeciedRoute Lesion Incidenceb Reference 

Nickel oxides and hydroxides 

Nickel monoxide 

(green) 

Ratlinhalation 

8 mg/m3: 1/8 lung lesion 

0.6 mg/m3: 016 lung lesion Horie et af. (1985) 

Nickel monoxide Rathhalation 

0.2 mg/m3: 0/2O lesion 

0.06 mg/m’: 0/40 lesion Glaser et af. (1986) 

RaVintrapleural 

3 1/32 local lesions 

Controls: 0132 local lesions Skaug et al. (1985) 

Rathntratracheal 

10 X 15 mg: 12/38 lung lesions 

10 X 5 mg: 10/37 lung lesions 

Controls: 0/40 lesions 

21/32 local lesions 

Pott et af. (1987) 

Ratlintraperitoneal 

Radintraperitoneal 

Rat/subperiosteal 

Rathntramuscular 

Rat/intramuscular 

Rathntramuscular 

Ratlintramuscular 

Ratlintramuscular 

Pott et al. (1989, 1992) 

Pott et af. (1987) 

Berry et af. (1984) 

Berry et al. (1984) 

Sunderman and McCully (1983) 

Sosinski (1975) 

Gilman ( 1966) 

Gilman ( 1962) 

2/20 local lesions 

0120 local lesions 

14/15 local lesions 

OD0 local lesions 

0120 local lesions 

46/47 local lesions 

100 mg: 15/36 local lesions 

25 mg: 12/34 local lesions 

Nickel monoxide Sunderman er al. (1984) 

(green) 

Rauintrarenal 0/12 local lesions 

Hamstedintratracheal 

Hamstedinhalation 

Mouse/intramuscular 33/50 and 23/52 local lesions 

Farrell and Davis (1974) 

Wehner et af. (1975, 1979) 

Nickel monoxide Gilman (1962) 

1 /5 1 osteosarcoma 

1/49 lung lesions 

Controls: 4/50 lung lesions 

Nickel hydroxide 

Kasprzak et af. (1983) 

Gilman (1966) 

Rathntramuscular 

Rat/intramuscular 15/20 local lesions 

Colloidal: 0/13 local lesions 

Crystalline: 3/20 local lesions 

Dried gel: 5/19 local lesions 

0/10 local lesions 

Sosinski (1975) 

Nickel trioxide Judde et af. (1987) 

Ratlintracerebral 

Rat/intramuscular 

3/20 local lesions 

(continued) 
Introduction 25 

in Experimental Animals (continued) 

Summary of Studies Used to Evaluate the Carcinogenicity of Nickel Compounds 

TABLE 3

Nickel Compound SpecieslRoute Lesion Incidence Reference 

Nickel disulfide 

Nickel sulfides 

Nickel sulfide 

Nickel sulfide 

&Nickel sulfide 

(amorphous) 

Nickel sulfide 

&Nickel sulfide 

Nickel sulfide 

(amorphous) 

Nickel subsulfide 

(amorphous) 

Ratlintramuscular 

Ratlintrarenal 

Ratlintramuscular 

Ratlintramuscular 

Ratlintramuscular 

Ratlintrarenal 

Ratlintrarenal 

Ratlintrarenal 

Ratlinhalation 

Ratlintratracheal 

Rat/subcutaneous 

Ratlintrapleural 

Ratlintramuscular 

Ratlintramuscular 

Ratlsubcutaneous 

5.6 mg: 0/10 local lesions 

2/10 local lesions 

12/14 local lesions 

3/25 local lesions 

14/14 local lesions 

22.4 mg: 0/10 local lesions 

0/15 local lesions 

8/14 local lesions 

0/18 local lesions 

15/208 benign lung lesions 

14/208 malignant lung lesions 

3.75 mg: 12/40 lung lesions 

1.88 mg: 13/45 lung lesions 

0.94 mg: 7/47 lung lesions 

3.3 mg: 37/39 local lesions 

28/32 local lesions 

Controls: 

25/28 local lesions 

18/19 local lesions 

10 mg: 37/40 local lesions 

10 mg powder: 

1119 local lesion 

14/17 local lesions 

10 mg diffusion chamber: 

19/20 local lesions 

14/17 local lesions 

500 mg discs: 

5/7 local lesions 

500.mg fragments: 

Sunderman and Maenza (1976) 

Sunderman et al. (1984) 

Sunderman (1984) 

Sunderman (1984) 

Sunderman ( 1984) 

Sunderman et al. (1984) 

Sunderman et al. (1984) 

Jasmin and Riopelle (1976) 

Ottolenghi et al. (1975) 

Potter al. (1987) 

Mason (1972) 

Skaug et al. (1985) 

Gilman and Herchen (1963) 

Gilman (1 962) 

Shibata et al. (1989) 

(continued) 
Nickel Subsulfide, NTP TR 453 

in Experimental Animals (continued) 

Summary of Studies Used to Evaluate the Carcinogenicity of Nickel Compounds 

TABLE 3 

Nickel Compound SpeciedRoute Lesion Incidence Reference 

Nickel sulfides (continued) 

Nickel subsulfide 

(disc) 

Nickel subsulfide 

Nickel subsulfide 

a-Nickel subsulfide 

Ratlintramuscular 

Removal of disc after 206 days: 

Removal of disc after 128 days: 

Removal of disc after 64 days: 

10110 local lesions 

7/10 local lesions 

4/10 local lesions 

Ratlintramuscular 

Ratlintramuscular 

Ratlintramuscular 

Ratlintramuscular 

Rarlintramuscular 

Hooded: 14/23 local lesions 

NIH black: 28/28 local lesions 

10 mg: 34/40 local lesions 

3.3 mg: 38/39 local lesions 

Hooded: 11/20 local lesions 

Fischer: 59/63 local lesions 

20 mg: 9/9 local lesions 

5 mg: 8/20 local lesions 

5 mg: 29/30 local lesions 

2.5 mg: 28/30 local lesions 

1.2 mg: 23/30 local lesions 

0.6 mg: 7/30 local lesions 

Ratlintramuscular 

20 mg: 9/9 local lesions 

0.63 mg: 7/29 local lesions 

Rathtramuscular 9/9 local lesions 

Ratlintramuscular 10/20 local lesions 

Rathntramuscular 2/100 local lesions 

Rat/intramuscular 19/20 local lesions 

Ratlintraperitoneal 9/37 local lesions 

Herchen and Gilman (1964) 

Daniel (1966) 

Mason (1972) 

Sunderman and Maenza (1976) 

Yamashiro et al. (1980) 

Sunderman et al. (1976) 

Sunderman (1981) 

Gilman (1966) 

Shibata et al. (1989) 

Judde et al. (1987) 

Berry et al. (1984) 

Sunderman (1984) 

Ratlintraperitoneal 

Rathtraperitoneal 27/42 local lesions 

25 mg: 25/34 local lesions 

12 mg: 25/35 local lesions 

6 mg: 20/36 local lesions 

0120 local lesions Berry ef al. (1984) 

10120 local lesions Berry et al. (1984) 

In glycerin: 7/16 local lesions Jasmin and Riopelle (1976) 

In saline: 11/24 local lesions 

Pott et al. (1989, 1992) 

Pott et al. (1987) 

Rat/intrarenal 

Ratlintrafemoral 

Ratlsubperiosteal 

(continued) 
Introduction 27 

in Experimental Animals (continued) 

Summary of Studies Used to Evaluate the Carcinogenicity of Nickel Compounds 

TABLE 3 

Nickel Compound SpecieslRoute Lesion Incidence Reference 

Nickel sulfides (continued) 

a-Nickel subsulfide 

Nickel subsulfide 

Nickel subsulfide 

a-Nickel subsulfide 

a-Nickel subsulfide 

Rathntrarenal 

Radintraocular 

Radintratesticular 

Radtransplacental 

tracheal grafts 

into subcutaneous implanted 

Rat/pellet implantation 

Mouse/intratracheal 

Radintra-fat 

Radintra-articular 

Mousehbcutaneous 

Mousdintramuscular 

Mouselintramuscular 

Mouselintramuscular 

Hamster/intramuscular 

Hamstdintratracheal 

Hamsterkopical 

(continued) 

Nickel subsulfide Rabbithntramuscular 

Sunderman et al. (1979) 

Long-Evans: 0/12 local lesions 

Fischer 344: 9/32 local lesions 

NIH black: 6/12 local lesions 

Wistar Lewis: 711 1 local lesions 

14/15 local lesions 

16/19 local lesions 

No difference in lesion 

incidence 

15 mg: 45/64 local lesions 

5 mg: 9/60 local lesions 

No increase in lung 

9/20 local lesions 

16/19 local lesions 

lesion incidence 

C57B16: 5/10 local lesions 

10 mg: 4/8 local lesions 

5 mg: 4/8 local lesions 

C3H: 9/18 local lesions 

Swiss: 27/45 local lesions 

10 mg: 7/8 local lesions 

5 mg: 4/8 local lesions 

Controls: 0/14 local lesions 

0/62 lung lesions 

DBA/2: 6/10 local lesions 

10 mg: 12/17 local lesions 

5 mg: 4/15 local lesions 

1,080 mg total: 0113 local lesions 

540 mg total: 0/15 local lesions 

108 mg total: 017 local lesions 

54 mg total: 0/6 local lesions 

16 local lesions Hildebrand and Biserte (1979a,b) 

Damjanov et al. (1978) 

Sunderman (1983a) 

Albert et al. (1980); 

Sunderman et al. (1981) 

Yarita and Nettesheim (1978) 

Fisher e? af. (1986) 

Shibata et al. (1989) 

Shibata et al. (1989) 

Oskarsson et al. (1979) 

Gilman (1962) 

Oskarsson et al. (1979) 

Sunderman (1983a) 

Sunderman (1983b) 

Muhle et al. (1992) 

Sunderman (1983a) 
28 Nickel Subsulfide, NTP TR 453 

TABLE 3

in Experimental Animals (continued) 

Summary of Studies Used to Evaluate the Carcinogenicity of Nickel Compounds 

~~ ~~ 

Nickel Compound SpeciedRoute 

_____ ~~ 

Lesion Incidence 

________ ~~ 

Reference 

Nickel ferrosulfide 

Nickel subsulfide 

a-Nickel subsulfide 

Nickel sulfides (continued) 

Rathntrarenal 

Rat/intramuscular 

Salamander/intraocular 

Rabbitlintramuscular 

Basic nickel carbonate Rat/intraperitoneal 

Nickel salts 

tetrahydrate 

Nickel acetate Mouse/intraperitoneaI 

tetrahydrate 

Nickel acetate 

Nickel ammonium 

sulfate 

Nickel carbonate 

Nickel chloride 

hexahydrate 

Nickel chloride 

Nickel chromate 

Nickel fluoride 

Nickel sulfate 

1/12 local lesions 

15/15 local lesions 

7/8 local lesions 

0/4 local lesions 

50 mg: 3/33 lung lesions 

25 mg: 1/35 lung lesions 

Controls: 1/33 lung lesions 

360 mg: 12/19 lung lesions 

180 mg: 7/14 lung lesions 

72 mg: 8/18 lung lesions 

Sunderman et al. (1984) 

Sunderman (1984) 

Okamoto (1987) 

Sunderman (1983b) 

Pott et al. (1989, 1992) 

Stoner et al. (1976) 

Rathtramuscular 1/35 local lesions Payne (1964) 

1.5 lung lesionslanimal 

Mouse/intraperitoneaI Controls: 0.32 lung 

lesionslanimal 

Pokier et al. (1984) 

Rathntraperitoneal 

50 mg: 5/31 lung lesions 

25 mg: 3/35 lung lesions 

Controls: 1/33 lung lesions 

0/35 local lesions 

Pott et a!. (1989, 1992) 

Rathtramuscular Payne ( 1964) 

Rar/intramuscular 6/35 local lesions Payne ( 1964) 

Rathntraperitoneal 

Rat/intramuscular 

4/32 lung lesions 

Controls: 1/33 lung lesions 

0/35 local lesions 

Pott et al. (1989, 1992) 

Payne ( 1964) 

Rathtramuscular 1/16 local lesions Sunderman (1984) 

Rathntramuscular 

Rathtramuscular 

Rathntramuscular 

0/20 local lesions 

1/35 local lesions 

3/18 local lesions 

Gilman (1966) 

Payne (1964) 

Gilman (1966) 

Rathtramuscular 0/20 local lesions Kasprzak et al. (1983) 

(continued) 
Introduction 29 

in Experimental Animals (continued) 

Summary of Studies Used to Evaluate the Carcinogenicity of Nickel Compounds 

TABLE 3

Nickel Compound SpeciedRoute Lesion Incidence Reference 

Nickel salts (continued) 

hexahydrate 

Nickel sulfate Radintramuscular 0132 local lesions Gilman (1962) 

heptahydrate 

Nickel sulfate Radintraperitoneal 

6/30 lung lesions 

Controls: 1/33 lung lesions Pott et al. (1989, 1992) 

Other 

Nickel carbonyl Radinhalation 

1/32 pulmonary lesions 

60 mg/m' for 32 weeks: 

1/64 pulmonary lesions 

30 mg/m' for 32 weeks: 

1/80 pulmonary lesion 

250 mg/m' once: 

Sunderman et al. (1957, 1959) 

Radinhalation 

1/71 lung lesions 

Controls: 0/32 lung lesions Sunderman and Donnelly (1965) 

Radintravenous 19/120 lung lesions Lau et a/. (1972) 

a From IARC (1990) 

Number of animals with lesion per effective number 

throughout the world (Doll, 1984). 

nickel mines, or other nickel industrial operations 

Canada; or from other studies of nickel refineries, 

Nickel Company (INCO) refineries in Ontario, 

Wales; Kristiansand, Norway; the International 

in nickel refineries and mines in Clydach, South 

to nickel came from studies on occupational exposure 

Information on the hazards associated with exposure 

type of nickel exposures encountered based on 

Norseth, 1994). Doll et al. (1990) also estimate the 

Negusante, 1994; Nieboer and Templeton, 1994; 

more recent summaries (Courtin, 1994; McIlveen and 

world can be found in Doll et al. (1990) and in other 

in 10 different mines or refineries throughout the 

and the calcining, smelting, and refining operations 

tions. A complete analysis on the type of ore mined 

after exposure to nickel in mining or refinery operareviewed the epidemiological evidence for cancer 

Nickel Carcinogenesis in Man (Doll et al., 1990) 

(USEPA, 1986) and the International Committee on 

The United States Environmental Protection Agency 

studied did not have nickel-related cancers. 

cohorts of nickel workers, and many of the workers 

nickel. This study focused primarily on ''high-risk'' 

a few relatively recent measurements of total airborne 

knowledge of the nickel process procedures used and 

was particularly noted in refinery work involving 

An increased risk for lung and nasal sinus cancers 

exposure to nickel (USEPA, 1986; Doll et al., 1990). 

ments were made in factory design that reduced 

respirators and masks was introduced, and improveused to extract copper was reduced, the use of 

reduced, the arsenic contamination in sulfuric acid 

1925, the copper and sulfate content of the matte was 

in workers who were employed prior to 1925. After 

1933, nasal sinus and lung cancers were first noted 

opened in 1902 and used a nickel-copper matte. In 

Clydach Nickel Refinery (Mond Nickel Works) 

(Bridge, 1933; Doll, 1958; Morgan, 1958). The 

from refinery workers at Clydach, South Wales 

rise to lung and nasal sinus cancers was obtained 

The first indication that some form of nickel can give 
30 Nickel Subsulfide, NTP TR 453 

(Doll et al., 1990). 

converted impure nickel-copper matte to an oxide 

roasting, sintering, and calcining processes that 

concentrations were highest. 

workers in the reduction area where nickel carbonyl 

furnace areas. There was no increased risk to 

those workers employed in the copper sulfate and 

plant and found the highest incidence of cancer in 

nasal sinus cancers found in workers in the Clydach 

Pet0 et al. (1984) analyzed the incidence of lung and 

1990). 

cancer risk, but no nasal sinus cancers (Doll et al., 

mining operations in Ontario had an increase in lung 

is primarily petlandite (NiFeS,). Men working in 

and the Port Colborne refinery) that use an ore that 

include the Sudbury area mines (Copper Cliff Smelter 

1989a,b; Muir et al., 1994). Facilities operated 

Canada) mines and refineries (Roberts et al., 

from studies of workers in the INCO (Ontario, 

Other evidence for nasal sinus and lung cancer come 

1978). 

and urine nickel concentrations (H~rgetveit et al., 

electrolysis workers had the highest average plasma 

cancer (USEPA, 1986; Doll et al., 1990). The 

ting the soluble forms of nickel as the cause for the 

working in the electrolysis department, thus implicasinus and lung cancer risks were increased in men 

sulfate and nickel chloride. In this cohort, nasal 

Electrolysis workers are also exposed to nickel 

dust containing nickel subsulfide and nickel oxide. 

roasting and smelting operations are exposed to dry 

an electrolysis process to refine the ore. Workers in 

receives nickel-copper matte from Canada and uses 

The Falconbridge refinery in Kristiansand, Norway, 

more than a year (Doll et al., 1990). 

men exposed to sulfidic nickel at 4 mg nickel/m3 for 

evidence for an increased incidence in lung cancer in 

although the data from this cohort provided weak 

increased incidence in lung cancer in this population, 

Committee reported no clear evidence for an 

and/or nickel matte from New Caledonia. The Doll 

which received nickel-copper matte from Canada 

workers at a refinery in Huntington, West Virginia, 

Enterline and Marsh (1982) studied cancer rates in 

1990). 

cancer in this group of nickel workers (Doll et al., 

increased incidence in lung or upper respiratory tract 

Doll Committee also reported little evidence for an 

laterites including silicate and limonite ores. The 

et al., 1994). Nickel at this site is mined from nickel 

lung or upper respiratory tract cancers (Goldberg 

New Caledonia showed no increased incidence of 

nickel mining, smelting, and refinery operations in 

Results of epidemiology studies of workers in the 

Co., England (Doll et al., 1990). 

nickel refinery, Finland; and Henry Wiggin Alloy 

Gaseous Diffusion Plant, Tennessee; Outokumpu Oy 

the Hanna Nickel Smelting Co., Oregon; Oak Ridge 

West Virginia; and New Caledonia mines) as well as 

refinery, Kristiansand, Norway; Huntington Alloys, 

and Coniston], Ontario, Canada; Falconbridge 

mines and refineries [Copper Cliff, Port Colborne, 

Falconbridge Nickel Mines, Ontario, Canada; INCO 

(nickel refinery operations, Clydach, South Wales; 

Committee include the six cohorts mentioned above 

The ten cohorts of nickel workers studied by the Doll 

greater than 10 mg nickel/m3. 

exposure to less soluble forms at concentrations 

concentrations in excess of 1 mg nickel/m3 and to 

exposure to water-soluble nickel compounds at 

risks in ''high-risk'' cohorts are primarily related to 

from these studies suggests that respiratory cancer 

individual nickel species. However, the evidence 

provide exposure-specific estimates of risks for 

lung or nose. These investigators were not able to 

was likely to produce cancers elsewhere than in the 

exposure to metallic nickel or any of its compounds 

cancer risks. There was no evidence to suggest that 

was associated with increased lung and nasal sinus 

of nickel. There was no evidence that metallic nickel 

risks associated with exposure to less soluble forms 

lung and nasal sinus cancer and that it may enhance 

exposure to soluble nickel salts increased the risk of 

nasal sinus cancer risks. There was evidence that 

nickel was also associated with increased lung and 

nickel, exposure to large concentrations of oxidic 

oxidic and sulfidic nickel. In the absence of sulfidic 

cancer risk was attributed to exposure to a mixture of 

lung and nasal sinus cancer. Much of the respiratory 

gested that more than one form of nickel gives rise to 

the overall conclusion by the Doll Committee sugThe results within the individual cohorts varied, but 
Introduction 31 

sure to nickel (ATSDR, 1992). 

genic effect in humans specifically after oral expoThere are no studies evaluating the potential carcinoet al., 1991). 

marker of oxidatively damaged DNA (Littlefield 

8-hydroxy-2’-deoxyguanosine (8-OH-dG), a bioin vitro studies, nickel ions induce increases in 

radicals (Tkeshelashvili et al. , 1993); and 3) in 

by either ultraviolet irradiation or oxygen-free 

CC 4 TT mutations consistent with damage to DNA 

bacterial DNA, nickel ions produce tandem double 

et al., 1993); 2) in forward mutation assays with 

nickel (11)-thiol complexes and molecular oxygen (Shi 

radicals, suggesting free radicals are generated by 

generates hydroxyl radicals and carbon-centered alkyl 

of nickel ions with cysteine under aerobic conditions 

damage and cancer. Studies show that 1) incubation 

and the subsequent oxidative reactions lead to DNA 

studies suggest that nickel generates free radicals, 

intracellular oxidants (Costa et al., 1994). Recent 

proteins and/or the ability of nickel to generate 

ability of nickel ions to interact with chromatin 

cancer by nickel compounds may be related to the 

1992). The mechanisms involved in the induction of 

understood (Sunderman, 1989; Costa, 1991; Snow, 

mechanism for this carcinogenic activity is not fully 

the IARC as Group 1 (human) carcinogens, the 

While nickel and nickel compounds are classified by 

water-soluble nickel salts (Costa et al., 1994). 

than would be achieved by the cellular uptake of 

ions and results in a higher concentration of nickel 

whose acidity accelerates the dissolution of nickel 

cytized nickel particles are internalized in vacuoles 

passively diffuse through the cell membrane. Phagocytized than are the water-soluble nickel salts, which 

insoluble nickel compounds are more readily phagofindings may be related to the fact that waterpounds are the most potent carcinogens. These 

nickel compounds, the water-insoluble nickel comAfter subcutaneous or intramuscular injection of 

show that water-soluble nickel compounds adminLeonard and Jacquet (1984) reviewed studies which 

Experimental Animals 

AND DEVELOPMENTAL TOXICITY 

REPRODUCTIVE 

posed to in drinking water or in the diet. 

istered at higher doses than humans would be exstudies, the nickel compounds were generally admintial to cause embryotoxicity in rodents. In these 

istered orally or by peritoneal routes have the potenwater) (NAS, 1975). 

found in drinking water in the United States (48 pg/L 

are higher than the average concentration of nickel 

(Dostal et al., 1989). The doses used in these studies 

the milk and was found in the plasma of the pups 

23 mg/kg) to lactating rats, nickel was excreted in 

(10 to 100 pmol NiC1,- 6H,O/kg body weight or 

hexahydrate was given as a single subcutaneous dose 

phy (Olsen and Jonsen, 1979). When nickel chloride 

the embryo as measured by whole-body autoradiograta, and a marked uptake of nickel was observed in 

pregnant mice, nickel was found to cross the placen- 

[63Ni]-labeled nickel chloride was injected into 

brain, and heart. In another experiment, when 

various fetal tissues including liver, kidney, lung, 

weight) to pregnant mice, the label was detected in 

oral bolus dose (10 pmol or 0.58 mg/kg body 

[63Ni]-labeled nickel chloride was administered as an 

excreted in the milk of lactating animals. When 

nickel compounds can cross the placenta or be 

Studies in rodents have indicated that water-soluble 

1993). 

the proportion of dead pups per litter (Smith et al., 

gestation and lactation periods caused an increase in 

prior to mating and then during two successive 

of nickel compound) to female rats for 11 weeks 

(50 and 250 ppm, estimated to deliver 7 or 31 mg/kg 

Nickel chloride administered in the drinking water 

1985). 

maternal plasma glucose concentration (Mas et al., 

rhage. At these doses, there was an increase in 

cephalus, hydronephrosis, heart defects, and hemorskeletal retardation (poor ossification), hydroPorton rats on day 8, 12, or 16 of pregnancy caused 

2, or 4 mg/kg body weight) to pregnant Wistar 

death. Nickel chloride injected intraperitoneally (1, 

gestation also showed developmental toxicity or fetal 

by intramuscular or intraperitoneal injection during 

Other studies in rodents administered nickel chloride 

on day 8 of gestation to Fischer rats reduced the 

Nickel chloride injected intramuscularly (1 6 mg/kg) 
32 Nickel-Subsulfide, NTP TR 453 

day 8 (Gilani and Marano, 1980). 

phthalmia, and reduced body size when examined at 

abnormalities in the limb development, microembryo including exencephaly, everted viscera, 

and 4 after fertilization caused malformations in the 

doses of 0.02 to 0.8 mg per egg on days 0, 1, 2, 3, 

1978). Nickel chloride injected into chicken eggs at 

fetal body weights on day 20 (Sunderman et al., 

mean number of live pups per dam and diminished 

lungs, and hydronephrosis (Sunderman et al., 1983). 

ities including anophthalmia, microphthalmia, cystic 

live pups, and increased incidences of fetal abnormalincrease in fetal mortality, diminished body weight of 

tion (1 1 mg/kg) on day 7 of gestation caused an 

administered to pregnant dams by intravenous injecgroups (Sunderman et al., 1980). Nickel carbonyl 

neonatal mortality was increased in the xposed 

ments where dams were allowed to deliver the pups, 

palate, and fused ribs. In another series of experiobserved included cystic lung, exencephaly, cleft 

uated on day 15 of gestation. Teratogenic effects 

days 4, 5, 6, 7, or 8 of gestation; dams were evalcarbonyl by inhalation (0.06 mg/L for 15 minutes) on 

Groups of pregnant hamsters were exposed to nickel 

toxicity (Weischer et al., 1980). 

through 20. There was no evidence of embryoto 1.6 mg nickel/m3 by inhalation on gestation days 1 

In a study of nickel oxide, Wistar rats were exposed 

Jacquet, 1984). 

stand the mechanisms for these effects (Leonard and 

maternal toxicity. Further work is needed to undermammalian embryo, or indirect effects hrough 

mutagenic properties of nickel, direct effects on the 

nickel may be related to several factors including the 

barrier and enter the fetus. The embryotoxicity of 

in rodents. The metal can cross the feto-maternal 

nickel salts have the potential to cause embryotoxicity 

These and other studies how that water-soluble 

noted a suggested increased risk of pregnancy comwater-soluble nickel salts in electrolysis departments 

refinery workers in Russia who were exposed to 

(ATSDR, 1992). A preliminary study of nickel 

ductive effects in humans after exposure to nickel 

Until recently, there have been few studies of reproHumans 

1994). 

plications in female workers (Chashschin et al., 

GENETIC TOXICITY 

(Bryan, 1981; Evans et al., 1982) where they are 

once inside the cell, aggregate near the nucleus 

of nickel compounds. Insoluble nickel compounds, 

1982) are also important factors in the phagocytosis 

Mollenhauer, 1980) and surface charge (Costa et al., 

genetic damage. Particle size (Costa and 

efficiently phagocytized into the cell and cause 

crystalline, rather than in the amorphous state to be 

that the nickel sulfide compounds must be in the 

tured rodent cells, Costa and Heck (1983) concluded 

on the results of cell transformation studies in culmulate in high concentration within the cell. Based 

cannot be efficiently phagocytized and do not accuconcentration of nickel (11) ions. Soluble nickel salts 

the cell, and the consequent higher intracellular 

1991), longer retention of these compounds within 

nickels into the cell through phagocytosis (Costa, 

derives from the more efficient entry of insoluble 

forms of nickel. Presumably, this differential activity 

genetic toxicity assays than the soluble or amorphous 

crystalline nickel compounds are more active in 

Morita et al., 1991; Lee et al., 1993). Insoluble 

and Umeda, 1979; Amacher and Paillet, 1980; 

although these responses are usually weak (Nishimura 

ribosyl transferase locus in hamster V79 cells), 

in mouse lymphoma cells and hypoxanthine phosphomammalian cell forward mutation assays (TK locus 

addition, positive results were demonstrated in 

on DNA polymerases (Christie et al., 1991). In 

competing with magnesium for essential binding sites 

errors in mammalian cells in vitro, possibly by 

inhibit DNA replication and to increase replication 

strand breaks. Nickel salts have been shown to 

exchanges, chromosomal aberrations, and DNA 

malian cells inthe form of sister chromatid 

pounds that induce chromosomal damage in mamresults in in vitro assays designed to identify combacterial mutagens, but they often give positive 

Nickel compounds are not typically detected as 

Costa (1991), Snow (1992), and Costa et al. (1994). 

Coogan et al. (1989), Christie and Katisifis (1990), 

the production of these effects were presented by 

compounds and the possible mechanisms involved in 

Recent detailed reviews of the mutagenicity of nickel 
Introduction 33 

proceed to effect DNA damage (Costa et al., 1994). 

dissolved by lysosomes, releasing nickel (11) ions that 

described below. 

three nickel compounds under study by the NTP are 

in Figure 1. The genetic toxicity data for each of the 

of nickel ions within mammalian cells, is presented 1 

based upon the current understanding of the activities 

proposed mechanisms of nickel-induced genotoxicity, 

1991). A schematic representation of some of the 

cultured Chinese hamster cell lines (Klein et al., 

cells and to promote immortality in transformed 

controlling senescence in cultured Chinese hamster 

region were postulated to cause the loss of a gene 

X chromosome and subsequent deletions in this 

1991); binding of nickel ions to the long arm of the 

(Sen and Costa, 1986a,b; Sen et al., 1987; Costa, 

X chromosome in cultured Chinese hamster cells 

of the chromosomes such as the long arm of the 

Nickel binds preferentially to heterochromatic regions 

protein rather than directly to DNA (Costa, 1991). 

primarily from the binding of nickel to chromosomal 

genic activity of nickel (11) ions probably derives 

(Eichorn and Shin, 1968) and therefore, the mutato protein is far greater than to purified DNA 

Coogan et al. (1989). The binding affinity of nickel 

proteins, and other nuclear structures are reviewed by 

volved in the binding of nickel ions to DNA, nuclear 

Costa, 1985; Sen and Costa, 1986a). Factors inWetterhahn, 1982; Lee et al., 1982; Patierno and 

nickel (11)-induced DNA damage (Ciccarelli and 

crosslinks is implicated in the gneration of 

1994). The formation of persistent DNA-protein 

et al., 1992; Tkeshelashvili et al., 1993; Sugiyama, 

Kawanishi, 1989; Nieboer et al., 1989; Cotelle 

nuclear proteins (Biggart and Costa, 1986; Inoue and 

acids, glutathione, and amino acid side chains of 

the binding of nickel ions to ligands such as amino 

hydrogen peroxide or other oxidants subsequent to 

produced by the oxidation of Ni+’ to Ni+3 by 

lived reactive oxygen species inside the nucleus, 

mechanisms. It may follow the generation of shortbeen attributed to one or more of the following 

The DNA damage resulting from nickel exposure has 

nickel oxide did not result in growth inhibition due to 

in some in vitro test systems. Although exposure to 

however, there are clear indications of genotoxicity 

The mutagenicity data for nickel oxide are limited; 

tions of the metal (Popp et al., 1992). 

for example, were correlated with urine concentranoted after exposure to chromium-containing fumes, 

blood, whereas increased DNA-crosslink frequencies 

correlated with nickel concentrations in urine or 

sium. Also, the genetic effects noted were not 

well as other metals such as chromium and magneand welding fumes contain other nickel compounds as 

genetic endpoints is confounded because smelter dusts 

weak. The link between nickel oxide and these 

blood lymphocytes of workers, but the evidence is 

DNA-crosslinks (Costa et al., 1993) in peripheral 

chromosomal aberrations (Waksvik et al., 1984) and 

oxide-containing fumes or smelter dusts may induce 

epidemiology studies indicate that exposure to nickel 

testing insoluble nickel oxide. Data from human 

compounds and may not have been suitable for 

used in this test was designed for water-soluble 

Allison, 1972); however, the experimental protocol 

nickel oxide for 24, 48, or 72 hours (Paton and 

human fibroblast or leukocyte cultures exposed to 

induction of chromosomal aberrations was detected in 

nickel oxide activity (Kargacin et al., 1993). No 

site, the gpt gene, in V79 cells as the target for 

were reported in mutation assays using a different 

(Kargacin et al., 1993). However, positive effects 

in hamster V79 cells after exposure to nickel oxide 

bryo cells (Miura et al., 1989) or at the HPRT locus 

ouabain resistance locus in C3H/10T1,’ mouse emincrease in gene mutations was detected at the 

5 pg/mL nickel oxide (Costa et al., 1982). No 

in cycling Chinese hamster ovary cells incubated with 

(determined by flow cytometric analysis) was induced 

subtilis (Kanematsu et al., 1980), an S-phase block 

DNA damage in repair-deficient strains of Bacillus 

lethal mutations in germ cells of male Drosophila 

200, 300, and 400 ppm, induced sex-linked recessive 

hexahydrate, administered by injection at doses of 

(McGregor et al., 1988). In addition, nickel sulfate 

absence of S9 metabolic activation enzymes 

TK+’- cells treated with 500 to 1,000 pg/mL in the 

did induce mutations in L5178Y mouse lymphoma 

et al., 1993). However, nickel sulfate hexahydrate 

with nickel sulfate hexahydrate (Christie, 1989; Lee 

mutants were observed in hamster V79 cells treated 

results reported for nickel oxide) no increases in gpt 

ium (Arlauskas et al., 1985), and (in contrast to 

mutations in Escherichia coli or Salmonella typhimurNickel sulfate hexahydrate did not induce gene 
34 Nickel Subsulfide, NTP TR 453 

1978). 

cells was noted in treated animals (Mathur et al., 

Also, no change in the mitotic index of bone marrow 

that provided 3 or 6 mg nickel/kg body weight. 

after injection of nickel sulfate hexahydrate at doses 

served in rat bone marrow or spermatogonial cells 

no induction of chromosomal aberrations was obnickel sulfate hexahydrate (Fornace, 1982). In vivo, 

pigmentosum fibroblasts treated with 250 pg/mL 

strand breaks was detected in human xeroderma 

in vitro. However, no induction of DNA single 

et al., 1981) treated with nickel sulfate hexahydrate 

well as human peripheral lymphocytes (Larramendy 

(Larramendy et al., 1981; Ohno et al., 1982), as 

aberrations was observed in hamster cells 

tion of sister chromatid exchanges and chromosomal 

males (Rodriguez-Arnaiz and Ramos, 1986). Induchydrate (400 ppm) resulted in the production of X0 

loss, only the highest dose of nickel sulfate hexain male Drosophila, the test for sex chromosome 

lethal mutations. In another test for germ cell effects 

time of treatment showed no evidence of increased 

obtained from sperm cells undergoing meiosis at the 

post-meiotic cell stages were affected; the broods 

(Rodriguez-Arnaiz and Ramos, 1986). The pre- and 

Chinese hamster ovary cells for 24 hours with 

(Robison et al., 1983). Treatment of cultured 

repair in cultured Syrian hamster embryo cells 

Sonneborn et al., 1986) and unscheduled DNA 

in Paramecium tetraurelia, without S9 (Smith- 

(Arrouijal et al., 1990). It induced lethal mutations 

results in a standard plate incorporation assay 

tion protocol (Zeiger et al., 1992) and negative 

equivocal results in one study that used a preincubamutation assay, crystalline nickel subsulfide gave 

mainly positive. In the Salmonella typhimurium gene 

formed with this insoluble nickel compound were 

subsulfide. However, results of in vitro tests perthird nickel compound in the present studies, nickel 

there are limited published mutagenicity data for the 

As with the two nickel compounds discussed above, 

conducted. 

peritoneal injection), and no confirmatory study was 

(250 mg/kg nickel subsulfide administered by intrastudy, however, employed only a single dose 

positive results (Arrouijal et al., 1990). This second 

bone marrow micronucleus test, reportedly produced 

Sunderman, 1980). A second in vivo study, a mouse 

by intrarenal injection, was negative (Hui and 

rats administered 10 pg/rat (6 mg/100 g body weight) 

subsulfide, a measure of DNA synthesis inhibition in 

in vitro. One reported in vivo test with nickel 

(Arrouijal et al., 1992) in human lymphocytes 

aberrations (Arrouijal et al., 1990) and micronuclei 

significant dose-related increases in chromosomal 

(Saxholm et al., 1981). Nickel subsulfide induced 

tid exchanges incultured human lymphocytes 

ovary cells (Rossetto et al., 1994) and sister chromatransferase mutations in cultured Chinese hamster 

a weak inducer of hypoxanthine phosphoribosyl 

1982). Nickel subsulfide, in the absence of S9, was 

alkaline sucrose gradient techniques (Robison et al., 

the number of DNA strand breaks detected by 

10 pg/mL nickel subsulfide resulted in an increase in 

sulfide studies are presented in this Technical Report. 

nickel compounds. The results of the nickel subtoxicology and carcinogenicity information on these 

1996b) were performed to provide comparative 

subsulfide, and nickel sulfate hexahydrate (NTP, 

studies of nickel oxide (NTP, 1996a), nickel 

et al., 1975). The NTP toxicity and carcinogenicity 

which lung tumors were observed in rats (Ottolenghi 

was selected for study based on a previous study in 

the workplace in the United States. Nickel subsulfide 

selected as compounds that are commonly found in 

Nickel oxide and nickel sulfate hexahydrate were 

specific nickel compounds after inhalation exposure. 

mation on the toxic and carcinogenic properties of 

compounds for study because there was little inforThe National Cancer Institute nominated nickel 

STUDY RATIONALE 
Introduction 35 

0 

0 Ni+* 

(5) 

radicals oxygenated 

DNA’-/\b DNA 

et af., 1991). 

cause deletion of all or part of this region, leading to an immortalization of the cell and clonal expansion (Conway and Costa, 1989; Klein 

heterochromatic regions of the long arm of the X chromosome, which may contain a senescence gene and a tumor suppressor gene, can 

Ni+’ ions binding to heterochromatin (Lee et ai., 1982; Patierno and Costa, 1985; Sen and Costa, 1986a). 6. Binding of nickel ions to the 

(Ni+’ + Ni+’), releasing active oxygen species (Tkeshelashvili et al., 1993; Sugiyama, 1994). 5. DNA-protein crosslinks are produced by 

the nuclear membrane (Costa and Heck, 1983). 4. Oxidative damage is induced in DNA by nickel ions bound to nuclear proteins 

et al., 1982). 3. Lysosomal breakdown of insoluble nickel compounds releases large quantities of Ni+’ ions which concentrate adjacent to 

et al., 1993). 2. Insoluble nickel compounds such as nickel subsulfide are phagocytized into the cell and move toward the nucleus (Costa 

1. Soluble nickel compounds such as nickel chloride diffuse into the cell; Ni+2 ions are rapidly bound to cytoplasmic proteins (P) (Lee 

Possible Mechanisms of Nickel-Induced Genotoxicity 

FIGURE 1 
36 Nickel Subsulfide, NTP TR 453 
37 

MATERIALS AND METHODS 

PROCUREMENT 

Sciences (NIEHS). 

at he National Institute of Environmental Health 

in support of the nickel subsulfide studies are on file 

MO) (Appendix K). Reports on analyses performed 

laboratory, Midwest Research Institute (Kansas City, 

analyses were conducted by the analytical chemistry 

during the 2-year studies. Identity and purity 

three lots (identified as lot M082288) was used 

16-day and 13-week studies, and a blend of the 

Boston, MA). Lot HF-1517 was used during the 

micronizer prior to use (Sturtevant Mills, Inc., 

were subject to particle size reduction with a 4-inch 

three lots (HF-1517,1080786, and 1072588). All lots 

Nickel Company, Ltd. (Toronto, Ontario), in 

Nickel subsulfide was obtained from International 

OF NICKEL SUBSULFIDE 

AND CHARACTERIZATION 

phorus (335 ppm), and chromium (300 ppm). 

were silicon (1,200 ppm), iron (470 ppm), phosequal to 2,620 ppm; the major inorganic impurities 

spectrometry indicated total impurities of less than or 

at least 97 % . For lot M082288, spark source mass 

overall purity of lot HF- 15 17 was determined to be 

purity of 97.0% f 1.1 % nickel subsulfide. The 

chromatography. Chelometric titration indicated a 

sulfate, sulfite, and thiosulfate were detected by ion 

HF-15 17, only trace amounts (less than 70 ppm) of 

indicated approximately 0.08% water. For lot 

nickel subsulfide. Karl Fischer water analysis 

were in agreement with the theoretical values for 

slightly low. Elemental analysis results for sulfur 

both lots, elemental analysis values for nickel were 

consistent with that expected for the structure. For 

(Figure Kl), and the ultraviolet/visible spectrum was 

ance was observed with infrared spectroscopy 

matography, and chelometric titration. No absorbnitric acid, Karl Fischer water analysis, ion chroanalysis, melting point, spectroscopy, solubility in 

terized as high purity nickel subsulfide by elemental 

Thc chemical, a gray or black powder, was characof lot M082288 was determined to be at least 98 % . 

98.0 % f 1.7 % nickel subsulfide. The overall purity 

Chelometric titration indicated a purity of 

amber glass bottles at room temperature. 

ensure stability, the bulk chemical was stored in 

extreme conditions necessary for dissolution. To 

properties as evidenced by the high melting point and 

nickel subsulfide because of its inert refractoiy 

A chemical stability study was not performed for 

during the studies. 

change in purity of the bulk chemical was observed 

every 3 to 4 months during the 2-year studies. No 

Inc. (Golden, CO), prior to and after all studies and 

tal analysis was performed by Huffman Laboratories, 

Periodic monitoring of the bulk chemical by elemenAEROSOL GENERATION 

delivery system is shown in Figure K3. 

suspended particles in the enclosure. The aerosol 

was circulated through HEPA filters to remove 

tion assembly was enclosed in a walk-in hood. Air 

ered to the exposure chambers. The aerosol generato achieve the desired concentrations and was delivnickel subsulfide aerosol was mixed with diluting air 

reduce the electrical charge on the aerosol. The 

Kr-85 discharger was placed in the generator to 

fluid bed generators (FBGs) during all studies. A 

Nickel subsulfide aerosol was generated from 2-inch 

AND EXPOSURE SYSTEM 

rate was 5.99 to 21.63 (rats) and 12.02 to 

hour, respectively. In the 2-year studies, the air flow 

sponded to 10 f 2 and 12 * 2 air changes per 

flow rate in the 16-day and 13-week studies correwas monitored with calibrated rotameters. The air 

mice in these studies (Figure K4). The air flow rate 

Aberdeen, MD) were used to expose the rats and 

chambers (HI000 and H2000, Hazleton Systems, 

Stainless steel, multi-tiered, whole-body exposure 
38 Nickel Subsulfide, NTP TR 453 

to reduce the aerosol particle size. 

cyclone device was placed in the aerosol delivery line 

entered the chamber. During the 2-year studies, a 

aerosol entrance to further mix the aerosol as it 

with a flow rate of 60 ft3/min was placed below the 

2107FL-1002, Newark Electronics, Chicago, IL) 

the chamber, and a small boxer fan (Model WS 

diluted in a radial diluter prior to introduction into 

increase the uniformity of mixing, the aerosol was 

spatial variation of aerosol concentration and to 

18.06 (mice) air changes per hour. To reduce the 

MONITORING 

AEROSOL CONCENTRATION 

sure hour during the 13-week and 2-year studies. 

16-day studies and at least 5 minutes of every expoleast 10 minutes of every exposure hour during the 

RAM-S was used to monitor each chamber for at 

aerosol generation system during exposures. The 

concentrations and to determine the need to adjust the 

was used to monitor the stability of the aerosol 

monitor (Model RAM-S, GCA, Co., Bedford, MA) 

concentration was maintained. A continuous aerosol 

Figures K9 and K10. Good control of aerosol 

trations for the 2-year studies are presented in 

Figures K7 and K8. Weekly mean exposure concentrations for the 13-week studies are presented in 

6-hour filter sample. Daily mean exposure concenexposure day of the 2-year studies by collecting one 

ticles in the control chambers were monitored each 

background concentrations of total suspended parflow rate), during each 6-hour exposure day. The 

was determined from two 3-hour samples (4.5 L/min 

exposure chamber, except chamber 2 (0.15 mg/m3) 

three 2-hour samples (3 L/min flow rate) from each 

concentrations were determined gravimetrically from 

In the 13-week and 2-year studies, the aerosol 

CHARACTERIZATION 

CHAMBER ATMOSPHERE 

2-year studies. 

during the 13-week studies, and 8 minutes during the 

10 minutes during the 16-day studies, 15 minutes 

of the target (Tw), determined with a RAM-S, was 

aerosol concentration in the chambers to reach 90% 

from 0 % to 8.8 % for all chambers. The time for the 

the 2-year studies. The spatial variations ranged 

week of exposure, and was checked quarterly during 

in the chambers and with animals during the first 

sured prior to the start of the studies without animals 

concentration in the exposure chambers was mea- 

(Tables K1 through K4). Uniformity of aerosol 

standard deviation ranging from 1.63 to .47 

testing. 

observation for evidence of disease, and serologic 

randomly selected for parasite evaluation, gross 

five male and five female rats and mice were 

of the studies. Before initiation of the studies, 

and were approximately 7 weeks old on the first day 

tined for 19 to 20 days (rats) or 21 to 22 days (mice) 

approximately 4 weeks old. Animals were quaran- 

(Frederick, MD). On receipt, the rats and mice were 

obtained from Frederick Cancer Research Center 

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

16-DAY STUDIES 

the end of the studies. Details of the study design 

animals were weighed initially, on day 4 or 5, and at 

of the study and on day 5 for rats and mice. The 

ly. Clinical findings were recorded prior to the start 

out the study. Rats and mice were housed individualsure periods; water was available ad libitum throughFood was available ad libitum, except during expoweek for 12 exposure days during a 16-day period. 

target concentration) (10 minutes) per day, 5 days per 

exposed for 6 hours plus T, (time to reach 90% of 

3.65, or 7.33 mg nickel/m3). The animals were 

subsulfide/m3 (equivalent to 0, 0.44, 0.88, 1.83, 

concentrations of 0,0.6, 1.2, 2.5, 5, or 10 mg nickel 

were exposed to nickel subsulfide by inhalation at 

Groups of five male and five female rats and mice 

ranged from 1.65 to 2.99 pm with the geometric lung were analyzed to determine nickel content 

was similar for all exposure concentrations and 0.44, 1.83, or 7.33 mg nickel/m3). The kidney and 

as expressed as mass median aerodyriamic diameter 2.5, or 10 mg nickel subsulfide/m3 (equivalent to 0, 

studies for each exposure chamber. The particle size and three female rats and mice exposed to 0, 0.6, 

13-week studies and monthly during the 2-year A tissue burden study was conducted on three male 

cascade impactor once during the 16-day and 

Aerosol size distribution was determined with a and animal maintenance are summarized in Table 4. 
Materials and Methods 39 

found in Appendixes H and I. 

tissue burden studies in the lung and kidney are 

formula given by Keith et al. (1983). Results of the 

basis for each set of samples analyzed according to a 

the analytical method were calculated on a cumulative 

spectroscopy. Limits of detection and quantitation of 

content using -electrothermal atomic absorption 

Co., Bedford, MA) and analyzed for their nickel 

were then diluted with deionized water (Millipore 

and heated in a microwave oven. The digestates 

of nitric and sulfuric acids and hydrogen peroxide 

sure. Tissue samples were digested with a mixture 

exsanguination the morning following the last exporats and mice were killed using carbon monoxide and 

(Table 4). For determination of nickel concentration, 

examined. 

examined. Table 4 lists the tissues and organs 

and mice in the other exposure groups were also 

groups of rats and mice. Selected organs from rats 

5 (mice), and 10 mg nickel subsulfide/m3 exposure 

histopathologic examinations were performed on 0, 

stained with hematoxylin and eosin. Complete 

paraffin, sectioned to a thickness of 5 pm, and 

buffered formalin, processed, trimmed, embedded in 

examination were fixed and preserved in 10 % neutral 

and thymus were weighed. Tissues for microscopic 

brain, heart, right kidney, liver, lung, right testis, 

A necropsy was performed on all animals. The 

studies. 

exposure concentrations to be used in the 2-year 

nickel subsulfide and to determine the appropriate 

cumulative toxic effects of repeated exposure to 

The 13-week studies were conducted to evaluate the 

ISWEEK STUDIES 

analyses were performed on five male nd 

dence of disease. At the end of the studies, serologic 

parasite evaluation and gross observation for evifive female rats and mice were randomly selected for 

Before initiation of the studies, five male and 

mately 7 weeks old on the first day of exposure. 

19 (males) or 20 days (females) and were approxi4 weeks old. Animals were quarantined for 

(Frederick, MD). On receipt, the rats and mice were 

obtained from Frederick Cancer Research Facility 

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

of the NTP Sentinel Animal Program (Appendix M). 

five female control rats and mice using the protocols 

are summarized in Table 4. 

Details of the study design and animal maintenance 

weekly thereafter, and at the end of the studies. 

weights were recorded prior to the start of the study, 

housed individually. Clinical findings and body 

ad libitum throughout the study. Rats and mice were 

during exposure periods; water was available 

holidays). Feed was available ad libitum, except 

5 days per week for 13 weeks (excluding three 

exposed for 6 hours plus T, (15 minutes) per day, 

0.88, or 1.83 mg nickel/m3). The animals were 

subsulfide/m3 (equivalent o 0, 0.11, 0.22, 0.44, 

trations of 0, 0.15, 0.3, 0.6, 1.2, or 2.5 mg nickel 

exposed to nickel subsulfide by inhalation at concenGroups of 10 male and 10 female rats and mice were 

found in Appendixes H and I. 

kidney, and testes of rats and the lung of mice are 

Results of the tissue burden studies in the lung, 

used were those described for the 16-day studies. 

13 weeks of exposure. Tissue burden methodologies 

group were examined for nickel burden after 

of female rats and male and female mice in each 

examined for nickel burden at 13 weeks. The lungs 

and testes of six male rats in each group were also 

9, and 13 weeks of exposure (Table 4). The kidneys 

each group were examined for nickel burden after 4, 

1.83 mg nickel/m3). The lungs of six male rats in 

nickel subsulfide/m3 (equivalent to 0, 0.11, 0.44, or 

six female mice exposed to 0, 0.15, 0.6, or 2.5 mg 

18 male and 18 female rats and six male and 

In addition, a tissue burden study was conducted on 

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

thelial cells were determined and used to ascertain 

nucleated epithelial cells, and large squamous epicells were stained. Relative numbers of leukocytes, 

saline, if necessary, and samples of vaginal fluid and 

vaginal vaults of the females were moistened with 

7 consecutive days prior to the end of the studies, the 

NTP General Statement of Work (April, 1987). For 

Table 4. Methods used were those described in the 

groups. The parameters evaluated are listed in 

0.15, 0.6, and 2.5 mg nickel subsulfide/m3 exposure 

10 male and 10 female rats and mice from the 0, 

were performed at the end of the 13-week studies on 

Sperm morphology and vaginal cytology evaluations 
40 Nickel Subsulfide, NTP TR 453 

tion are given in Appendix J. 

tive tissue evaluations and estrous cycle characterizacounted with a hemacytometer. Results of reproducHomogenization-resistant spermatid nuclei were 

buffered saline containing 10 % dimethyl sulfoxide. 

nea and homogenizing the left testis in phosphatecount was determined by removing the tunica albugiquantify spermatogenesis, testicular spermatid head 

scopically with the aid of a hemacytometer. To 

65" C. Sperm density was then determined microbated in the saline solution and then heat fixed at 

'Cauda were finely minced, and the tissue was incuepididymis was placed in buffered saline solution. 

tion of sperm motility estimates, each right cauda 

fields per slide by two observers. Following compleand nonmotile spermatozoa were counted for five 

the buffer on the slides, and the numbers of motile 

sperm effluxing from the incision were dispersed in 

at the distal border of the cauda epididymis. The 

was applied to slides and a small incision was made 

Test yolk (rats) or modified Tyrode's buffer (mice) 

epididymal body (corpus epididymis) and weighed. 

(cauda epididymis) was then removed from the 

isolated and weighed. The tail of the epididymis 

right cauda, right testis, and right epididymis were 

for sperm morphology, count, and motility. The 

and metestrus). Male rats and mice were evaluated 

parameters measured are listed in Table 4. 

disc for reticulocyte quantitation. The hematology 

whole blood and new methylene blue and a Miller 

smears prepared by incubating equal volumes of 

counts were determined by light microscopy, using 

films stained with Wright's stain. Reticulocyte 

mined by light microscopic examination of blood 

morphologic evaluation of blood cells were determodel S-550. Leukocyte differential counts and 

tions were performed on a Coulter Electronics 

EDTA as the anticoagulant. Hematology determinanations was placed in tubes containing potassium 

hematology analysis. Blood for hematology determied from all surviving animals by cardiac puncture for 

At the end of the 13-week studies, blood was collectand mice were weighed. Tissues for microscopic 

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

morbidity or moribundity. The brain, heart, right 

and on any animals that were killed early due to 

exposure groups surviving to the end of the studies 

A necropsy was performed on all rats and mice in all 

examined. 

exposure groups. Table 4 lists the tissues and organs 

selected organs from rats and mice in the other 

and 2.5 mg nickel subsulfide/m3 rats and mice and on 

histopathologic examination was performed on all 0 

and stained with hematoxylin and eosin. A complete 

in paraffin, sectioned to a thickness of 5 to 6 pm, 

buffered formalin, processed and trimmed, embedded 

examination were fixed and preserved in 10 % neutral 

mice were evaluated for tissue burden. 

pathology, and another five male and five female 

evaluated for hematology parameters and histotissue burden; five male and five female mice were 

ated for hematology parameters, histopathology, and 

exposure, five male and five female rats were evaluevaluated for. tissue burden. After 15 months of 

five female mice from each exposure group were 

and, additionally, as many as five male and 

for histopathology; seven male and seven female rats 

and mice from each exposure group were evaluated 

7 months of exposure, five male and five female rats 

or 105 (mice) weeks, excluding holidays. After 

(8 minutes) per day, 5 days per week for 104 (rats) 

0.44, or 0.88 mg nickel/m3) for 6 hours plus T, 

or 1.2 mg nickel subsulfide/m3 (equivalent to 0, 

subsulfide by inhalation at concentrations of 0, 0.6, 

80 male and 80 female mice were exposed to nickel 

0, 0.11, or 0.73 mg nickel/m3) and groups of 

0, 0.15, or 1 mg nickel subsulfide/m3 (equivalent to 

to nickel subsulfide by inhalation at concentrations of 

Groups of 63 male and 63 female rats were exposed 

Study Design 

%YEAR STUDIES 

approximately 6 weeks old and mice approximately 

evaluations, and at the end of the studies. Rats were 

study start, at he 7-month and 15-month interim 

4 times during the study for viral screening: prior to 

evidence of disease. Serology samples were collected 

ed for parasite evaluation and gross observation for 

Five male and five female rats and mice were select12 days (mice) before the beginning of the studies. 

studies. Animals were quarantined for 10 (rats) or 

Laboratories (Gilroy, CA) for use in the 2-year 

female B6C3F, mice were obtained from Simonsen 

Taconic Farms (Germantown, NY) and male and 

Male and female F344/N rats were obtained from 

Source and Specification of Animals 
Materials and Methods 41 

nel Animal Program (Appendix M). 

studies according to the protocols of the NTP Sentihealth of the animals was monitored during the scopically are listed in Table 4. 

7 weeks old at the beginning of the studies. The organ were xamined. Tissues examined microlaboratory pathologist, and the pathology data were 

Microscopic evaluations were completed by the study 

racy, the slide and tissue counts were verified, and 

animal records and tables were compared for accu- and contaminants is provided in Appendix L. 

given in Table 4. Information on feed composition 

and pathology tables were evaluated by an indepen- the study. Further details of animal maintenance are dent quality assessment laboratory. The individual 

audit. The slides, individual animal data records, periods; water was available ad libitum throughout 

for inventory, slide/block match, and wet tissue 

was available ad libitum, except during exposure 

residual wet tissues were sent to the NTP Archives chambers were cleaned and rotated weekly. Feed 

System. The microscopic slides, paraffin blocks, and Rats and mice were housed individually. Cages and 

entered into the Toxicology Data Management Animal Maintenance 

of the studies. 

the first 13 weeks, monthly thereafter, and at the end 

and body weights were recorded initially, weekly for 

toxicity, mortality, or moribundity. Clinical findings 

The animals were observed twice daily for signs of 

Clinical Examinations and Pathology 

are listed in Table 4. 

anticoagulant. The hematology parameters measured 

placed in tubes containing potassium EDTA as the 

blood was drawn from the retro-orbital sinus and 

mice were anesthetized with carbon dioxide and 

At 15 months, five male and five female rats and 

Appendixes H and I. 

The results of the tissue burden studies are found in 

using the same methods used in the 16-day studies. 

1.2 mg/m3 after 7 and 15 months and were analyzed 

or 1 mg/m3 and from mice exposed to 0, 0.6, or 

burden were collected from rats exposed to 0, 0.15, 

Lung and kidney samples for determination of tissue 

adrenal gland, kidney, ovary), samples from each 

microscopic examination. For all paired organs (i.e., 

6 pm, and stained with hematoxylin and eosin for 

embedded in paraffin, sectioned to a thickness of 5 to 

neutral buffered formalin, processed, trimmed, 

major tissues were fixed and preserved in 10% 

were examined for grossly visible lesions, and all 

were weighed. At necropsy, all organs and tissues 

and thymus of all animals at both interim evaluations 

mice. The brain, right kidney, liver, lung, spleen, 

A complete necropsy was performed on all rats and 

also reviewed. 

interstitial cell adenomas of the testis in rats, were 

all organs in rats and mice, with the exception of 

nonneoplastic lesions. All diagnosed neoplasms from 

bronchial lymph nodes in mice for all neoplastic and 

adrenal gland medulla in rats and the lung, nose, and 

lung, bronchial and mediastinal lymph nodes, and 

studies, a quality assessment pathologist reviewed the 

the histotechnique was evaluated. For the 2-year 

the guidelines of McConnell et al. (1 986). 

were evaluated separately or combined according to 

ogy data, the diagnosed lesions for each tissue type 

et al. (1985). For subsequent analyses of the patholby Maronpot and Boorman (1982) and Boorman 

these review procedures have been described, in part, 

contractor pathologists and the PWG. Details of 

Thus, the final diagnoses represent a consensus of 

laboratory pathologist, the diagnosis was changed. 

the PWG consensus differed from the opinion of the 

without any knowledge of exposure groups. When 

logic pathology. This group examined the tissues 

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

presented by the chair to the PWG for review. The 

pathologist, or lesions of general interest were 

ses between the laboratory and quality assessment 

administration, examples of disagreements in diagnocontaining examples of lesions related to chemical 

ogists existed. Representative histopathology slides 

between the laboratory and quality assessment patholother tissues for which a disagreement in diagnosis 

chair, who reviewed the selected tissues and any 

ted to the NTP Pathology Working Group (PWG) 

The quality assessment report and slides were submit- 
42 Nickel Subsulfide, NTP TR 453 

for the survival analyses are two sided. 

to identify dose-related trends. All reported P values 

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

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

Statistical analyses for possible dose-related effects 

dying from natural causes were not censored. 

were censored from the survival analyses; animals 

found dead of other than natural causes or missexed 

and is presented in the form of graphs. Animals 

product-limit procedure of Kaplan and Meier (1958) 

The probability of survival was estimated by the 

Survival Analyses 

STATISTICAL METHODS 

competing risks (Kaplan and Meier, 1958). 

of the study in the absence of mortality from all other 

incidence that would have been observed at the end 

i.e., the Kaplan-Meier estimate of the neoplasm 

rate for each group and each site-specific neoplasm, 

C3, and D3 also give the survival-adjusted neoplasm 

which a necropsy was performed. Tables A3, B3, 

nominators consist of the number of animals on 

occurrence (e.g., leukemia or lymphoma), the deor when neoplasms had multiple potential sites of 

and mammary gland) before microscopic evaluation, 

certain tissues (e.g., skin, intestine, harderian gland, 

examination was required to detect neoplasms in 

microscopically. However, when macroscopic 

numbers of animals affected at each site examined 

an? D3) and all nonneoplastic lesions are given as the 

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

scopically. For calculation of statistical significance, 

number of animals with that site examined microsuch lesions at a specific anatomic site and the 

and D5 are given as the number of animals bearing 

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

The incidence of neoplasms or nonneoplastic lesions 

Calculation of Incidence 

Both linear and quadratic terms in time were incorpologistic function of chemical exposure and time. 

approach, neoplasm prevalence was modeled as a 

and thus did not affect the risk of death. In this 

ered as the result of death from an unrelated cause 

assumed that the diagnosed neoplasms were discovmethod used was logistic regression analysis, which 

not rapidly lethal. Thus, the primary statistical 

considered to be incidental to the cause of death or 

The majority ‘of neoplasms in these studies were 

Analysis of Neoplasm Incidences 

neoplasm incidence (McKnight and Crowley , 1984). 

ences also provides a comparison of the time-specific 

this comparison of the time-specific neoplasm prevalHaseman (1986). When neoplasms are incidental, 

further described and illustrated by Dime and 

prevalence analysis of Dime and Lagakos (1983), 

method of adjusting for intercurrent mortality is the 

score test for the regression coefficient of dose. This 

groups were compared on the basis of the likelihood 

The neoplasm incidence of exposed and control 

if the fit of the model was not significantly enhanced. 

rated initially, and the quadratic term was eliminated 

neoplasm-bearing animals. 

1979), procedures based on the overall proportion of 

Armitage trend test (Armitage, 1971 ; Gart et al., 

plasms, and the Fisher exact test and the CochranTarone, 1975), appropriate for rapidly lethal neomethods include the life table test (Cox, 1972; 

tests are summarized in the appendixes. These 

statistical analysis were used, and the results of these 

In a.ddition to logistic regression, other methods of 

to Haseman ( 1984). 

further discussion of these statistical methods, refer 

to evaluate slected nonneoplastic lesions. For 

described in the preceding paragraphs were also used 

reported P values are one sided. The procedures 

were used in the analysis of neoplasm incidence, and 

overall dose-related trend. Continuity-corrected tests 

of each exposed group with controls and a test for an 

Tests of significance included pairwise comparisons 

proportion of affected animals. 

test was used, a procedure based on the overall 

detected at the interim evaluation, the Fisher exact 

function of chemical exposure and time. For lesions 

neoplastic lesion prevalence was modeled as a logistic 

was a logistic regression analysis in which nonnot rapidly lethal, the primary statistical analysis used 

considered to be incidental to the cause of death or 

Because all nonneoplastic lesions in this study were 

Analysis of Nonneoplastic Lesion Incidences 

approximately normal distributions, were analyzed 

ables. Organ and body weight data, which have 

control groups in the analysis of continuous varicance of pairwise comparisons between exposed and 

Two approaches were employed to assess the signifiAnalysis of Continuous Variables 
Materials and Methods 43 

exposure levels. 

simultaneous equality of measurements across 

(Morrison, 1976) to the transformed data to test for 

applying a multivariate nalysis of variance 

assumptions. Treatment effects were investigated by 

bring the data into closer conformance with normality 

estrous stage), an arcsine transformation was used to 

observation period that an animal was in a given 

cytology data are proportions (the proportion of the 

(Hollander and Wolfe, 1973). Because the vaginal 

significance using the Mann-Whitney U test 

test). Average severity values were analyzed for 

a monotonic dose-related trend (Dunnett’s or DUM’S 

pairwise comparisons than a test that does not assume 

(Williams’ or Shirley’s test) was more appropriate for 

trends and to deternine whether a trend-sensitive test 

was used to assess the significance of the dose-related 

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

multiple comparison methods of Shirley (1977) and 

distributions, were analyzed using the nonparametric 

tissue burden data, which have typically skewed 

Hematology, spermatid, epididymal spermatozoa, and 

of Dunnett (1955) and Williams (197 1, 1972). 

using the parametric multiple comparison procedures 

effects. 

for neoplasms appearing to show compound-related 

et al., 1984, 1985) are included in the NTP reports 

from the NTP historical control database (Haseman 

certain instances. Consequently, neoplasm incidence 

the overall assessment of neoplasm incidence in 

evaluation, historical control data can be helpful in 

first and most appropriate control group used for 

Although the concurrent control group is always the 

Historical Control Data 

QUALITY ASSURANCE METHODS 

at NIEHS. The audit findings were reviewed and 

findings are presented in the reports and are on file 

Report were conducted. Audit procedures and 

preliminary review draft of this NTP Technical 

pathology specimens, final pathology tables, and 

completeness and accuracy of the pathology data, 

assurance contractor. Separate audits covering 

audited retrospectively by an independent quality 

submitted to the NTP Archives, these studies were 

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

Laboratory Practice Regulations (21 CFR, Part 58). 

compliance with Food and Drug Administration Good 

The 13-week and 2-year studies were conducted in 

preparation of this Technical Report. 

resolved or were otherwise addressed during the 

assessed by NTP staff, so all comments had been 

GENETIC TOXICOLOGY 

studies and the results are given in Appendix E. 

cytes in peripheral blood. The protocols for these 

murium and the frequency of micronucleated erythromutations in various strains of Salmonella typhiby testing the ability of the chemical to induce 

The genetic toxicity of nickel subsulfide was assessed 

Straw, 1981; Crawford, 1985). 

the somatic mutation theory (Miller and Miller, 1977; 

electrophilic theory of. chemical carcinogenesis and 

to predict carcinogenicity in animals, based on the 

mechanisms of chemically induced DNA damage and 

toxicity tests were originally developed to study 

and in vivo genetic toxicity tests. These genetic 

and responses of the chemical in short-term in vitro 

genicity in experimental animals from the structure 

database that would permit the evaluation of carcinopart of a larger effort by the NTP to develop a 

The genetic toxicity studies of nickel subsulfide are 

yet defined. 

chromosome aberration or micronucleus tests is not 

genicity of a positive response in bone marrow 

Salmonella test alone. The predictivity for carcinoSalmonella test improved the predictivity of the 

That is, no battery of tests hat included the 

plementarity among the in vitro genetic toxicity tests. 

were rodent carcinogens), and that there is no comcarcinogenicity (89% of the Salmonella mutagens 

currently the most predictive in vitro test for rodent 

studies show that a positive response in Salmonella is 

the chemical being investigated. Data from NTP 

DNA and chromosome effects that can be induced by 

other tests can provide information on the types of 

et al., 1987; Zeiger et al., 1990), although these 

correlate well with rodent carcinogenicity (Tennant 

1991). Other in vitro genetic toxicity tests do not 

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

with the induction of carcinogenicity in rats and mice 

city and Salmonella mutagenicity is highly correlated 

genicity in rodents. The combination of electrophilireactivity), mutagenicity in Salmonella, and carcinopotential electrophilicity (structural alert to DNA 

There is a strong correlation between a chemical’s 
44 Nickel Subsulfide, NTP TR 453 

Experimental Design and Materials and Methods in the Inhalation Studies of Nickel Subsulfide 

TABLE 4 

16-Day Studies 13-Week Studies 2-Year Studies 

(Albuquerque, NM) 

Research Institute 

Lovelace Inhalation Toxicology 

Study Laboratory 

Mice: B6C3F, 

Rats: F344lN 

Strain and Species 

(Frederick, MD) 

Frederick Cancer Research Facility 

Animal Source 

Mice: 21 or 22 days 

Rats: 19 or 20 days 

Time Held Before Studies 

7 weeks 

Average Age When Studies Began 

Mice: 3 or 4 April 1985 

Rats: 1 or 2 April 1985 

Date of First Dose 

6 hourslday, 5 dayslweek for 16 days 

Duration of Dosing 

Mice: 18 or 19 April 1985 

Rats: 16 or 17 April 1985 

Date of Last Dose 

(Albuquerque, NM) 

Research Institute 

Lovelace Inhalation Toxicology 

Mice: B6C3F, 

Rats: F344lN 

(Frederick, MD) 

Frederick Cancer Research Facility 

19 days (males) or 20 days (females) 

7 weeks 

Mice: 2 December (males) or 

Rats: 25 November (males) or 

3 December 1985 (females) 

26 November 1985 (females) 

3 holidays) for 13 weeks 

6 hourslday, 5 dayslweek (excluding 

Mice: 4-6 March (males) or 

Rats: 25-26 February (males) or 

6-7 March 1986 (females) 

27-28 February 1985 (females) 

(Albuquerque, NM) 

Research Institute 

Lovelace Inhalation Toxicology 

Mice: 

Rats: 

Mice: 

Rats: 

Mice: 

Rats: 

Mice: 

Rats: 

Mice: 

Rats: 

B6C3F, 

F344/N 

(Gilroy, CA) 

Simonsen Laboratories 

(Germantown, NY) 

Taconic Farms 

12 days 

10 days 

I weeks 

6 weeks 

29 September 1988 

31 October 1988 

holidays) 

or 105 weeks (mice) (excluding 

6 hours/day, 5 daydweek for 104 (rats) 

Mice: 28 September 1990 

Rats: 26 October 1990 
Materials and Methods 45 

(continued) 

Experimental Design and Materials and Methods in the Inhalation Studies of Nickel Subsulfide 

TABLE 4 

16-Day Studies 13-Week Studies 2-Year Studies 

Mice: 19 or 20 April 

Rats: 18 6r 19 April 

Necropsy Dates 

1985 

1985 

9 weeks 

Average Age at Necropsy 

Tissue burden study: 3 males and 

Core study: 5 males and 5 females 

Size of Study Groups 

3 females 

Rats: 26-27 February (males) or Rats 

7-8 March 1986 (females) 

(females) 

28 February and 1 March 1986 

Terminal sacrifice: 

15-Month interim evaluation: 

7-Month interim evaluation: 

2, 5-6 November 1990 (males) and 

31 January-1 February 1990 

17-18 May 1989 

(females) 

29 October-1 November 1990 

Mice: 5-6 March (males) or 

Mice 

Terminal sacrifice: 

15-Month interim evaluation: 

7-Month interim evaluation: 

5-9 October 1990 (males) or 

17-18 January 1990 

12-13 April 1989 

1-4 October 1990 (females) 

20 weeks 

Tissue burden study: 

Core study: 10 males and 10 females 

6 male and 6 female mice 

18 male and 18 female rats; 

Rats 

7-Month interim evaluation: 

72 weeks 

35 weeks 

7-Month interim evaluation: 

Terminal sacrifice: 11 1-1 12 weeks 

1.5-Month interim evaluation: 

Mice 

75 weeks 

35 weeks 

Terminal sacrifice: 112 weeks 

15-Month interim evaluation: 

7-Month interim evaluation: 

Core study: 

5 males and 5 females 

15-Month interim evaluation: 

5 males and 5 females 

61 (0 mg/m’), 60 (0.6 mg/m’), and 

53 male and 53 female rats; 

2-Year study: 

60 (1.2 mglm’) male and 58, 60, 

7-MOnth interim evaluation: 

Tissue burden study: 

and 60 female mice 

as many as 5 male and 

7 male and 7 female rats; 

5 female mice 

15-Month interim evaluation: 

5 males and 5 females 
46 Nickel Subsulfide, NTP TR 453 

Experimental Design and Materials and Methods in the Inhalation Studies of Nickel Subsulfide 

TABLE 4 

(continued) 

16-Day Studies 13-Week Studies 2-Year Studies 

mean body weights. 

groups of approximately equal initial 

Animals were distributed randomly into Same as 16-day studies 

Method of Distribution 

1 

Animals per Cage 

chamber unit 

Toe clip, ear tag, and location within 

Method of Animal Identification 

weekly 

exposure periods, changed at least 

available ad libitum, except during 

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

NM-07 open formula meal diet 

Diet 

per day 

available ad libitum and checked twice 

(Edstrom Industries, Waterford, WI), 

supply) via automatic watering system 

Tap water (Albuquerque municipal , Water Distribution 

units rotated and changed weekly 

Systems, Inc., Aberdeen, MD); cage 

Stainless steel wire cages (Hazleton 

Cages 

MI); changed twice daily 

Specialties Paper, Inc., Kalamazoo, 

Techboard untreated paper (Shepherd 

Bedding/Cage Board 

as needed 

(Flanders, Washington, DC); changed 

High-efficiency particulate air filter 

Room/Chamber Air Supply Filter 

1 

chamber unit 

Toe clip, ear tag, and location within 

Same as 16day studies 

Same as 16day studies 

Same as 16day studies 

Same as 16day studies 

Same as 16day studies 

rotated weekly 

Products, Maywood, NJ), changed and 

stainless steel inhalation chambers (Lab 

Hazleton 1OOO and 2OOO multitiered, 

Chambers 

Same as 16day studies 

Same as 16-day studies 

1 

Mice: tail tattoo and ear tag 

Rats: tail tattoo 

needed and cleaned weekly 

exposure periods; troughs filled when 

available ad libitum, except during 

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

NIH-07 open formula meal diet 

Same as 16-day studies 

Same as 16day studies 

Same as 16day studies 

Same as 16day studies 

Same as 16day studies 
Materials and Methods 47 

(continued) 

Experimental Design and Materials and Methods in the Inhalation Studies of Nickel Subsulfide 

TABLE 4 

16-Day Studies 13-Week Studies 2-Year Studies 

Chamber air: 10 f 2 changedhow 

Fluorescent light: 12 hourslday 

'Relative humidity: 13% to 44% 

Temperature: 21.1" to.28.4" C 

Chamber Environment 

Doses 

nickellrn' 

0.88, 1.83, 3.65, or 7.33 mg 

10 mg nickel subsulfide/m' (0, 0.44, 

Core study: 0, 0.6, 1.2, 2.5, 5, or 

1.83, or 7.33 mg nickel/m') 

10 mg nickel subsulfide/m3 (0, 0.44. 

Tissue burden study: 0, 0.6, 2.5, or 

initially and after 5 days of exposure. 

Clinical observations were recorded 

exposure, and at the end of the studies. 

weighed initially, after 5 days of 

Observed twice daily; animals were 

Type and Frequency of Observation 

Intraperitoneal T,, euthanasia solution 

Method of Sacrifice 

and thymus. 

right kidney, liver, lung, right testis, 

Organs weighed were the brain, heart, 

Necropsy performed on all animals. 

Necropsy 

None 

Clinical Pathology 

Temperature: 18.6" to 24.1" C (rats), 

Chamber air: 12 f 2 changedhow 

Fluorescent light: 12 hours/day 

Relative humidity: 15% to 38% 

18.6" to 24.2" C (mice) 

nickellm') 

0.11. 0.22, 0.44, 0.88, or 1.83 mg 

2.5 mg nickel subsulfide/m' (0, 

Core study: 0, 0.15, 0.3, 0.6, 1.2, or 

0.11, 0.44, or 1.83 mg nickel/m3) 

2.5 mg nickel subsulfide/m' (0, 

Tissue burden study: 0, 0.15, 0.6, or 

and at the end of the studies. 

recorded initially, weekly thereafter, 

weighed and clinical observations were 

Observed twice daily; animals were 

anesthesia 

Exsanguination under halothane 

and thymus. 

right kidney, liver, lung, right testis, 

Organs weighed were the brain, heart, 

Necropsy performed on all animals. 

segmented neutrophils. 

erythrocytes, reticulocytes, and 

concentration, monocytes, nucleated 

volume, mean cell hemoglobin 

leukocytes, lymphocytes, mean cell 

erythrocytes, hematocrit, hemoglobin, 

Hematology: eosinophils, 

hematology analysis. 

by cardiac puncture after 13 weeks for 

Blood was collected from all animals 

Chamber air: 5.99 to 21.63 (rats) and 

Fluorescent light: 12 hourslday 

Relative humidity: 11 % to 93% 

Temperature: 17.6" to 30.5" C 

12.02 to 18.06 (mice) changeshour 

Rats: 0, 0.15, or 1 mg nickel 

burden study) 

nickel/m') (core study and tissue 

subsulfide/m' (0, 0.11, or 0.73 mg 

burden study) 

nickel/m3) (core study and tissue 

subsulfide/m' (0, 0.44, or 0.88 mg 

Mice: 0, 0.6, or 1.2 mg nickel 

the end of the studies. 

13 weeks, monthly thereafter, and at 

recorded initially, weekly for 

weighed and clinical observations were 

Observed twice daily; animals were 

anesthesia 

Exsanguination under carbon dioxide 

thymus. 

kidney, liver, lung, spleen, and 

interim evaluations were brain, right 

Organs weighed at the 7- and 15-month 

Necropsy performed on all animals. 

segmented neutrophils. 

monocytes, reticulocytes, and 

cell hemoglobin concentration, 

volume, mean cell hemoglobin, mean 

leukocytes, lymphocytes, mean cell 

erythrocytes, hematocrit, hemoglobin, 

Hematology: eosinophils, 

after 15 months of exposure. 

5 female rats and mice in each group 

retroorbital sinus of 5 male and 

Blood was collected from the 
48 Nickel Subsulfide, NTF' TR 453 

(continued) 

Experimental Design and Materials and Methods in the Inhalation Studies of Nickel Subsulfide 

TABLE 4 

16-Day Studies 13-Week Studies 2-Year Studies 

and respiratory tract lymph nodes. 

groups of rats and mice: lung, nose, 

examined from all other exposure 

and uterus. The following organs were 

thyroid gland, trachea, urinary bladder, 

glandular portions), testis, thymus, 

spleen, stomach (forestomach and 

salivary gland, seminal vesicle, skin, 

preputial gland (rats), prostate gland, 

parathyroid gland, pituitary gland, 

pancreas and pancreatic islets, 

(3 sections), ovary and oviduct, 

mesenteric), mammary gland, nose 

mandibular, mediastinal, and 

liver, lung, lymph nodes (bronchial, 

colon, and rectum), kidney, larynx, 

and ileum), large intestine (cecum, 

small intestine (duodenum, jejunum, 

esophagus, gallbladder (mice), heart, 

clitoral gland (rats), epididymis, 

bone marrow, nasal, and rib), brain, 

gland, bone (vertebra, spinal cord, 

tissues examined included: adrenal 

gross lesions and tissue masses, the 

10 mg/m3 rats and mice. In addition to 

performed on 0, 5 (mice), and 

Complete histopathology was 

Histopathology 

and respiratory tract lymph nodes. 

groups of rats and mice: lung, nose, 

examined from all other exposure 

and uterus. The following organs were 

thyroid gland, trachea, urinary bladder, 

glandular portions), testis, thymus, 

spleen, stomach (forestomach and 

salivary glands, seminal vesicles, skin, 

preputial gland (rats), prostate gland, 

parathyroid gland, pituitary gland, 

pancreas and pancreatic islets, 

(3 sections), ovary and oviduct, 

mesenteric), mammary gland, nose 

mandibular, mediastinal, and 

liver, lungs, lymph nodes (bronchial, 

colon, and rectum), kidneys, larynx, 

and ileum), large intestine (cecum, 

small intestine (duodenum, jejunum, 

esophagus, gallbladder (mice), heart, 

clitoral gland (rats), epididymis, 

bone marrow and rib of mice), brain, 

(vertebra, spinal cord, and femur and 

included: adrenal gland, bone 

tissue masses, the tissues examined 

mice. In addition to gross lesions and 

performed on 0 and 2.5 mg/m3 rats and 

Complete histopathology was 

urinary bladder, and uterus. 

vesicle, thymus, thyroid gland, trachea, 

testis with epididymis and seminal 

stomach (forestomach and glandular), 

spinal cord and sciatic nerve, spleen, 

prostate gland, salivary gland, skin, 

pituitary gland, preputial gland, 

ovary, pancreas, parathyroid gland, 

cavity and turbinates (3 sections), 

adjacent skin, muscle (rats), nasal 

and mediastinal), mammary gland and 

(mandibular, mesenteric, bronchial, 

mainstem bronchi, lymph nodes 

ileum), kidney, larynx, liver, lung and 

small intestine (duodenum, jejunum, 

large intestine (cecum, colon, rectum), 

gallbladder (mice), heart and aorta, 

eyes (if grossly abnormal), femur, 

(3 sections), clitoral gland, esophagus, 

included: adrenal gland, brain 

regional lymph nodes, tissues examined 

to gross lesions and tissue masses with 

examined. For all others, in addition 

mediastinal), and nasal turbinates were 

lymph nodes (bronchial and 

7 months, only the kidney, lung, 

15-month interim evaluations. At 

5 female rats and mice at the 7- and 

end of the studies and on 5 male and 

performed on all rats and mice at the 

Complete histopathology was 

and I) Appendixes H and I) 

Lung andlor kidney (see Appendixes H Lung, kidney, and/or testis (see 

Tissue Burden Analyses 

None 

Sperm Morphology and Vaginal Cytology Evaluations 

stages and estrous cycle length. 

were relative frequency of estrous 

evaluations. The parameters evaluated 

2.5 mg/m' groups for vaginal cytology 

female animals in the 0, 0.15, 0.6, and 

prior to the end of the studies from all 

collected for up to 7 consecutive days 

were weighed. Vaginal samples were 

cauda, right epididymis, and right testis 

morphology, and motility. The right 

meters evaluated were sperm density, 

morphology evaluations. The para2.5 mg/mJ groups for sperm 

animals in the 0, 0.15, 0.6, and 

samples were collected from all male 

At the end of the studies sperm 

and I) 

Lung and/or kidney (see Appendixes H 

None 
49 

RESULTS 

RATS 

 DAY STUDY 

females did not gain weight; the final mean body 10 mg/m3 females. 

10 mg/m3 lost weight, and 5 mg/m3 males and 5 and 10 mg/m3 females and dehydration in 5 and 

study (Table 5). Male and female rats exposed to included labored respiration in 10 mg/m3 males and 

on day 14; all other rats survived until the end of the day 5 of exposure, clinical findings of toxicity 

One male‘exposed to 10 mg nickel subsulfide/m3 died significantly lower than those of the controls. On 

weights of these groups and 2.5 mg/m3 females were 

Survival and Body Weights of Rats in the 16-Day Inhalation Study of Nickel Subsulfide 

TABLE 5

Mean Body Weightb (g) 

Change Relative to Controls 

Final Weight 

Dose Survivala Initial Final 

(mg/m3) (%I 

Male 

10 

5 

2.5 

1.2 

0.6 

0 

Female 

10 

5 

2.5 

1.2 

0.6 

0 

4/5c 

515 

515 

515 

515 

515 

515 

515 

515 

515 

515 

515 

158 f 4 

155 f 3 

160 f 2 

159 f 3 

159 f 4 

161 f 3 

113 f 2 

115 f 3 

116 f 2 

116 f 2 

116 f 1 

115 f 3 

113 f 5** 

155 f 8** 

198 f 6 

226 f 6 

236 f 4 

216 f 13 

86 f 1** 

117 f 6** 

138 f 1* 

147 f 5 

149 f 4 

151 f 4 

38f 6 

66f 5 

775 5 

55 f 15 

0 f 9** 

-45 f 5** 

22 f 1* 

31 f 3 

33 f 3 

36 f 3 

-28 f 2** 

2 f 6** 

52 

72 

92 

105 

109 

57 

78 

91 

97 

99 

a Number of animals surviving at 16 dayslnumber initially in group 

** P10.01 

* Significantly different (P10.05) from the control group by Williams’ or Dunnett’s test 

Day of death: 14. Final weights and weight changes are based on 5 animals. 

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

Absolute and relative lung weights of 2.5, 5, and (Table Fl). Absolute brain, liver, and thymus 

10 mg/m3 males and all exposed groups of females weights of 5 mg/m3 males and 10 mg/m3 males and 

were significantly greater than those of the controls; females and the relative thymus weight of 10 mg/m3 

the absolute lung weight of 1.2 mg/m3 males was males were significantly less than those of the 

also significantly greater than that of the controls controls. 
50 Nickel Subsulfide, NTP TR 453 

inflammatory response involved most of the airways 

bronchiolar epithelial cells. At 5 and 10 mg/m3, this 

vacuolation, necrosis, and detachment of some of the 

bronchial epithelium was increased. There was also 

bronchioles, and the prominence of goblet cells in the 

alveoli. There was mucus in the lumen of 

neutrophils in the lumen of terminal bronchioles and 

necrotic cellular debris, macrophages, and a few 

inflammation was characterized by the presence of 

of inflammation with necrosis. At 5 and 10 mg/m3, 

the lung of rats exposed to nickel subsulfide consisted 

males and females (Table 6). Microscopic lesions in 

ologic lesions were present in the lung and nose of 

during the 16-day study. Chemical-related histopaththymus size; rats in these groups did not gain weight 

females exposed to 5 or 10 mg/m3 was a decrease in 

The only other gross observation in males and 

were present in the lungs of most 10 mg/m3 rats. 

addition, gray or red foci up to 1 mm in diameter 

expected when the thoracic cavity is opened. In 

to 10 mg/m3 did not collapse to the extent normally 

At necropsy, the lungs of males and females exposed 

nodes. 

lymphoid depletion in the spleen, thymus, and lymph 

changes included testicular degeneration and 

that occurred during the course of the study. These 

reduction in body weight gain or body weight loss 

secondary to generalized toxicity and the marked 

exposure groups were considered nonspecific and 

lesions present in a few animals from the higher 

meatus in the nasal passages. Other histopathologic 

lial layer, primarily in the anterior of the dorsal 

Atrophy consisted of thinning of the olfactory epitheepithelium was present in all exposed groups. 

phages. Minimal to mild atrophy of the olfactory 

perivascular accumulation of lymphocytes and macromacrophages and interstitial infiltrates consisting of 

primarily of focal increases in the number of alveolar 

lesions in the lung were less diffuse and consisted 

0.6, 1.2, and 2.5 mg/m3 groups, the inflammatory 

and were considered to be nickel subsulfide. In the 

pigment were present within alveolar macrophages 

10 mg/m3, aggregates of black, slightly refractile 

throughout the lung. In rats exposed to 5 or 

Incidences of Selected Nonneoplastic Lesions in Rats in the 16-Day Inhalation Study of Nickel Subsulfide 

TABLE 6

0 mg/mJ 0.6 mg/m3 1.2 mg/m3 2.5 mg/m3 5 mg/m3 10 mg/m3 

Male J 

5 5 5 5 

0' 5** (1.O)C 5** (1.0) 5** (2.0) 5** (1.8) 5** (3.0) 

5 5 

Nose 

Olfactory Epithelium Atrophy 0 4* (1.0) 4* (1.0) 5** (1.0) 5** (1.0) 5** (1.0) 

5 5 5 5 5 5 

Female 

Lung 

Inflammation 0 5** (1.0) 5** (1.0) 

5 5 5 

5** (2.4) 

5 

5** (1.2) 5** (2.6) 

5 5 

Nose 

Olfactory Epithelium Atrophy 0 

5 5 5 5 5 5 

2 (1.0) 5** (1.0) 5** (2.0) 5** (1.2) 5** (1.2) 

a Number of animals with organ examined microscopically 

** PSO.01 

* Significantly different (P 50.05) from the control group by the Fisher exact test 

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

Number of animals with lesion 
Results 51 

kidneys of exposed males and females were 

(Tables 7 and Hl). Nickel concentrations in the 

significantly greater than those of the controls 

of these 2.5 and 10 mg/m3 males and females were 

exposure concentration, and the absolute lung weights 

greater than those of the controls and increased with 

10 mg/m3 males and females were significantly 

Nickel concentrations in the lungs of 0.6, 2.5, and 

exposure concentration for the 13-week study. 

10 mg/m3, 2.5 mg/m3 was selected as the highest 

the severity of lung lesions in rats exposed to 5 and 

less than that of the controls. (Table H2). Because of 

kidney weight of 10 mg/m3 males was significantly 

10 mg/m3 males and females; however, the absolute 

increased with exposure concentration in 2.5 and 

significantly greater than those of the controls and 

Lung Weight and Lung Burden in Rats in the 16-Day Inhalation Study of Nickel Subsulfidea 

TABLE 7 

0 mg/m3 0.6 mg/m3 2.5 mg/m3 10 mg/m3 

n 

Male 

pg Nilg control lung 

pg Nilg lung 

pg Ni/lung 

Absolute lung wt (g) 

Female 

pg Ni/g control lung 

pg Nilg lung 

pg Ni/lung 

Absolute lung wt (g) 

~ 

3 

0.827 f 0.009 - b 

0.810 f 0.169 - 

- 

- 

3 

1.000 f 0.000 

9 f 1.0* 

7 f 0.9* 

7 f 0.9* 

0.883 f 0.041 

12 f 2.0* 

9 f 1.3* 

8 f 1.3* 

3 

30 f 0.6** 

18 f 0.3** 

25 f 0.6** 

1.370 f 0.012** 

1.263 f 0.078* 

36 f 3.0** 

19 f 1.6** 

24 f 1.9** 

3 

127 f 3.3** 

67 f 8.7** 

103 f 2.9** 

1.577 f 0.150** 

133 f 13.3** 

77 f 12.1** 

87 f 9.3** 

1.167 f 0.087 

a Mean rt standard error; statistical tests were performed on unrounded data. 

** P<O.Ol 

* Significantly different (PsO.05) from the control group by Dunnett’s or Williams’ test (lung weight) or Shirley’s test (lung burden 

parameters) 

Results were below 0.093 pg Ni (the limit of detection). 
52 Nickel Subsulfide, NTP TR 453 

females during weeks 2 through 7. 

included labored respiration in 2.5 mg/m3 males and 

the controls. Chemical-related clinical findings 

of all other exposure groups were similar to those of 

than those of the controls; final mean body weights 

gain of 2.5 mg/m3 males were significantly lower 

The final mean body weight and mean body weight 

All rats survived until the end of the study (Table 8). 

WWEEK STUDY 

account for the neutrophilia. Increased tissue 

inflammation in the exposure groups, and this could 

There was microscopic evidence of pulmonary 

neutrophil counts in all exposed males and females. 

neutrophilia was indicated by greater segmented 

females were affected more than males. A mature 

general, there were minimal to mild differences, and 

Hematology results are presented in Table G1. In 

neutrophil count. 

controls and was a reflection of the increased 

in 2.5 mg/m3 males was greater than that in the 

resulting in neutrophilia. The total leukocyte c,ount 

can increase the intravascular life span of neutrophils 

increased bone marrow production and release and 

demand for granulocytes due to inflammation causes 

were minimally greater than those in the controls. 

trations in 1.2 and 2.5 mg/m3 males and females 

those of the controls. Mean cell hemoglobin concenmean cell volumes that were minimally less than 

controls. These differences were accompanied by 

females were minimally greater than those in the 

2.5 mg/m3 males and 0.3, 0.6, 1.2, and 2.5 mg/m3 

males and females, and erythrocyte counts in 1.2 and 

hemoglobin concentrations in 1.2 and 2.5 mg/m3 

Hematocrit values in 2.5 mg/m3 males and females, 

TABLE 8 

Survival and Body Weights of Rats in the 13-Week Inhalation Study of Nickel Subsulfide 

Mean Body Weightb (g) 

("/.I 

Change Relative to Controls 

Final Weight 

Dose Survivala Initial Final 

(mg/m3) 

Male 

0 10110 

0.15 10110 

0.3 10110 

0.6 10/10 

1.2 10110 

2.5 10/10 155 f 4 

157 f 3 

153 f 5 

149 f 6 

157 f 4 

157 f 4 

330 f 8* 

350 f 4 

338 f 7 

337 f 4 

355 f 6 

353 f 4 

174 f 6** 

193 f 4 

186 f 7 

188 * 4 

198 f 6 

:96 f 2 

Female 

0 10110 

0.15 10110 

0.3 10110 

0.6 10/10 

1.2 10/10 

2.5 10110 119 f 2 

118 f 2 

118 f 2 

121 f 3 

121 f 3 

118 f 3 

198 f 3 

201 f 3 

202 f 2 

209 f 4 

203 f 3 

200 f 3 

79 f 3 

83 f 2 

84 * 3 

87 * 3 

82 2 

83 f 3 

93 

99 

96 

95 

100 

99 

100 

101 

104 

101 

a Number of animals surviving at 13 weekshumber initially in group 

** P10.01 

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

Weights and weight changes are given as mean f standard error. 
Results 53 

lower mean cell volume). 

that occurred (e.g., the minimal erythrocytosis and 

metals and could account for some of the differences 

normal biological activities associated with transition 

the presence of excess nickel may have altered 

tion, and iron is essential for heme synthesis. Thus, 

erythrocytosis due to increased erythropoietin producare cobalt and iron. Cobalt excess can induce 

reticulocyte counts. Nickel is a transition metal, as 

panied by anemia or by corresponding differences in 

in nucleated erythrocyte counts were not accomwere greater than those in the controls. Differences 

erythrocyte counts in 1.2 and 2.5 mg/m3 females 

copper, and pyridoxine deficiency. Total nucleated 

ated with metabolic alterations related to iron, 

ropoiesis. Lower mean cell volume has been associerythrocytosis or with a very mild increase in erythaffected, and this is consistent with mild relative 

observed increases. Reticulocyte counts were not 

exposure groups, and this could account for the 

nary lesions were observed microscopically in all 

reduced atmospheric oxygen. In this study, pulmoerythrocyte/hemoglobin oxygen transport, and 

pulmonary or cardiovascular disease, altered 

Secondary erythrocytosis has been observed with 

result of tissue hypoxia (secondary erythrocytosis). 

or with increased erythropoietin production as the 

consistent with dehydration (relative erythrocytosis) 

concentrations, and erythrocyte counts could be 

bodies). The higher hematocrit values, hemoglobin 

or hematocrit, or an artifact (e.g., lipemia, Heinz 

in vitro), alterations in the hemoglobin concentration 

been related to erythrocyte hemolysis (in vivo or 

Increases in mean cell hemoglobin concentration have 

were observed (Table Jl). 

vaginal cytology between exposed and control rats 

No significant differences in sperm morphology or 

groups were significantly greater than those of the 

Absolute and relative lung weights of all exposed 

most 0.6, 1.2, and 2.5 mg/m3 males and females. 

region of the dorsal meatus of the nasal passages of 

cells. Olfactory epithelial atrophy was present in the 

amount of cytoplasm in the apical portion of the 

which was attributable primarily to a decrease in the 

in the normal thickness of this neuroepithelial layer, 

epithelium consisted of a minimal to mild reduction 

0.6, 1.2, or 2.5 mg/m3. Atrophy of the olfactory 

hyperplasia was present in groups exposed to 0.3, 

cortical region of the lymph nodes. Lymphoid 

the number of lymphocytes, primarily in the paraattributed to lymphoid hyperplasia and an increase in 

lymph nodes associated with the respiratory tract was 

around blood vessels. The gross enlargement of the 

minimal to mild interstitial infiltrates of lymphocytes 

Other inflammatory changes in the lung included 

mation, the alveolar type I1 cells were enlarged. 

neutrophils. Often within these focal areas of inflammatory cell infiltrate consisting of macrophages and 

focal thickening of alveolar septa from an inflamphages. At 0.3, 0.6, 1.2, and 2.5 mg/m’, there was 

minimal increase in the number of alveolar macromacrophage hyperplasia consisted primarily of a 

inflammatory lesions in the lung. At 0.15 mg/m3, 

sure-related increase in the incidence and severity of 

males and females (Table 9). There was an expochial and mediastinal ymph nodes, and noses of 

pathologic lesions were observed in the lungs, bronin one 0.3 mg/m3 female. Chemical-related histoMediastinal and bronchial lymph nodes were enlarged 

most males and females exposed to 0.6 mg/m3. 

or both of these lymph nodes was also observed in 

rats exposed to 1.2 or 2.5 mg/m3; enlargement of one 

of the bronchial and mediastinal lymph nodes of most 

with exposure concentration. There was enlargement 

incidence and the severity of this finding increased 

parenchyma of rats in all exposed groups. The 

2-mm, white foci were scattered throughout the lung 

and mediastinal lymph nodes. Numerous, 1- to 

gross lesions were present in the lung and bronchial 

controls (Table F2). At necropsy, chemical-related 
Nickel Subsulfide, NTP TIP 453 

of Nickel Subsulfide 

Incidences of Selected Nonneoplastic Lesions in Rats in the 13-Week Inhalation Study 

TABLE 9 

0 mg/m3 0.15 mg/m3 0.3 mg/m3 0.6 mglm' 1.2 mg/m3 2.5 mg/m3 

Lunga 

Male 

Inflammation, Chronic Active 0 2 (1.0) 9** (1.3) lo** (1.8) lo** (2.9) lo** (3.7) 

Interstitial Infiltrate 0 0 lo** (1.9) 9** (2.1) 8** (1.2) 

Alveolar Macrophage Hyperplasiab 0 lo** (1.1)' lo** (1.5) lo** (1.6) lo** (3.4) lo** (3.8) 

10 10 10 10 10 

0 0 1 (1.0) lo** (1.2) lo** (2.6) lo** (3.3) 

6 9 9 10 10 10 

1 (1.0) 

10 

Lymph Node, Bronchial 

Hyperplasia 

Lymph Node, Mediastinal 7 10 

0 0 

10 10 

Olfactory Epithelium Atrophy 0 0 1 (1.0) 5* (1.0) lo** (1 .O) lo** (1.6) 

10 10 10 10 

0 6* (1.5) 9** (2.5) 9** (2.6) 

9 9 9 9 

Hyperplasia 

Nose 

Female 

Lung 

Inflammation, Chronic Active 0 3 (1.0) 9** (1.0) lo** (1.9) lo** (2.6) lo** (3.8) 

Interstitial Infiltrate 0 0 2 (1.0) 9** (1.7) lo** (2.4) 5* (1.6) 

Alveolar Macrophage Hyperplasia 0 lo** (1 .O) lo** (1.7) lo** (1.8) lo** (2.9) lo** (3.8) 

10 10 10 

1 (1.0) 2 (1.0) 2 (1.0) 9** (1.2) 9** (2.8) 9** (3.2) 

Lymph Node, Bronchial 7 8 8 10 9 9 

Hyperplasia 

Lymph Node, Mediastinal 8 9 

0 0 

10 10 

Olfactory Epithelium Atrophy 0 0 0 8** (1.1) 9** (1.0) lo** (2.3) 

10 10 10 10 

1 (1.0) 8** (1.2) lo** (2.5) lo** (3.1) 

10 10 10 10 

Hyperplasia 

Nose 

a Number of animals with organ examined microscopically 

** P<O.Ol 

* Significantly different (PSO.05) from the control group by the Fisher exact test 

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

Number of animals with lesion 

Nickel concentrations in the lung increased with lungs of 0.15, 0.6, and 2.5 mg/m3 females were also 

exposure concentration (Tables 10 and H3). At 4, 9, significantly greater than that of the controls at 

and 13 weeks on study, nickel concentrations in the 13 weeks. The nickel concentration in the kidneys of 

lungs of 0.15, 0.6, and 2.5 mg/m3 males were 2.5 mg/m3 males was significantly greater than that 

significantly greater than those of the controls. of the controls, and the nickel concentration in the 

However, by 13 weeks, nickel concentrations had testes of 0.6 mg/m3 males was significantly less than 

reached a steady state. Nickel concentrations in the that of the controls (Table H4). 
Results 55 

Lung Weight and Lung Burden in Rats in the 13-Week Inhalation Study of Nickel Subsulfidea 

TABLE 10 

0 mg/m3 0.15 mg/m3 0.6 mg/m3 2.5 mg/m3 

Male 

n 6 6 6 6 

4 weeks 

pg Nilg control lung 

pg Nilg lung 

3 f 0.2** 

3 f 0.2** 6 f 0.7** 15 f 1.4** 

8 f 0.9** 23 f 2.2** 

9 weeks 

pg Nilg control lung 

pg Nilg lung 

13 weeks 

pg Nilg control lung 

pg Ni/g lung 

pg Nillung 

Absolute lung wt (8) 

6 f 0.2** 

5 f 0.2** 7 f 0.6** 16 f 1.0** 

12 f 1.2** 31 f 1.7** 

1.15 f 0.03 

- 

- 

- 

1.56 f 0.01** 

7 f 0.3** 

5 f 0.2** 

8 f 0.3** 

2.23 f 0.08** 2.38 f 0.03** 

17 f 1.0** 42 f 1.3** 

7 f 0.4** 18 f 0.5** 

14 f 0.9** 36 f 1.2** 

Female 

n 6 6 6 5 

13 weeks 

pg Nilg control lung 

pg Nilg lung 

pg Nillung 

Absolute lung wt (g) 0.849 f 0.026 - 

- 

- 

1.234 f 0.053** 

7 f 0.3** 

5 f 0.1** 

6 f 0.2** 

1.570 f 0.034** 1.607 f 0.045** 

11 f 0.3** 28 f 1.4** 

7 f 0.1** 17 f 0.7** 

13 f 0.4** 33 f 1.7** 

a Mean f standard error; statistical tests were performed on unrounded data. 

** Significantly different (P10.01) from the control group by Williams’ test (lung weight) or Shirley’s test (lung burden parameters) 

Results were below 0.218 pg Ni (the limit of detection). 

giving toxicological lesions comparable to the highest 

the highest nickel subsulfide exposure concentration replicate the study by Ottolenghi et al. (1975). 

in the lung at 0.3 mg/m3, 0.15 mg/m3 was selected as selected for the 2-year nickel subsulfide study to 

incidence and severity of chronic active inflammation sulfate hexahydrate 2-year studies; 1 mg/m3 was 

Dose Selection Rationale: Based on the increased exposure concentration in the nickel oxide and nickel 
56 Nickei Subsulfide, NTP TR 453 

of the controls. 

of exposed male and female rats was similar to that 

Kaplan-Meier survival curves in Figure 2. Survival 

and female rats are shown in Table 11 and in the 

Estimates of 2-year survival probabilities for male 

Survival 

%YEAR STUDY 

throughout the second year of the study. Chemical1 mg/m3 were lower than those of the controls 

Figure 3). Mean body weights of rats exposed to 

throughout the study (Tables 12 and 13 and 

0.15 mg/m3 were similar to those of the controls 

Mean body weights of males and females exposed to 

Body Weights and Clinical Findings 

breathing following exposure periods. 

related clinical findings included rapid and shallow 

reduced atmospheric oxygen. 

erythrocyte/hemoglobin oxygen transport, and 

with pulmonary or cardiovascular disease, altered 

sis). Secondary erythrocytosis has been observed 

as a result of tissue hypoxia (secondary erythrocytorocytosis) or with increased erythropoietin production 

could be consistent with dehydration (relative erythgreater than those in the control's. These differences 

erythrocyte count in 1 mg/m3 males were mildly 

concentrations in 1 mg/m3 males and females and the 

Table G2. Hematocrit values and hemoglobin 

Hematology results at 15 months are presented in 

Hematology 

Survival of Rats in the 2-Year Inhalation Study of Nickel Subsulfide 

TABLE 11 

Male 

Animals initially in study 63 63 63 

Mean survival (days)c 

Percent probability of survival at end of studyb 

Animals surviving to study termination 

Natural deaths 

Moribund 

15-Month interim evaluationa 

7-Month interim evaluationa 

25 

13 

5 

35 

5 

5 

635 663 

40 

21 

4 

28 

5 

5 

34 

18 

2 

33 

5 

5 

658 

Female 

Survival analysisd P=0.579N P=0.122N P=0.260N 

Animals initially in study 63 63 63 

Mean survival (days) 

Percent probability of survival at end of study 

Animals surviving to study termination 

Natural deaths 

Moribund 

Accidental deatha 

15-Month interim evaluationa 

7-Month interim evaluationa 

658 

47 

25 

4 

24 

0 

5 

5 

0 

5 

5 

674 

47 

25 

1 

27 

1 

5 

5 

670 

54 

28 

1 

23 

Survival analysis P=0.563N P= 1.000N P=0.633N 

a Censored from survival analyses 

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

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, 

Mean of all deaths (uncensored, censored, and terminal sacrifice) 

Kaplan-Meier determinations based on the number of animals alive on the first day of terminal sacrifice 
Results 57 

0.9 

4 

A 0.8 

L 

In 

5 0.7 

> 

z 

0 

g o.6 

2 

n 

0 

a 

2 0.5 m 

0.4 

0.3 

0.2 f I 

0 15 30 45 60 75 90 105 

I I I I I I 

WEEKS ON STUDY 

I .O 

0.9 

LL 

0 

0.4 

0.3 

0.2 

.............................. 

.................... 

... ... 

... ......... 

m OUC/YJ 

0 0.15 UC/Ul 

I 1 1 I 

105 15 30 45 60 75 90 I 1 

WEEKS ON STUDY 

!O 

0 

FIGURE 2 

Kaplan-Meier Survival Curves for Rats Administered Nickel Subsulfide by Inhalation for 2 Years 
58 Nickel Subsulfide, NTP TR 453 

Mean Body Weights and Survival of Male Rats in the 2-Year Inhalation Study of Nickel Subsulfide 

TABLE 12 

Weeks O malm3 

Study (g) Survivors (8) 

0.15 me/m3 

controls) Survivors 

1 me/m3 

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

controls) Survivors (g) 

101 

97 

93 

89 

85 

81 

77 

73 

69a 

65 

61 

57 

53 

49 

45 

41 

37 

33 

29a 

25 

21 

17 

13 

12 

11 

10 

9 

8 

7 

6 

5 

4 

3 

2 

1 

455 

47 1 

474 

487 

492 

498 

503 

503 

501 

498 

494 

487 

483 

476 

468 

452 

448 

433 

413 

40 1 

377 

360 

333 

325 

316 

304 

293 

279 

268 

253 

238 

217 

195 

166 

120 

Mean for weeks 

53-101 

14-52 

1-13 

488 

425 

254 

17 

23 

28 

32 

36 

41 

47 

50 

50 

55 

55 

55 

56 

57 

57 

57 

57 

58 

58 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

446 

45 8 

459 

462 

468 

476 

479 

478 

476 

473 

475 

466 

466 

458 

448 

436 

43 3 

416 

394 

385 

366 

35 1 

325 

314 

310 

298 

286 

273 

263 

249 

234 

213 

190 

162 

118 

468 

410 

249 

98 

98 

98 

98 

98 

99 

98 

98 

98 

97 

98 

98 

97 

97 

95 

95 

96 

95 

95 

95 

95 

96 

96 

97 

96 

96 

96 

97 

96 

95 

96 

97 

97 

97 

97 

96 

96 

98 

25 

28 

30 

38 

44 

47 

50 

51 

51 

57 

57 

58 

58 

58 

58 

58 

58 

58 

58 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

386 

405 

418 

427 

435 

444 

450 

45 1 

452 

45 1 

455 

448 

448 

441 

435 

424 

419 

403 

383 

37 1 

353 

337 

316 

305 

299 

290 

279 

264 

255 

242 

228 

210 

187 

160 

117 

436 

396 

242 

85 

86 

88 

88 

88 

89 

90 

90 

90 

91 

92 

92 

93 

93 

93 

94 

94 

93 

93 

93 

94 

94 

95 

94 

95 

95 

95 

95 

95 

96 

96 

97 

96 

96 

98 

89 

93 

95 

24 

27 

31 

36 

40 

44 

49 

50 

51 

56 

56 

58 

58 

58 

58 

58 

58 

58 

58 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

a Interim evaluations occurred during weeks 29 and 66. 
Results 59 

Mean Body Weights and Survival of Female Rats in the 2-Year Inhalation Study of Nickel Subsulfide 

TABLE 13 

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

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

Weeks 0 mg/m3 0.15 me/m3 1 mdm3 

101 

97 

93 

89 

85 

81 

77 

73 

69a 

65 

61 

57 

53 

49 

45 

41 

37 

33 

29a 

25 

21 

17 

13 

12 

11 

10 

9 

8 

7 

6 

5 

4 

3 

2 

1 

332 

336 

330 

331 

329 

325 

318 

3 14 

308 

304 

299 

287 

284 

274 

265 

253 

248 

238 

233 

228 

217 

210 

200 

195 

193 

186 

181 

175 

170 

164 

156 

146 

137 

124 

100 

Mean for weeks 

1-13 164 

14-52 24 1 

53-101 315 

29 

30 

37 

41 

42 

44 

45 

47 

48 

55 

55 

55 

56 

57 

57 

57 

58 

58 

58 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

320 

324 

314 

315 

318 

313 

3 10 

306 

300 

298 

294 

283 

277 

271 

260 

250 

243 

232 

228 

223 

213 

206 

195 

190 

189 

182 

178 

173 

168 

163 

156 

146 

136 

123 

100 

306 

236 

161 

96 

96 

95 

95 

97 

96 

98 

97 

97 

98 

98 

99 

98 

99 

98 

99 

98 

97 

98 

98 

98 

98 

98 

98 

98 

98 

98 

98 

99 

99 

100 

100 

99 

100 

100 

97 

98 

98 

29 

32 

34 

40 

45 

49 

50 

50 

50 

57 

58 

58 

58 

58 

58 

58 

58 

58 

58 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

260 

274 

276 

279 

282 

285 

286 

285 

283 

28 1 

278 

268 

266 

259 

252 

243 

239 

228 

222 

218 

208 

202 

192 

188 

185 

180 

175 

170 

166 

160 

153 

144 

134 

120 

99 

277 

230 

159 

78 

82 

84 

84 

86 

88 

90 

91 

92 

93 

93 

94 

94 

94 

95 

96 

96 

96 

95 

96 

96 

96 

96 

96 

96 

97 

97 

97 

97 

97 

98 

99 

98 

97 

99 

88 

95 

97 

32 

32 

34 

42 

45 

47 

49 

50 

51 

56 

57 

57 

57 

57 

57 

57 

58 

58 

58 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

63 

a Interim evaluations occurred during weeks 29 and 66. 
Nickel Subsulfide, NTP TR 453 

550 

500 

450 

0 

2 400 

x 

v) 

JSO 

0 

+ 

i;l so0 

r 

3 * 

2 200 

P 

n 250 

w 

I 

150 

100 

50 

...... ............ ........... 

.... 

A 

................ ... ............................................................ .................... ................. 

............ ............................ 

.. .. .. ............... 

R 

I 

A 1 YG/YJ 

0 0.1 5 YG/Y3 

. 0 YG/W 

I 

15 so 45 60 75 90 

WEEKS ON STUDY 

550 

.................. 

............................. 

..... 

.. 0 0.1 5 UC/Y3 

FEMALE RATS 

0 I I I I I 

1s 30 I 

1 

WEEKS ON STUDY 

45 60 75 90 

Growth Curves for Rats Administered Nickel Subsulfide by Inhalation for 2 Years 

mGURE 3 
Results 61 

rats and Appendix B for female rats. 

this section are presented in Appendix A for male 

historical incidences for the neoplasms mentioned in 

at least 5 % in at least one animal group, and 

primary neoplasms that occurred with an incidence of 

animal tumor diagnoses, statistical analyses of 

neoplasms and nonneoplastic lesions, individual 

and other organs. Summaries of the incidences of 

adrenal medulla, nose, and bronchial lymph node, 

neoplasms and/or nonneoplastic lesions of the lung, 

biologically noteworthy changes in the incidences of 

This section describes the statistically significant or 

Pathology and Statistical Analyses 

alveolar/bronchiolar adenoma or carcinoma (combronchiolar carcinoma in males and females, and 

alveolar/bronchiolar adenoma in males, alveolar/ 

sure-related increased trends in the incidences of 

the controls (Tables F3 and F4). There were expoat 15 months) were significantly greater than those of 

(except the relative lung weight of 0.15 mg/m3 males 

exposed males and females at 7 and 15 months 

Lung: The absolute and relative lung weights of 

panying inflammatory reaction. 

disproportionately severe compared to the accoma distance from inflammatory cell aggregates or were 

only used when proliferations were either located at 

can be stimulated by inflammation, this diagnosis was 

lumens (Plate 3). Because such proliferative changes 

occasionally formed small projections within alveolar 

areas where alveoli were lined by cuboidal cells that 

continuum toward neoplasia and consisted of discrete 

epithelial hyperplasia was part of the morphologic 

of these neoplasms at necropsy. Focal alveolar 

exposed rats obscured the gross identification of most 

ty of the inflammatory alterations in the lungs of the 

macroscopically in histologic specimens. The severiconnective tissue. Adenomas were usually visible 

prominent (10% or more) amount of dense fibrous 

and areas of squamous differentiation; several had a 

had incorporated inflammatory cells, cellular debris, 

to low columnar epithelial cells. A few adenomas 

solid structures of moderately pleomorphic cuboidal 

were discrete proliferations of alveolar, tubular, or 

(NTP, 1993). Most alveolar/bronchiolar adenomas 

other studies of inhaled particulates such as talc 

plasms have been seen in lung neoplasms in rats in 

ation seen in some of the alveolar/bronchiolar neofibrous tissue component and the squamous differentistratified squamous epithelium (Plates 1 and 2). The 

tissue component; four had areas of differentiation to 

carcinomas had a prominent (10% or more) fibrous 

found in control rats. Five alveolar/bronchiolar 

ations of alveolar/bronchiolar epithelium typically 

from the typical papillary or solid neoplastic proliferneoplasms in exposed rats differed morphologically 

deeply basophilic. Some of the alveolar/bronchiolar 

carcinomas were pleomorphic and often stained 

papillary, and solid formations. Neoplastic cells in 

or low columnar epithelium in mixtures of tubular, 

Most alveolar/bronchiolar carcinomas had cuboidal 

two 0.15 mg/m3 males (Tables 14, A5, and B5). 

observed in the lungs of three 1 mg/m3 females and 

1 mg/m3 female and squamous metaplasia was 

addition, one squamous cyst was diagnosed in a 

increased with increasing exposure concentration. In 

proteinosis, and bronchial lymphoid hyperplasia 

controls. The severity of macrophage hyperplasia, 

males were also significantly greater than those of the 

tial infiltration in exposed males and 1 mg/m3 fethan those of the controls. The incidences of interstihyperplasia, and fibrosis were significantly greater 

hyperplasia and proteinosis, bronchial lymphoid 

of chronic active inflammation, alveolar macrophage 

exposed males and females at 2 years, the incidences 

significantly greater than those of the controls. In 

and interstitial infiltration in exposed females were 
62 Nickel Subsulfide, NTP TR 453 

of Nickel Subsulfide 

Incidences of Neoplasms and Nonneoplastic Lesions of the Lung in Rats in the 2-Year Inhalation Study 

TABLE 14 

0 mg/m3 0.15 mg/m3 1 mg/m3 

Lunga 

7-Month Interim Evaluation 

Male 

Interstitium, Infiltration Cellular 

Bronchus, Hyperplasia, Lymphoid 

Alveolus, Proteinosis 

Alveolus, Hyperplasia, Macrophage 

Inflammation, Chronic Activeb 

Lung 

15-Month Interim Evaluation 

Interstitium, Infiltration Cellular 

Alveolus, Proteinosis 

Alveolus, Hyperplasia, Macrophage 

Inflammation, Chronic Active 

2-Year Study 

Squamous Metaplasia 

Interstitium, Infiltration Cellular 

Bronchus, Hyperplasia, Lymphoid 

Alveolus, Proteinosis 

Alveolus, Hyperplasia, Macrophage 

Alveolar Epithelium, Hyperplasia, Focal 

Inflammation, Chronic Active 

Fibrosis 

Lung 

5 

3 (1.0) 

0 

0 

0 

1 (1.O)C 

2 (1.0) 

0 

1 (1.0). 

0 

5 

0 

17 (1.2) 

0 

1 (1.0) 

9 (1.1) 

2 (3.0) 

9 (1.3) 

2 (2.0) 

53 

5 (1.0) 

1 (2.0) 

3 (1.0) 

5** (2.0) 

5* (1.2) 

5 

4 (1.3) 

5** (1.6) 

5* (2.0) 

5** (1.4) 

5 

2 (1.0) 

31* (1.6) 

lo** (1.7) 

36** (1.3) 

48** (2.4) 

6 (2.2) 

53** (2.5) 

48** (1.8) 

53 

5 (1.8) 

4* (1.0) 

5** (2.2) 

5** (3.4) 

5* (2.0) 

5 

4 (2.3) 

5** (4.0) 

5* (2.2) 

5** (1.2) 

5 

0 

39** (1.9) 

14** (2.1) 

51** (3.6) 

52** (3.0) 

11* (2.1) 

51** (2.6) 

40** (1.7) 

53 

Alveolar/bronchiolar Adenoma (Multiple) 0 0 2 

Alveolarlbronchiolar Adenoma 

(Single or Multiple) 0 3 6* 

Alveolar/bronchiolar Adenoma or Carcinomad 

Alveolar/bronchiolar Carcinoma 0 3 7* 

Logistic regression test" 

First incidence 

Terminal rateg 

Adjusted ratef 

Overall ratee 

0/13 (0%) 

0.0% 

0/53 (0%) 

P=O.O03 P=O.O20 

549 

1/21 (5%) 

19.7% 

6/53 (11%) 

P=O.OOl 

62 1 

7/18 (39%) 

48.1% 

11/53 (21 %) 

- I 

7-Month Interim Evaluation 

Female 

Interstitium, Infiltration Cellular 

Bronchus, Hyperplasia, Lymphoid 

Alveolus, Proteinosis 

Alveolus, Hyperplasia, Macrophage 

Inflammation, Chronic Active 

Lung 

2 (1.0) 

0 

0 

0 

0 

5 

5 (1.0) 

1 (1.0) 

2 (1.0) 

5** (2.0) 

5** (1.2) 

5 

5 (1.4) 

4* (1.0) 

5** (3.6) 

5** (2.6) 

5** (1.2) 

5 

(continued) 
Results 63 

of Nickel Subsulfide (continued) 

Incidences of Neoplasms and Nonneoplastic Lesions of the Lung in Rats in the 2-Year Inhalation Study 

TABLE 14 

O mg/m3 1 mg/m3 

15-Month Interim Evaluation 

Female (continued) 

Interstitium, Infiltration Cellular 

Bronchus, Hyperplasia, Lymphoid 

Alveolus, Proteinosis 

Alveolus, Hyperplasia, Macrophage 

Inflammation, Chronic Active 

Lung 

Lung 

2-Year Study 

Squamous Metaplasia 

Interstitium, Infiltration Cellular 

Bronchus, Hyperplasia, Lymphoid 

Alveolus, Proteinosis 

Alveolus, Hyperplasia, Macrophage 

Alveolar Epithelium, Hyperplasia, Focal 

Inflammation, Chronic Active 

Fibrosis 

cyst 

Alveolarlbronchiolar Carcinoma 

Alveolar/bronchiolar Carcinoma (Multiple) 

Alveolar/bronchiolar Adenomd 

(Single or Multiple) 

Squamous Cell Carcinoma 

Alveolarlbronchiolar Adenoma or Carcinod 

0 

0 

0 

1 

0 

5 

0 

28 

0 

2 

8 

2 

7 

0 

0 

53 

0 

2 

0 

2 

0 

4* (1.0) 

0 

5** (1.8) 

5* (2.6) 

5** (2.4) 

5 

(1.0) 

0 

36 (1.6)

15** (1.4) 

49** (2.1) 

51** (2.5) 

10* (2.1) 

51** (2.5) 

50** (1.7) 

0 

53 

0 

5 

(1.3) 

(1 .O) 

(1 .O) 

(2.0) 

(1.1) 

1 

5 

0 

5** (1.8) 

1 (1.0) 

5** (4.0) 

5* (2.6) 

5** (1.2) 

5 

3 (2.0) 

43** (1.5) 

18** (1.9) 

53** (3.7) 

52** (2.7) 

11** (1.6) 

51** (2.3) 

44** (1.9) 

1 (1.0) 

53 

1 

5 

0 

9* 

4 

Alveolar/bronchiolar Adenoma or Carcinoma or Squamous Cell Carcinomd*k 

Logistic regression test 

First incidence 

Terminal rate 

Adjusted rate 

Overall rate 

P=O.O30 

729 (T) 

2/25 (8%) 

8.0% 

2/53 (4%) 

P=O.142 

594 

4/25 (16%) 

20.0% 

6/53 (1 1 %) 

P=O.O31 

594 

6/28 (21 %) 

27.2 % 

9/53 (17%) 

* Significantly different (P sO.05) from the control group by the Fisher exact test (interim evaluations) or the logistic regression test 

(T)Terminal sacrifice 

C 

b 

a 

d 

h 

g 

f 

e 

I 

j 

k 

(2-year study) 

** PSO.01 

range 0%-10% 

Historical incidence for 2-year NTP inhalation studies: 8/700 (1.1% f 1.5%); range 0%-4%. Feed studies: 25/1,201 (2.1% f 2.2%); 

Includes one adenoma NOS, NOS = nonspecified site 

Not applicable; no neoplasms in animal group 

the pairwise comparisons between the controls and that exposure group. The logistic regression test regards these lesions as nonfatal. 

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

Observed incidence in animals surviving until the end of the study 

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

Number of animals with neoplasm per number of animals with organ examined microscopically 

(3.8% f 3.8%); range 0%-10% (includes squamous cell carcinoma). Feed studies: 39/1,200 (3.3% f 2.0%); range 0%-8% 

Historical incidence for 2-year NTP inhalation studies with untreated control groups (mean f standard deviation): 27/703 

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

Number of animals with lesion 

Number of animals with organ examined microscopically 
64 Nickel Subsulfide, NTP TR 453 

differentiated squamous epithelium. 

focal replacement of pneumocytes by wellkeratin. Squamous cell metaplasia consisted of a 

epithelium without atypia and a lumen containing 

a wall of well-differentiated stratified squamous 

rats. The squamous cyst in the 1 mg/m3 female had 

to the inflammatory processes in the lungs of exposed 

clefts. The fibrosis was considered to be secondary 

occasionally had slit-like spaces typical of cholesterol 

dense fibrous tissue within alveolar septa and lumens, 

septa. Fibrosis, consisting of varying amounts of 

vascular cuffs and infiltrating the adjacent alveolar 

phocytes with fewer macrophages forming periinfiltrates were minimal to mild aggregates of lymcytes around major bronchi. Interstitial cellular 

minimal to mild increase in the number of lymphopresent. Bronchial lymphoid hyperplasia was a 

extent of lung affected and by the amount of material 

lumens. Severity of proteinosis was judged by the 

philic granular or flocculent material with alveolar 

osis was a moderate to marked presence of eosinointravascular pool of circulating monocytes. Proteinsource of these macrophages was probably the 

containing abundant pale vacuolated cytoplasm. The 

variably distributed large intra-alveolar macrophages 

moderate severity in exposed rats and consisted of 

(Plate 4). Macrophage hyperplasia was typically of 

and cell debris, often subjacent to the pleura 

distributed intra-alveolar macrophages, neutrophils, 

ate severity in exposed rats and consisted of variably 

Chronic active inflammation was typically of moderof focal decreased numbers and thickness of the 

epithelium was minimal in exposed rats and consisted 

respiratory epithelium. Atrophy of the olfactory 

occasionally accompanied by focal hyperplasia of 

neutrophils and cell debris within the nasal lumen, 

lamina propria. With increased severity, there were 

cytes, occasionally including plasma cells, in the 

consisted of infiltrates of neutrophils and lymphoChronic active inflammation varied in extent and 

meatus. 

pseudostratified olfactory epithelium in the dorsal 

cantly greater than that of the controls at 2 years. 

medulla hyperplasia in 1 mg/m3 females was signifiother of these diagnoses. The incidence of adrenal 

adrenal glands of exposed male rats had one or the 

the medulla of each adrenal gland. Most of the 

chromocytoma were mutually exclusive diagnoses in 

(Tables 15 and B4b). Focal hyperplasia and pheohistorical controls ranges from inhalation studies 

nant pheochromocytoma (combined) exceeded the 

benign pheochromocytoma and of benign or maligand A4b). In 1 mg/m3 females, the incidences of 

neoplasms from NTP inhalation studies (Tables 15 

exceeded the historical control ranges for these 

cytoma (combined) in 0.15 and 1 mg/m3 males 

males, and of benign or malignant pheochromomales, of malignant pheochromocytoma in 1 mg/m3 

of benign pheochromocytoma in 0.15 and 1 mg/m3 

increased trend (Tables 15 and B3). The incidences 

also occurred with a significant exposure-related 

malignant pheochromocytoma (combined) in females 

of benign pheochromocytoma nd of benign or 

bined) in males (Tables 15 and A3). The incidences 

and benign or malignant pheochromocytoma (compheochromocytoma, malignant pheochromocytoma, 

exposure-related increases in the incidences of benign 

Adrenal Medulla: At 2 years, there were significant 

compress the adjacent parenchyma. 

staining characteristics. Hyperplastic lesions did not 

adrenal medullary cells with alterations in size and/or 

focal to multifocal clusters of increased numbers of 

invaded the periadrenal tissues. Hyperplasias were 

cytomas penetrated the adrenal gland capsule and 

mocytomas were bilateral. Malignant pheochromosion of the adrenal cortex. Many benign pheochroadrenal medullary cells producing distinct compresBenign pheochromocytomas were discrete masses of 
Results 65 

in the 2-Year Inhalation Study of Nickel Subsulfide 

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

TABLE 15 

O mg/m3 0.15 mg/m' 1 mg/m3 

Hyperplasiab 

Adrenal Medullaa 

15-Month Interim Evaluation 

Male 

Hyperplasia 

Adrenal Medulla 

2-Year Study 

0 

5 

26 (2.2) 

53 

Pheochromocytoma Benign (Includes Bilateral) 

Pheochromocytoma Benign, Bilateral 1 

Logistic regression testg 

First incidence (days) 

Terminal ratef 

Adjusted ratee 

Overall rated 

P<O.OOl 

559 

4/13 (31%) 

54.4% 

13/53 (25 X) 

Pheochromocytoma Malignant, Bilateral 0 

Pheochromocytoma Malignant (Includes Bilateral) 

Logistic regression test 

First incidence 

Terminal rate 

Adjusted rate 

Overall rate 

0113 (0%) 

0.0% 

0153 (0%) 

P<O.ool 

- h 

Pheochromocytoma Benign, Malignant, or Complex' 

Logistic regression test 

First incidence 

Terminal rate 

Adjusted rate 

Overall rate 

P<O.ool 

553 

4/13 (31 %) 

55.4 % 

14/53 (26%) 

0 

5 

52 

22 (1.9) 

I* 

P=O.o02 

455 

16/21 (76%) 

84.6% 

30/52 (58 %) 

0 

P=O.242 

60 1 

0/21 (0%) 

6.9% 

2/52 (4%) 

P=O.O03 

455 

16/21 (76%) 

84.6% 

30/52 (58 %) 

5 

1 (1.O)C 

lo** (2.4) 

53 

23** 

P<0.001 

552 

13/18 (72%) 

88.0% 

37/53 (70%) 

5 

P=O.O02 

703 

7/18 (39%) 

48.6% 

11/53 (21 X) 

P<O.ool 

552 

18/18 (100%) 

100.0% 

42/53 (79%) 

(continued) 
66 Nickel Subsutfide, NTP TR 453 

in the 2-Year Inhalation Study of Nickel Subsulfide (continued) 

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

TABLE 15 

O mg/m3 0.1s mg/m3 1 mg/m3 

Female 

Hyperplasia 

Adrenal Medulla 

2-Year Study 

Pheochromocytoma Benign (Includes Bilateral) 

Pheochromocytoma Benign, Bilateral 0 

5 (1.6) 

53 

11 (2.0) 

53 

24** 

16** (2.1) 

53 

1 

Logistic regression test 

First incidence 

Terminal rate 

Adjusted rate 

Overall rate 

Pheochromocytoma Malignant 

P<O.OOl 

72 1 

1/25 (4%) 

7.6% 

2/53 (4%) 

P=O.O83 

590 

5/25 (20%) 

24.4% 

7/53 (13%) 

PCO.001 

563 

22/28 (79%) 

85.5% 

36/53 (68 %) 

Logistic regression test 

First incidence 

Terminal rate 

Adjusted rate 

Overall rate 

P=O.628 

677 

0/25 (0%) 

3.1 % 

1/53 (2%) 

0125 (0%) 

0.0% 

0153 (0%) 

P=0.497N P=0.755N 

729 (T) 

1/28 (4%) 

3.6% 

1153 (2%) 

- 

Pheochromocytoma Benign, Malignant, or ComplexJ 

Logistic regression test 

First incidence 

Terminal rate 

Adjusted rate 

Overall rate 

P<O.OOl 

677 

1/25 (4%) 

10.4% 

3/53 (6%) 

P=O.166 

590 

5/25 (20%) 

24.4% 

7/53 (13 %) 

P<O.OOl 

563 

22/28 (79%) 

85.5% 

36/53 (68%) 

** Significantly different (P~0.01) from the control group by the Fisher exact test (interim evaluations) or the logistic regression test 

(T)Terminal sacrifice 

g 

f 

e 

d 

C 

b 

a 

i 

h 

j 

(5.3% f 2.8%); range 2%-12% 

Historical incidence for 2-year NTP inhalation studies: 39/608 (6.4% f 4.4%); range 2%-14%. Feed studies: 62/1,175 

(28.3% f 12.0%); range 8%-50%. Feed studies: 400/1,182 (33.8% f 10.9%); range 14%-63% 

Historical incidence for 2-year NTP inhalation studies with untreated control groups (mean f standard deviation): 1761623 

Not applicable; no neoplasms in animal group 

lower incidence in an exposure group is indicated by N. 

the painvise comparisons behveen the controls and that exposure group. The logistic regression test regards these lesions as nonfatal. A 

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

Observed incidence in animals surviving until the end of the study 

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

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

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

Number of animals with lesion 

Number of animals with adrenal medulla examined microscopically 

(2-year study) 
Results 67 

controls (Tables 16, A5, and B5). chial lymph node of exposed males at 15 months 

females were significantly greater than those of the Incidences of macrophage hyperplasia in the bronolfactory epithelial atrophy in 1 mg/m3 males and those of the controls (Tables 17, A5, and B5). 

inflammation of the nose in 1 mg/m3 females and of females at 2 years were significantly greater than 

Nose: At 2 years, the incidences of chronic active males at 7 and 15 months and in exposed males and 

hyperplasia of the bronchial lymph node in exposed controls. 

Bronchial Lymph Node: The incidences of lymphoid were also significantly greater than those of the 

and exposed males and females at 2 years 

of Nickel Subsulfide 

Incidences of Nonneoplastic Lesions of the Nose in Rats in the 2-Year Inhalation Study 

TABLE 16 

Male 

Nosea 

7-Month Interim Evaluation 

Inflammation, Chronic Active 

Nose 

15-Month Interim Evaluation 

Olfactory Epithelium, Atrophy 

Inflammation, Chronic Activeb 

Nose 

2-Year Study 

Olfactory Epithelium, Atrophy 

Inflammation, Chronic Active 

Female 

15-Month Interim Evaluation 

Inflammation, Chronic Active 

Nose 

7-Month Interim Evaluation 

Olfactory Epithelium, Atrophy 

Inflammation, Chronic Active 

Nose 

Nose 

2-Year Study 

Olfactory Epithelium, Atrophy 

Inflammation, Chronic Active 

0 

0 

5 

1 (1.0) 

5 

2 (1.0) 

12 (1.8) 

53 

5 

1 (1.0) 

0 

3 (1.3) 

5 

0 

6 (1.7) 

53 

0 

0 

5 

2 (1.0) 

5 

1 (1.0) 

10 (2.6) 

53 

2 (1.5) 

5 

0 

2 (1.0) 

5 

0 

9 (1.7) 

53 

5 

3 (I.OIC 

1 (1.0) 

0 

5 

9* (1.1) 

18 (2.3) 

52 

5 

4 (1.0) 

5 

2 (1.5) 

1 (1.0) 

16** (1.0) 

20** (2.0) 

52 

a Number of animals with organ examined microscopically 

** P10.01 

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

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

Number of animals with lesion 
68 Nickel Subsulfide, NTP TR 453 

of Nickel Subsulfide 

Incidences of Nonneoplastic Lesions of the Bronchial Lymph Node in Rats in the 2-Year Inhalation Study 

TABLE 17 

O mg/m3 0.15 mg/m’ 1 mg/m3 

Male 

Lymph Node, Bronchial 

15-Month Interim Evaluation 

Hyperplasia, Lymphoidb 

Lymph Node, Bronchiala 

7-Month Interim Evaluation 

Hyperplasia, Macrophage 

Hyperplasia, Lymphoid 

Lymph Node, Bronchial 

2-Year Study 

Hyperplasia, Macrophage 

Hyperplasia, Lymphoid 

Female 

Hyperplasia, Lymphoid 

Lymph Node, Bronchial 

7-Month Interim Evaluation 

Lymph Node, Bronchial 

15-Month Interim Evaluation 

Hyperplasia, Macrophage 

Hyperplasia, Lymphoid 

Lymph Node, Bronchial 

2-Year Study 

Hyperplasia, Macrophage 

Hyperplasia, Lymphoid 

0 

4 

0 

0 

5 

5 

5** (1.6) 

5 

4* (1.O)C 

4* (1.0) 

5** (1.6) 

5 4 

4* (2.0) 

4* (1.3) 

1 (1.0) 

5 (2.2) 

52 

14** (1.1) 

29** (2.1) 

51 

28** (1.2) 

34** (2.4) 

53 

2 (1.0) 

5 

2 (1.0) 

5 

5 (2.4) 

5 

1 (1.0) 

0 

5 

2 (1.0) 

1 (2.0) 

5 

4 (1.0) 

2 (1.5) 

5 

0 

11 (1.8) 

50 

16** (1.3) 

36** (1.9) 

49 

24** (1.3) 

36** (2.6) 

50 

a Number of animals with bronchial lymph node examined microscopically 

** PSO.01 

* Significantly different (PSO.05) from the control group by the Fisher exact test (interim evaluations) or the logistic regression test 

(2-year study) 

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

Number of animals with lesion 

phages with abundant pale granular cytoplasm within Table B3) of the mammary gland in exposed females. 

hyperplasia consisted of a few to several macro- carcinoma (combined) (24/53, 13/53, 10/53; 

of the lymph node was maintained. Macrophage 1063; Table B3) and of fibroadenoma, adenoma, and 

stages of differentiation and the overall architecture es in the incidences of fibroadenoma (22/53, 11/53, 

the lymph node. The lymphocytes were at different Other Organs: There were exposure-related decreasregion and usually accompanied by enlargement of 

number of lymphocytes, mainly in the paracortical reaction to inflammation in the lung. 

Lymphoid hyperplasia consisted of an increased the medulla of the lymph node, consistent with a 
Results 69 

Table B3). 

less than that of the controls (24/52, 30/53, 15/53; 

pituitary gland in 1 mg/m3 females was significantly 

The incidence of adenoma of the pars distalis of the 

those of the controls (Table H6). 

females at 15 months were significantly greater than 

females at 7 months and those of 0.15 and 1 mg/m3 

tions in the kidney of 0.15 and I mg/m3 males and 

the controls at 7 and 15 months. Nickel concentraand females were significantly greater than those of 

Absolute lung weights of 0.15 and 1 mg/m3 males 

concentrations in the lung had reached a steady state. 

(Tables 18 and H5). However, by 15 months, nickel 

increased with increasing exposure concentration 

the controls at 7 and 15 months, and generally 

exposed rats were significantly greater than those of 

In general, nickel concentrations in the lung of 

llssue Burden Analyses 

Lung Weight and Lung Burden in Rats in the 2-Year Inhalation Study of Nickel Subsulfide” 

TABLE 18 

Male 

7 7 7 

7-Month Interim Evaluation 

n 

pg Ni/g control lung 

pg Ni/g lung 

pg Ni/lung 

Absolute lung wt (8) 1.53 f 0.13 - b 

8 f 0.3** 

6 f 0.2** 

12 f 0.5** 

1.97 f 0.09** 

18 f 1.2** 

9 f OS** 

28 f 1.8** 

3.21 f 0.08** 

5 5 5 

15-Month Interim Evaluation 

n 

pg Ni/g control lang 

pg Nilg lung 

pg Ni/lung 

.4bsolute lung wt (g) 6.84 f 0.70** 

9 f 0.7** 

3 f 0.1 

21 f 1.5** 

2.27 f 0.09 

- 

- 

- 6 f 0.3** 

4 f 0.2** 

14 f 0.6** 

3.31 f 0.22** 

Female 

7 7 7 

7-Month Interim Evaluation 

n 

pg Ni/g control lung 

pg Ni/g lung 

pg Nillung 

Absolute lung wt (8) 1.10 f 0.03 

- 

- 

- 

1.48 f 0.04** 

8 f 0.4** 

6 f 0.2** 

9 f 0.4** 

21 f 0.6** 

9 f 0.4** 

23 f 0.7** 

2.44 f 0.07** 

5 5 5 

15-Month Interim Evaluation 

n 

pg Ni/g control lung 

pg Nilg lung 

pg Nillung 

Absolute lung wt (8) 

19 f 0.6** 

7 f 0.2** 

29 f 1.0** 

1.52 f 0.11 4.15 f 0.10** 

- 

- 

- 

2.52 f 0.25** 

6 f OS** 

4 f OS** 

9 f 0.8** 

” Mean f standard error; statistical tests were performed on unrounded data. 

** Significantly different (P10.01) from the control group by Williams’ test (lung weight) or DUM’S or Shirley’s test (lung burden 

interim evaluation. 

Results were below 0.035 pg Ni (the limit of detection) for the 7-month interim evaluation and below 0.119 pg Ni for the 15-month 

parameters) 
70 Nickel Subsulfide, NTP TR 453 

MICE 

16-DAY STUDY 

death of one female was accidental (Table 19). One 

subsulfide/m3 died before the end of the study; the 

All male and female mice exposed to 10 mg nickel 

labored respiration in 10 mg/m3 males and females. 

the controls. Clinical findings at day 5 included 

5 mg/m3 males were significantly lower than those of 

mean body weights and mean body weight gains of 

male also died before the end of the study. Final 

control male, one control female, and one 1.2 mg/m3 

TABLE 19 

Survival and Body Weights of Mice in the 16-Day Inhalation Study of Nickel Subsulfide 

Mean Body Weightb (g) 

Change Relative to Controls 

Final Weight 

Dose Survivala Initial Final 

Male 

10 

5 

2.5 

1.2 

0.6 

0 

Female 

10 

5 

2.5 

1.2 

0.6 

0 

015e 

515 

515 

415d 

515 

415' 

015g 

515 

515 

515 

515 

415' 

23.1 f 0.3 

22.6 f 0.3 

22.6 f 0.2 

22.4 f 0.3 

22.8 f 1.0 

23.3 f 0.3 

18.8 f 0.5 

19.6 f 0.4 

19.1 f 0.5 

18.8 f 0.3 

18.6 f 0.2 

19.6 f 0.3 

21.8 f 0.5* 

23.4 f 0.5 

22.8 f 1.8 

25.0 f 0.4 

25.3 f 0.3 

- 

20.1 f 0.6 

20.6 f 0.4 

21.1 f 0.4 

21.6 f 0.2 

20.3 f 2.0 

- 

0.7 f 0.3 

0.4 f 1.7 

2.2 f 0.7 

2.0 f 0.2 

-0.8 f 0.4* 

- 

0.5 f 0.6 

1.5 f 0.4 

2.3 f 0.4 

2.9 f 0.3 

0.7 & 2.1 

- 

86 

92 

90 

99 

99 

101 

104 

106 

- 

Day of death: 5, 6 (accidental death), 9, 10, 10. 

Day of death: 9. Final weights and weight changes are based on 5 animals. 

Day of death: 5, 6, 6, 7, 8. 

Day of death: 11. Final weights and weight changes are based on 5 animals. 

Day of death: 5. 

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

Number of animals surviving at 16 dayslnumber initially in group 

Significantly different (PSO.05) from the control group by Williams' or Dunnett's test 
Results 71 

matory cell infiltration as well as necrosis in the 

In 10 mg/m3 mice, inflammation included inflamcurred in 2.5, 5, and 10 mg/m3 males and females. 

females (Table 20). Inflammation in the lung oclung, .bronchial lymph node, and nose of males and 

related histopathologic lesions were observed in the 

which died before the end of the study. Chemicalin a few 5 mg/m3 mice and in most 10 mg/m3 mice, 

were limited to mottled or diffusely reddened lungs 

controls. At necropsy, chemical-related gross lesions 

females were significantly less than those of the 

relative thymus weights of 10 mg/m3 males and 

those of the controls (Table F5). Absolute and 

10 mg/m3 females were significantly greater than 

and 5 mg/m3 females, and the relative lung weight of 

absolute and relative lung weights of 10 mg/m3 males 

The absolute lung weight of 5 mg/m3 males, the 

observed included atrophy due to lymphoid depletion 

1.2, 2.5, 5, or 10 mg/m3. Other microscopic lesions 

the nasal passages of males and females exposed to 

atrophy of the olfactory epithelium was observed in 

1.2, 2.5, and 5 mg/m3 mice. Minimal to marked 

plasia was observed in the bronchial lymph node of 

alveolar septa of 5 mg/m3 mice. Lymphoid hyperalso minimal to mild focal areas of fibrosis within the 

phages in mice from the 5 mg/m3 group. There were 

black pigment were present in some alveolar macrovessels and within alveolar septa. A few granules of 

interstitial inflammatory cell infiltrates around blood 

increase in the number of alveolar macrophages and 

survived until the end of the study, there was an 

In 2.5 and 5 mg/m3 males and females, all of which 

vascular congestion, and edema throughout the lung. 

alveolar and bronchiolar epithelium, extensive 

Incidences of Selected Nonneoplastic Lesions in Mice in the 16-Day Inhalation Study of Nickel Subsulfide 

TABLE 20 

0 mg/m3 0.6 mg/m3 1.2 mg/m3 2.5 mg/m3 5 mg/m3 10 mg/m’ 

Male 

Lunga 

Fibrosis 

Inflammationb 

Lymph Node, Bronchial 

Hyperplasia 

Nose 

Olfactory Epithelium Atrophy 

Female 

Lung 

Fibrosis 

Inflammation 

Lymph Node, Bronchial 

Hyperplasia 

Nose 

Olfactory Epithelium Atrophy 

0 

0 

5 

0 

5 

0 

3 

0 

0 

5 

0 

5 

0 

3 

0 

0 

5 

0 

5 

0 

2 

0 

0 

5 

0 

5 

0 

3 

0 

0 

5 

0 

5 

5** (1.2)C 

3* (1.3) 2 (2.0) 

3 3 

3 (1.0) 

5 

5** (1.8) 

5 

0 

0 

5 

0 

4* (1.5) 

5 

1 (1.0) 3 (2.0) 

2 4 

5 

2 (1.0) 5** (1.0) 

5 

3 (1.3) 0 

5** (1.4) 5** (3.0) 

5 5 

2 (1.5) 0 

3 2 

5** (4.0) 5** (3.0) 

5 5 

2 (2.0) 0 

5** (2.0) 4** (2.5) 

5 4 

2 (3.0) 0 

3 2 

5** (4.0) 4** (3.0) 

5 4 

a Number of animals with organ examined microscopically 

** P10.01 

* Significantly different (P10.05) from the control group by the Fisher exact test 

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

Number of animals with lesion 
72 Nickel Subsulfide, NTP TR 453 

that lose weight during the study. 

observed in mice that are killed moribund or mice controls (Table 12). 

considered to be nonspecific findings typically females were significantly less than those of the 

females (1/5, 0/4, 0/5, 3/4); these lesions were however, absolute kidney weights of these males and 

2.5 mg/m3, 0/5; 5 mg/m3, 0/5; 10 mg/m3, 5/5) and significantly greater than those of the controls; 

4/4) and spleen of 10 mg/m3 males (0 mg/m3, 1 /5; the kidney of 10 mg/m3 males and females were 

5 mg/m3, 06; 10 mg/m3, 5/5) and females (1/5,0/5, than those of the controls. Nickel concentrations in 

in the thymus of 10 mg/m3 males (0 mg/m3, 1/5; 2.5 and 10 mg/m3 animals were significantly greater 

Although nickel concentrations in the lung of exposed Because of the severity of lung lesions in mice 

males and females were generally greater than those exposed to 5 or 10 mg/m3, 2.5 mg/m3 was selected 

of controls, the increase was not exposure related as the highest exposure concentration for the 13-week 

(Tables 21 and 11). Absolute lung weights of these study. 

Lung Weight and Lung Burden in Mice in the 16-Day Inhalation Study of Nickel Subsulfidea 

TABLE 21 

0 mg/m3 0.6 mg/m3 2.5 mg/m3 10 mg/m3 

Male 

n 3 3 3 2 

pg Nilg control lung 

pg Ni/g lung 

pg Ni/lung 

Absolute lung wt (g) 0.138 f 0.004 0.145 f 0.006 0.200 f 0.014** 0.335 f 0.005** - b 1 f 0.2* 4 f 0.9** 4 f 0.9* 

10 f 0.7* 20 f 3.2** 13 f 2.5* 

10 f 1.0* 30 f 6.8** 31 f 6.0* 

- 

- 

Female 

n 3 3 3 3 

pg Nilg control lung 

pg Ni/g lung 

pg Ni/lung 

Absolute lung wt (g) 

10 f 1.0* 31 f 4.1** 22* 3.9* 

8 i 0.4 20 f 2.6** 8 f 1.9 

1 f 0.1* 4 * 0.5** 3 f 0.5* 

0.125 f 0.002 0.153 f 0.011 0.197 f 0.006* 0.380 f 0.040** - 

- 

- 

a Mean f standard error; statistical tests were performed on unrounded data. 

** P10.01 

* Significantly different (P~0.05) from the control group by Williams’ test (lung weight) or Dum’s or Shirley’s test (lung burden parameters) 

Results were below 0.155 pg Ni (the limit of detection). 
Results 73 

clinical findings were observed. 

similar to those of the controls. No chemical-related 

mean body weights of all exposure groups were 

were not considered to be chemical related. Final 

not related to exposure level or sex of the animal and 

before the end of the study (Table 22). Deaths were 

0.6 mg/m3 female, and two 2.5 mg/m3 females died 

1.2 mg/m3 male, two 0.15 mg/m3 females, one 

Two control males, two 0.6 mg/m3 males, one 

13-WEEK STUDY 

(Table G3) were similar to those reported for rats at 

In general, hematology differences in mice 

controls. 

males were minimally greater than those of the 

rocyte counts in 0.3, 0.6, 1.2, and 2.5 mg/m3 fethe controls. Hemoglobin concentrations and eryth2.5 mg/m3 males were minimally greater than that of 

and exposure groups. Lymphocyte counts in 1.2 and 

and minimal differences occurred in fewer end points 

13 weeks. The mice were less affected than the rats, 

were observed (Table J2). 

vaginal cytology between exposed and control mice 

No significant differences in sperm morphology or 

Survival and Body Weights of Mice in the 13-Week Inhalation Study of Nickel Subsulfide 

TABLE 22 

Mean Body Weightb (9) 

("/.I 

Change Relative to Controls 

Final Weight 

Dose Survivala Initial Final 

(mg/m3) 

Male 

2.5 

1.2 

0.6 

0.3 

0.15 

0 

23.1 f 0.6 

21.6 f 0.4 

8/10' 23.0 f 0.3 

23.7 f 0.2 

9/10e 23.0 f 0.6 

8/10d 23.0 f 0.5 

29.0 f 0.3 

30.2 f 0.8 

30.8 f 0.5 

31.6 f 0.6 

30.5 f 0.5 

29.8 f 0.5 

5.4 f 0.4* 

7.1 f 0.5 

7.7 f 0.5 

8.5 f 0.4 

9.0 f 0.6 

6.8 f 0.3 

10110 

10110 

97 

101 

103 

106 

102 

10/10 

Female 

2.5 

1.2 

0.6 

0.3 

0.15 

0 

10/10 

10110 

8/10] 18.1 f 0.2 

18.3 f 0.3 

9/1@ 18.1 f 0.3 

17.8 f 0.3 

8/10f 18.0 f 0.2 

18.3 f 0.4 

24.9 f 0.5 

25.3 f 0.4 

25.4 f 0.4 

25.2 f 0.4 

25.4 f 0.5 

25.1 f 0.5 

6.8 f 0.4 

6.9 f 0.5 

7.3 f 0.3 

7.5 f 0.5 

7.6 f 0.4 

6.8 f 0.3 

10/1oh 

99 

101 

101 

100 

101 

* 

C 

b 

a 

h 

g 

f 

e 

d 

I Week of death: 4, 9. Final weights and weight changes are based on 7 animals. 

Final weights and weight changes are based on 9 animals. 

Week of death: 2 

Week of death: 5, 7 

Week of death: 5 

Week of death: 5, 5 

Week of death: 4, 4 

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

Number of animals surviving at 13 weekslnumber initially in group 

Significantly different (PsO.05) from the control group by Williams' or Dunnett's test 
74 Nickel Subsulfide, NTP TR 453 

present in the lung, bronchial lymph node, and nose 

mice. Chemical-related histopathologic lesions were 

the pleural surface of the lungs of two 1.2 mg/m3 

1.2 mg/m3. White, pinpoint foci were observed on 

2.5 mg/m3 and four female mice exposed to 

two male and three female mice exposed to 

enlargement of the bronchial lymph nodes in

chemical-related gross findings in mice consisted of 

cantly less than that of the controls. At necropsy, 

absolute liver weight of 2.5 mg/m3 males was signifigreater than those of the controls (Table F6). The 

2.5 mg/m3 males and females were significantly 

Absolute and relative lung weights of 1.2 and 

focal areas of chronic inflammation, which 

Perivascular interstitial infiltrates of lymphocytes and 

in the severity of alveolar macrophage accumulation. 

exposed to 1.2 or 2.5 mg/m3, there was an increase 

2.5 mg/m3 males and in exposed females. In mice 

vessels, were present in0.15, 0.6, 1.2, and 

infiltrates, predominantly lymphocytes around blood 

to 0.3 mg/m3 or greater. Minimal focal interstitial 

within the alveolar spaces occurred in mice exposed 

detectable increase in the number of macrophages 

ty of inflammatory lesions in the lung. A minimally 

exposure-related increase in the incidence and severiof male and female mice (Table 23). There was an 

Incidences of Selected Nonneoplastic Lesions in Mice in the 13-Week Inhalation Study of Nickel Subsulfide 

TABLE 23 

0 mg/m3 0.15 mg/m3 0.3 mg/m3 0.6 mg/m3 1.2 mg/m3 2.5 mg/m3 

Male 

Lunga 

Fibrosis 0 

Inflammation, Chronic Active 0 

Interstitial Infiltrate 0 

Alveolar Macrophage Hyperplasiab 0 

10 

0 0 

0 0 

1 (1.0) 0 

0 8** (1.0)' 

10 10 

0 

0 

2 (1.0) 

8** (1.0) 

10 

5* (1.6) 

5* (1.2) 

3 (1.2) 

9** (2.0) 

10 

lo** (2.1) 

7** (1.6) 

2 (1.5) 

lo** (2.2) 

10 

Lymph Node, Bronchial 

Olfactory Epithelium Atrophy 0 

8 

0 

3 

Hyperplasia 

Nose 

0 

4 

3 (1.0) 

5 

8** (2.1) 

8 

0 

10 

0 

10 10 

5* (1.0) 5* (1.0) 

9 

lo** (2.8) 

10 

Female 

0 0 

0 0 

2 (1.0) 3 (1.0) 

0 4* (1.0) 

10 10 

0 

0 

4* (1.0) 

9** (1.0) 

10 

1 (2.0) 

lo** (1.5) 

9** (1.4) 

lo** (2.4) 

10 

9** (1.6) 

7** (2.0) 

8** (1.7) 

lo** (2.6) 

Lung 10 

Fibrosis 0 

Inflammation, Chronic Active 0 

Interstitial Infiltrate 0 

Alveolar Macrophage Hyperplasia 0 

10 

0 

5 

6* (1.1) 

10 

5* (1.2) 

7 

5* (2.8) 

Lymph Node, Bronchial 6 

Olfactory Epithelium Atrophy 0 

8 

0 

4 

Hyperplasia 

Nose 

lo** (2.5) 

10 

0 

10 

0 

10 

1 (1.0) 

9 

a Number of animals with organ examined microscopically 

** PSO.01 

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

Tissue not examined at this exposure concentration 

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

Number of animals with lesion 
Results 75 

dorsal meatus of the nasal passages. 

cells. This olfactory lesion was most evident in the 

amount of cytoplasm in the apical portion of the 

from a decrease in cell number and a decrease in the 

consisted of a thinner neuroepithelial layer resulting 

lymph nodes. Atrophy of the olfactory epithelium 

cytes, primarily- in the paracortical region of the 

grossly. This consisted of an increase in lymphoing to the enlargement of these nodes observed 

observed in the bronchial lymph nodes, correspondalveolar septa. Microscopically, hyperplasia was 

increase in fibroblasts and collagen fibers in the 

was minimal to mild fibrosis characterized by a focal 

In some of the foci of chronic inflammation, there 

resulted in thicker alveolar septa, were also present. 

greater than those of the controls. 

2.5 mg/m3 males and females were significantly 

lung weights of these 0.6 mg/m3 males and 

exposure concentration (Tables 24 and 13). Absolute 

these concentrations increased with nickel subsulfide 

significantly greater than those of the controls, and 

0.15, 0.6, and 2.5 mg/m3 males and females were 

At 13 weeks, nickel concentrations in the lung of 

1.2 mg/m3. 

selected for the 2-year inhalation study was 

highest nickel subsulfide exposure concentration 

mation and fibrosis in the lung at 2.5 mg/m3, the 

incidences and severities of chronic active inflamDose Selection Rationale: Based on the increased 

Lung Weight and Lung Burden in Mice in the 13-Week Inhalation Study of Nickel Subsulfidea 

TABLE 24 

O mglm3 0.15 mg/m3 0.6 mglm3 2.5 mglm’ 

Male 

n 

pg Ni/g control lung 

pg Ni/g lung 

pg Nillung 

Absolute lung wt (g) 

Female 

n 

pg Ni/g control lung 

pg Ni/g lung 

pg Nillung 

Absolute lung wt (9) 

0.158 f 0.005 

6 

- b 

- 

- 

6 

0.163 f 0.004 

- 

- 

- 

5 6 6 

0.178 f 0.010 0.195 f 0.010* 0.258 f 0.013** 

1 f 0.2* 2 f 0.1** 4 f os** 

3 f 0.9* 11 f 0.8** 17 f 1.6** 

4 f 1.0* 14 f 0.8** 28 f 3.2** 

5 5 6 

0.139 f 0.007 0.170 f 0.011 0.298 f 0.012** 

1 f 0.1** 2 f 0.1** 7 f 0.5** 

6 f 0.4** 13 f 1.0** 23 f 1.4** 

5 f 0.4** 14 f 0.8** 41 f 2.9** 

a Mean f standard error; statistical tests were performed on unrounded data. 

** PSO.01 

* Significantly different (PsO.05) from the control group by Williams’ test (lung weight) or Shirley’s test (lung burden parameters) 

Results were below 0.255 pg Ni (the limit of detection). 
76 Nickel Subsulfide, NTP TR 453 

of the controls. 

of exposed male and female mice was similar to that 

Kaplan-Meier survival curves in Figure 4. Survival 

and female mice are shown in Table 25 and in the 

Estimates of 2-year survival probabilities for male 

Survival 

%YEAR STUDY Body Weights and Clinical Findings 

following exposure periods. 

in male and female mice included labored respiration 

Tables 26 and 27). Chemical-related clinical findings 

out he second year of the study (Figure 5 and 

females were less than those of the controls throughMean body weights of 0.6 and 1.2 mg/m3 males and 

TABLE 25 

Survival of Mice in the 2-Year Inhalation Study of Nickel Subsulfide 

0 mg/m3 

- 

0.6 mg/m3 1.2 mg/m3 

%Month interim evaluationa 

Animals initially in study 

Male 

Mean survival (days)c 

Percent probability of survival at end of studyb 

Animals surviving to study termination 

Natural deaths 

Moribund 

15-Month interim evaluationa 

7-Month interim evaluationa 

Animals initially in study 

Female 

Survival analysisd 

Mean survival (days) 

Percent probability of survival at end of study 

Animals surviving to study termination 

Natural deaths 

Moribund 

Missexeda 

15-Month interim evaluationa 

Survival analysis 

80 

43 

26 

8 

27 

10 

9 

P= 1.000 

563 

80 

590 

63 

36 

8 

14 

2 

10 

10 

P=0.979N 

80 

43 

25 

9 

26 

10 

10 

P=O.920 

544 

80 

58 

34 

11 

15 

0 

10 

10 

580 

P=O.658 

80 

43 

26e 

9 

25 

10 

10 

P= 1.000 

552 

80 

64 

38 

9 

13 

0 

10 

10 

587 

P= 1.000N 

~ 

a Censored from survival analyses 

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

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, 

Mean of all deaths (uncensored, censored, and terminal sacrifice) 

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

e Includes one animal that died during the last week of the study. 
Results 77 

................... 

I I I I I 4 

15 30 Is SO 75 90 1 

WEEKS ON STUDY 

............................ ; ......... 

........... :. ....... 

............................ i 

0.5 .... 

0.4 

75 0 15 SO 45 60 

WEEKS ON STUDY 

- 

..... 

i 

........ 

... 

..,. 

- 

90 

- 

...... 

...... 

.......... 

............. 

.......... 

............ 

- 

1 is 

0 

D 

Kaplan-Meier Survival Curves for Mice Administered Nickel Subsulfide by Inhalation for 2 Years 

FIGURE 4 
78 Nickel Subsulfide, NTP TR 453 

._ 

T, . i 

.. 

40 - ............................ :, .......... ................. 

0: 

I 

v) 

0 

I 

" 35- 

a ......... ................ .................. ................ .............. 

X 

t 

>- 

......... .i. 

I 

W 

4 

Z 

5 

20 - .......... .: 

IS I 

0 15 30 45 60 76 I I I I 

WEEKS ON STUDY 

- 

11 

... 

:. . 

... 

... 

.... 

i 

90 

I 

................... mi 

i,,; 

.........e. 8 ....... 

................................ 

................................ 

1 is 

45 

:. i i i : 40 - ......... .: ............... .... ................. ................ ................ 

I 

v) 

0 

z 

a 

g 35 - ..... ...... ... ... ............/.. ............ i ............... 

I 

IJO-' .. ........................ ; ................ : .................. : ................. : ................................ 

n 

......... ............. ..: .................. ................. 

I 

15- 

0 15 30 45 60 75 90 105 

I I I I I I I 

WEEKS ON STUDY 

0 
Results 79 

Mean Body Weights and Survival of Male Mice in the 2-Year Inhalation Study of Nickel Subsulfide 

TABLE 26 

Weeks 0 mg/m3 

Study (g) Survivors controls) Survivors (g) 

0.6 mg/m3 

controls) Survivors 

1.2 mdm3 

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

105 

101 

97 

93 

89 

85 

81 

77 

73 

69a 

65 

61 

57 

53 

49 

45 

41 

37 

33 

29a 

25 

21 

17 

13 

12 

11 

10 

9 

8 

7 

6 

5 

4 

3 

2 

1 

38.8 

39.3 

39.8 

40.8 

41.4 

41.5 

41.4 

41.4 

41.5 

41 .O 

42.6 

42.4 

41.5 

41.4 

41.3 

39.9 

39.5 

38.2 

37.1 

36.8 

35.7 

34.8 

33.6 

32.7 

32.2 

31.6 

30.3 

30.8 

30.6 

30.1 

29.4 

29.0 

28.1 

26.7 

25.5 

22.9 

Mean for weeks 

1-13 29.2 

14-52 37.4 

53-105 41.1 

26 

28 

33 

34 

37 

38 

45 

50 

53 

55 

66 

67 

68 

68 

68 

69 

69 

69 

69 

70 

79 

79 

79 

79 

79 

79 

80 

80 

80 

80 

80 

80 

80 

80 

80 

80 

35.7 

36.5 

36.7 

37.7 

38.5 

38.6 

38.9 

38.7 

38.7 

38.8 

40.5 

40.1 

39.8 

40.1 

40.4 

39.9 

38.7 

38.4 

37.2 

36.8 

35.9 

35.0 

33.7 

32.3 

31.9 

31.4 

30.8 

30.7 

30.3 

30.0 

29.1 

28.8 

27.8 

26.6 

25.4 

22.6 

38.5 

37.3 

29.1 

92 

93 

92 

92 

93 

93 

94 

94 

93 

95 

95 

95 

96 

97 

98 

100 

98 

101 

100 

100 

101 

101 

100 

99 

99 

99 

102 

100 

99 

100 

99 

99 

99 

100 

100 

99 

94 

100 

100 

25 

28 

30 

33 

35 

36 

42 

46 

50 

51 

64 

64 

65 

65 

66 

66 

66 

66 

67 

67 

I7 

78 

78 

78 

78 

78 

78 

78 

78 

78 

I8 

79 

79 

80 

80 

80 

35.5 

35.7 

36.7 

37.2 

37.6 

37.5 

38.4 

38.6 

38.7 

39.7 

40.7 

40.7 

40.0 

40.1 

40.7 

39.6 

39.3 

38.4 

37.4 

37.2 

36.3 

35.3 

34.1 

32.9 

32.4 

31.4 

31 .O 

30.8 

30.3 

30.0 

29.4 

29.1 

27.9 

26.6 

25.2 

22.7 

38.4 

37.6 

29.2 

99 

100 

101 

101 

101 

102 

101 

102 

101 

101 

99 

102 

100 

99 

100 

100 

100 

99 

100 

99 

99 

92 

91 

92 

91 

91 

90 

93 

93 

93 

97 

96 

96 

96 

98 

99 

26 

30 

31 

38 

41 

43 

44 

45 

47 

48 

61 

62 

63 

66 

66 

67 

67 

67 

68 

68 

78 

79 

80 

80 

80 

80 

80 

80 

80 

80 

80 

80 

80 

80 

80 

80 

93 

101 

100 

~~ 

a Interim evaluations occurred during weeks 28 and 68. 
80 Nickel Subsulfide, NTP TR 453 

TABLE 27 

Mean Body Weights and Survival of Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide 

Weeks O mg/m3 

Study (g) Survivors (g) controls) Survivors 

0.6 mg/m3 

controls) Survivors 

1.2 mg/m3 

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

(9) 

9 

8 

7 

6 

5 

4 

3 

2 

1 

105 

101 

97 

93 

89 

85 

81 

77 

73 

69a 

65 

61 

57 

53 

49 

45 

41 

37 

33 

29a 

25 

21 

17 

13 

12 

11 

10 

36.3 

36.4 

36.8 

37.3 

38.3 

38.4 

38.4 

39.0 

39.5 

39.5 

40.9 

40.5 

40.5 

40.4 

40.2 

38.7 

38.5 

36.7 

34.6 

34.3 

32.4 

31.2 

29.7 

28.2 

27.2 

27.2 

25.2 

26.0 

25.9 

25.2 

24.5 

23.9 

23.1 

21.8 

20.6 

18.5 

Mean for weeks 

53-105 

14-52 

1-13 

38.7 

35.1 

24.4 

36 

38 

44 

46 

47 

50 

50 

53 

53 

54 

64 

64 

65 

66 

66 

66 

68 

68 

68 

68 

78 

78 

78 

78 

78 

78 

78 

78 

80 

80 

80 

80 

80 

80 

80 

80 

32.6 

33.4 

33.6 

34.9 

35.8 

35.9 

36.6 

36.5 

37.0 

37.3 

38.8 

38.7 

38.6 

39.0 

38.9 

37.9 

37.1 

36.7 

35.0 

34.9 

32.9 

31.6 

29.8 

28.2 

27.3 

27.2 

26.3 

26.5 

25.9 

25.6 

24.5 

24.1 

23.2 

22.0 

20.7 

18.6 

36.3 

35.0 

24.6 

90 

92 

91 

94 

94 

94 

95 

94 

94 

94 

95 

96 

95 

97 

97 

98 

96 

100 

101 

102 

102 

101 

100 

100 

100 

100 

104 

102 

100 

102 

100 

101 

100 

101 

101 

101 

94 

100 

101 

34 

39 

41 

46 

49 

50 

50 

53 

55 

55 

65 

66 

67 

67 

67 

67 

67 

67 

67 

68 

78 

78 

79 

80 

80 

80 

80 

80 

80 

80 

80 

80 

80 

80 

80 

80 

31.3 

32.1 

32.7 

33.7 

33.8 

34.4 

34.8 

35.7 

36.4 

37.0 

37.8 

37.5 

37.0 

38.0 

37.5 

36.5 

36.3 

35.1 

33.6 

34.1 

32.3 

30.9 

29.5 

28.3 

27.5 

26.9 

26.3 

25.9 

25.7 

25.5 

24.7 

24.3 

23.3 

21.8 

20.8 

18.6 

35.2 

34.0 

24.6 

101 

101 

102 

101 

100 

101 

101 

86 

88 

89 

90 

88 

90 

91 

92 

92 

94 

92 

93 

91 

94 

93 

94 

94 

96 

97 

99 

100 

99 

99 

100 

101 

99 

104 

1 00 

99 

91 

97 

101 

40 

41 

43 

45 

48 

49 

53 

54 

54 

56 

66 

67 

67 

67 

68 

68 

68 

68 

69 

69 

79 

79 

79 

79 

79 

80 

80 

80 

80 

80 

80 

80 

80 

80 

80 

80 

a Interim evaluations occurred during weeks 29 and 69. 
Results 81 

females was greater than that in the controls. Inmatory process. The lymphocyte count in 1.2 mg/m3 

particularly if the increases are a result of an inflamnumbers can accompany increases in neutrophils, 

vascular neutrophil life span. Increases in monocyte 

nal and the circulating pools, or increased intramarrow; neutrophils redistributed between the margigranulopoiesis and/or rate of release from the bone 

Increased neutrophil counts can occur due to altered 

count was mildly greater than in the controls. 

was greater than in the controls, and the monocyte 

1.2 mg/m3 females, the segmented neutrophil count 

Hematology results are presented in Table G4. In 

Hematology 

in neutrophil, lymphocyte, and monocyte numbers. 

controls, and this difference reflected the differences 

1.2 mg/m3 females was greater than that in the 

and recirculation. The total leukocyte count in 

lymphocyte migration or homing, tissue migration, 

of lymphopoiesis or mechanisms involving altered 

creased lymphocyte numbers may reflect stimulation 

erythrocytosis). 

production as a result of tissue hypoxia (secondary 

erythrocytosis) or with increased erythropoietin 

ence could be consistent with dehydration (relative 

mildly greater than that of the controls; this differThe hematocrit value in 1.2 mg/m3 females was 
82 Nickel Subsulfide, NTP TR 453 

mice and Appendix D for female mice. 

this section are presented in Appendix C for male 

historical incidences for the neoplasms mentioned in 

of at least 5% in at least one animal group, and 

of primary neoplasms that occurred with an incidence 

individual animal tumor diagnoses, statistical analyses 

incidences of neoplasms and nonneoplastic lesions, 

nose, and bronchial lymph node. Summaries of the 

neoplasms and/or nonneoplastic lesions of the lung, 

biologically noteworthy changes in the incidences of 

This section describes the statistically significant or 

Pathology and Statistical Analyses 

(Tables 28 and C4). 

the historical control range from inhalation studies 

carcinoma (combined) in male controls was within 

The incidence of alveolar/bronchiolar adenoma or 

controls from inhalation studies (Tables 28 and D4). 

female controls exceeded the ranges in historical 

bronchiolar adenoma or carcinoma (combined) in 

alveolar/bronchiolar carcinoma and of alveolar/ 

those of the controls. However, the incidences of 

and 1.2 mg/m3 females were significantly less than 

or carcinoma (combined) in 0.6 mg/m3 males and 0.6 

and the incidences of alveolar/bronchiolar adenoma 

alveolar/bronchiolar carcinoma in 0.6 mg/m3 females 

2 years (Tables 28, C3, and D3). The incidence of 

females occurred with a significant negative trend at 

adenoma or carcinoma (combined) in males and 

females and the incidences of alveolar/bronchiolar 

The incidences of alveolar/bronchiolar carcinoma in 

controls at 7 and 15 months (Tables F7 and F8). 

females were significantly greater than those of the 

males (except 0.6 mg/m3 group at 7 months) and 

Lung: Absolute and relative lung weights of exposed 

(except in 0.6 mg/m3 females), macrophage hyperces of chronic active inflammation, bronchialization 

(Tables 28, C5, and D5). At 15 months, the incidensignificantly greater than those of the controls 

infiltration in 0.6 mg/m3 males and females were 

1.2 mg/m3 males and females, and of interstitial 

macrophage hyperplasia and proteinosis in 0.6 and 

mation in 1.2 mg/m3 males and females, of alveolar 

At 7 months, the incidences of chronic active inflamthan in 0.6 mg/m3 mice. 

bronchialization were more severe in 1.2 mg/m3 mice 

mation, macrophage hyperplasia, proteinosis, and 

of the controls at 2 years. Chronic active inflamexposed groups were significantly greater than those 

plasia, proteinosis, and interstitial infiltration in 

inflammation, bronchialization, macrophage hypermales, the incidences of fibrosis, chronic active 

of the controls. Except for fibrosis in 0.6 mg/m3 

exposed groups were significantly greater than those 

plasia, proteinosis, and interstitial infiltration in all 

within the lung parenchyma. 

consisted of foci of loosely arranged fibroblasts 

change considered secondary to inflammation, 

microscopic appearance only. Fibrosis, a minimal 

ization in this study corresponds to the overall light 

determine their cell of origin, the term bronchialobserved to have cilia and no attempts were made to 

hyperplasia. Because these cuboidal cells were not 

and therefore was not termed alveolar epithelial 

part of the morphologic continuum toward neoplasia 

alveolar epithelial hyperplasia, is not considered a 

alveoli. This hyperplastic change, unlike focal 

extended from terminal bronchioles into adjacent 

study consisted of multiple foci where cuboidal cells 

bronchiolar lymphoid tissue. Bronchialization in this 

of lymphocytes in alveolar septa adjacent to peria focus of inflammation and/or an increased number 

occasional macrophages in alveolar septa adjacent to 

Interstitial infiltrating cells were lymphocytes with 

granular to hyaline material within alveolar lumens. 

accumulations of varying amounts of eosinophilic 

of circulating monocytes. Proteinosis consisted of 

these macrophages were from the intravascular pool 

to vacuolated cytoplasm (Plate 5). Most probably, 

varied, typically with abundant eosinophilic granular 

in intra-alveolar macrophages, the number of which 

phage hyperplasia consisted of a widespread increase 

eosinophilic, rod-shaped crystalline material. Macroadmixed neutrophils, cellular debris, and/or brightly 

macrophages and lymphocytes; rarely, there were 

cells within alveolar lumens. Typically, these were 

to locally extensive accumulations of inflammatory 

Chronic active inflammation consisted of multifocal 
Results 83 

of Nickel Subsulfide 

Incidences of Neoplasms and Nonneoplastic Lesions of the Lung in Mice in the 2-Year Inhalation Study 

TABLE 28 

O mg/m3 0.6 mg/m3 1.2 mg/m3 

Male 

Lunga 

7-Month Interim Evaluation 

0 

0 

0 

0 

5 

Interstitium, Infiltration Cellular 

Alveolus, Proteinosis 

Alveolus, Hyperplasia, Macrophage 

Inflammation, Chronic Activeb 

15-Month Interim Evaluation 

Interstitium, Infiltration Cellular 

Alveolus, Proteinosis 

Alveolus, Hyperplasia, Macrophage 

Bronchialization 

Inflammation, Chronic Active 

Lung 

0 

0 

0 

0 

0 

5 

2-Year Study 

Interstitium, Infiltration Cellular 

Alveolus, Proteinosis 

Alveolus, Hyperplasia, Macrophage 

Bronchialization 

Inflammation, Chronic Active 

Fibrosis 

Lung 

10 (1.0) 

0 

6 (2.7) 

3 (2.7) 

1 (1.0) 

0 

61 

Alveolarlbronchiolar Adenoma 6 

AlveolarlBronchiolar Adenoma or Carcinomad 

Alveolar/bronchiolar Carcinoma 7 

Logistic regression tesa 

First incidence (days) 

Terminal rateg 

Adjusted ratef 

Overall ratee 

P=0.035N 

552 

8/26 (3 1 %) 

40.7% 

13/61 (21%) 

Female 

7-Month Interim Evaluation 

Interstitium, Infiltration Cellular 

Alveolus, Proteinosis 

Alveolus, Hyperplasia, Macrophage 

Inflammation, Chronic Active 

Lung 

1 (1.0) 

0 

1 (1.0) 

0 

5 

5** (1.0) 

5** (1.0) 

5** (1.0) 

3 (1.3)' 

5 

4* (1.8) 

5** (3.4) 

5** (1.4) 

4* (1.5) 

5** (1.2) 

5 

55** (1.7) 

57** (2.9) 

57** (2.2) 

53** (1.8) 

52** (1.7) 

3 (1.0) 

59 

3 

2 

P=0.038N 

66 1 

4/25 (16%) 

18.5% 

5/59 (8 %) 

5 

2 (1.0) 

5* (1.0) 

4* (1.0) 

5* (1.4) 

3 (1.0) 

5** (1.6) 

5** (1.4) 

5** (1.6) 

5 

5** (2.6) 

5** (1.8) 

5 

5** (1.4) 

5** (3.0) 

5** (2.2) 

55** (1.8) 

57** (3.0) 

58** (2.5) 

54** (2.1) 

53** (2.0) 

16** (1.0) 

58 

2 

4 

P=0.058N 

66 1 

4/25 (16%) 

21.4% 

6/58 (10%) 

4 (1.5) 

5** (1.4) 

5* (1.2) 

5** (1.2) 

5 

(continued) 
84 Nickel Subsulfide, NTP TR 453 

of Nickel Subsulfide (continued) 

Incidences of Neoplasms and Nonneoplastic Lesions of the Lung in Mice in the 2-Year Inhalation Study 

TABLE 28 

O mg/m3 0.6 mg/m3 1.2 mg/m3 

Female (continued) 

Lung 

15-Month Interim Evaluation 

1 (1.0) 

0 

0 

0 

0 

5 

5* (2.2) 

5** (2.8) 

5** (1.6) 

2 (1.0) 

4* (2.5) 

5 

5* (1.8) 

5** (3.6) 

5** (2.8) 

5** (1.6) 

5** (1.4) 

5 

Interstitium, Infiltration Cellular 

Alveolus, Proteinosis 

Alveolus, Hyperplasia, Macrophage 

Bronchialization 

Inflammation, Chronic Active 

2-Year Study 

Interstitium, Infiltration Cellular 

Alveolus, Proteinosis 

Alveolus, Hyperplasia, Macrophage 

Bronchialization 

Inflammation, Chronic Active 

Fibrosis 

Lung 

Alveolar/bronchiolar Adenoma 

19 (1.2) 

0 

5 (1.8) 

3 (2.3) 

1 (2.0) 

0 

58 

3 

53** (2.6) 

54** (2.8) 

57** (2.6) 

53** (1.8) 

46** (1.7) 

7** (1.0) 

59 

1 

58** (2.2) 

59** (3.4) 

60** (2.9) 

58** (2.1) 

58** (2.0) 

17** (1.1) 

60 

1 

Alveolar/bronchiolar Adenoma or Carcinoma' 

Alveolar/bronchiolar Carcinoma 7 1 2 

Logistic regression test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

P=0.027N 

683 

8/36 (22%) 

24.1 % 

9/58 (16%) 

P=0.028N 

552 

1/34 (3 %) 

4.8% 

2/59 (3 %) 

P=0.050N 

733 (T) 

3/38 (8%) 

7.9% 

3/60 (5 %) 

* Significantly different (P50.05) from the control group by the Fisher exact test (interim evaluations) or the logistic regression test 

(T)Terminal sacrifice 

a Number of animals with organ examined microscopically 

** P<O.Ol 

(2-year study) 

(21.5% f 8.0%); range 10%-42%. Feed studies: 249/1,319 (18.9% f 7.6%); range 4%-32% 

Historical incidence for 2-year NTP inhalation studies with untreated control groups (mean f standard deviation): 205/952 

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

Number of animals with lesion 

negative trend or a lower incidence in an exposure group is indicated by N. 

the painvise comparisons between the controls and that exposure group. The logistic regression test regards these lesions as nonfatal. A 

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

Observed incidence in animals surviving until the end of the study 

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

(7.7% f 5.3%); range 2%-26% 

e Number of animals with neoplasm per number of animals with organ examined microscopically 

I Historical incidence for 2-year NTP inhalation studies: 97/944 (10.3% f 3.7%); range 0%-16%. Feed studies: 102/1,319 
Results 85 

controls. 

females were significantly less than that of the 

degeneration of olfactory epithelium in exposed 

controls at 2 years. At 2 years, the incidences of 

females were significantly greater than that of the 

acute inflammation of the nose in 0.6 and 1.2 mg/m3 

controls (Tables 29, C5, and DS). The incidences of 

2 years were significantly greater than those of the 

and in 0.6 and 1.2 mg/m3 males and females at 

epithelium in 1.2 mg/m3 males at 7 and 15 months 

Nose: The incidences of atrophy of the olfactory 

on or adjacent to the surface respiratory epithelium. 

neutrophils, sometimes admixed with cellular debris, 

Acute inflammation consisted of focal aggregates of 

within the cytoplasm of olfactory epithelial cells. 

droplets of brightly eosinophilic homogenous material 

in B6C3F, mice, was a minimal accumulation of 

(Plate 6). Degeneration, a normal age-related lesion 

decrease in the thickness of olfactory epithelial cells 

Atrophy of olfactory epithelium was a minimal focal 

of Nickel Subsulfide 

Incidences of Nonneoplastic Lesions of the Nose in Mice in the 2-Year Inhalation Study 

TABLE 29 

O mg/m3 0.6 mg/m3 1.2 mg/m3 

Nosea 

7-Month Interim Evaluation 

Male 

1 (1.0) 

0 

5 

0 

5 

0 

1 (1.O)C 4* (1.0) 

5 

Olfactory Epithelium, Atrophy 

Nose 

15-Month Interim Evaluation 

Olfactory Epithelium, Degeneration 

Olfactory Epithelium, Atrophyb 

0 

5 

3 (1.0) 

5 

4* (1.0) 

5 

Nose 

2-Year Study 

3 (1.0) 

1 (1.0) 

0 

61 

27** (1.1) 

0 

59 

6 (1.0) 

55** (1.2) 

3 (2.0) 

59 

Olfactory Epithelium, Degeneration 

Olfactory Epithelium, Atrophy 

Inflammation, Acute 

8 (1.0) 

Nose 

7-Month Interim Evaluation 

Female 

4 (1.0) 

0 

5 

O* 

0 

5 

O* 

1 (1.0) 

5 

Olfactory Epithelium, Degeneration 

Olfactory Epithelium, Atrophy 

Nose 

15-Month Interim Evaluation 

1 (2.0) 

5 

0 

5 

0 

5 

Olfactory Epithelium, Degeneration 

Nose 

2-Year Study 

27 (1.1) 

1 (1.0) 

0 

58 59 

41** (1.1) 

14** (1.7) 

60 

Olfactory Epithelium, Degeneration 

Olfactory Epithelium, Atrophy 

Inflammation, Acute 

3** (1.0) 

11** (1.0) 

11** (1.8) 

12** (1.0) 

a Number of animals with organ examined microscopically 

** PSO.01 

* Significantly different (PsO.05) from the control group by the Fisher exact test (interim evaluations) or the logistic regression test 

(2-year study) 

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

Number of animals with lesion 
86 Nickel Subsulfide, NTP TR 453 

those of the controls. 

females at 2 years were significantly greater than 

15 months, and in 0.6 and 1.2 mg/m3 males and 

15 months, in 0.6 and 1.2 mg/m3 females at 

phage hyperplasia in 1.2 mg/m3 males at 7 and 

(Tables 30, C5, and D5). The incidences of macrosignificantly greater than those of the controls 

and 1.2 mg/m3 males and females at 2 years were 

0.6 and 1.2 mg/m3 females at 15 months, and in 0.6 

hyperplasia in 1.2 mg/m3 males at 15 months, in 

Bronchial Lymph Node: The incidences of lymphoid 

probably secondary to inflammation in the lung. 

macrophages in the bronchial lymph node were 

within the medulla of the lymph node. Increased 

phages with abundant pale foamy cytoplasm typically 

plasia consisted of scattered aggregates of macrolymph node was maintained. Macrophage hyperdifferentiation and the overall architecture of the 

node. The lymphocytes were at different stages of 

an increase in the size and cellularity of the lymph 

increased number of cortical lymphocytes resulting in 

Lymphoid hyperplasia was characterized by an 

in the 2-Year Inhalation Study of Nickel Subsulfide 

Incidences of Nonneoplastic Lesions of the Bronchial Lymph Node in Mice 

TABLE 30 

O mg/m3 0.6 mg/m3 1.2 mg/m3 

%Month Interim Evaluation 

Male 

Hyperplasia, Macrophage 

Hyperplasia, Lymphoidb 

Lymph Node, Bronchiala 

Lymph Node, Bronchial 

15-Month Interim Evaluation 

Hyperplasia, Macrophage 

Hyperplasia, Lymphoid 

Lymph Node, Bronchial 

2-Year Study 

Hyperplasia, Macrophage 

Hyperplasia, Lymphoid 

%Month Interim Evaluation 

Female 

Hyperplasia, Macrophage 

Hyperplasia, Lymphoid 

Lymph Node, Bronchial 

Lymph Node, Bronchial 

15-Month Interim Evaluation 

Hyperplasia, Macrophage 

Hyperplasia, Lymphoid 

Lymph Node, Bronchial 

2-Year Study 

Hyperplasia, Macrophage 

Hyperplasia, Lymphoid 

0 

0 

3 

0 

0 

4 

40 

4 (2.3) 

1 (3.0) 

0 

0 

5 

0 

0 

5 

0 

10 (1.9) 

50 

4 

2 (1.O)C 

2 (1.0) 

3 (1.0) 

3 (1.7) 

3 

47** (1.7) 

40** (2.1) 

53 

1 (1.0) 

1 (1.0) 

4 

5** (1.2) 

5** (2.6) 

5 

44** (1.5) 

46** (2.2) 

57 

5* (1.0) 

3 (1.0) 

5 

5 

5** (1.2) 

4* (1.3) 

54 

50** (1.8) 

49** (2.2) 

1 (1.0) 

0 

5 

5** (1.0) 

5** (2.4) 

5 

47** (1.8) 

52** (2.4) 

59 

a Number of animals with bronchial lymph nodeexamined microscopically 

** PSO.01 

* Significantly different (PSO.05) from the control group by the Fisher exact test (interim evaluations) or the logistic regression test 

(2-year study) 

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

Number of animals with lesion 
Results 87 

Tissue Burden Analyses 

Nickel concentrations in the lung of exposed groups and females at 7 and 15 months were significantly 

of mice were significantly greater than those of the greater than those of the controls. Nickel concentracontrols at 7 and 15 months, and these concentrations tions in the kidney of 0.6 mg/m3 males and 

increased with increasing exposure concentration and 1.2 mg/m3 males and females were significantly 

with time (Tables 3 1 and 14). In these groups, greater than those of the controls at 15 months 

absolute lung weights of 0.6 and 1.2 mg/m3 males (Table 15). 

Lung Weight and Lung Burden in Mice in the 2-Year Inhalation Study of Nickel Subsulfidea 

TABLE 31 

0 mg/m3 0.6 mg/m3 1.2 mg/m3 

n 

Male 

7-Month Interim Evaluation 

pg Nilg control lung 

pg Nilg lung 

pg Nillung 

Absolute lung wt (g) 

15-Month Interim Evaluation 

pg Nilg control lung 

pg Nilg lung 

pg Nillung 

Absolute lung wt (g) 

Female 

7-Month Interim Evaluation 

pg Nilg control lung 

pg Nilg lung 

pg Nillung 

Absolute lung wt (8) 

15-Month Interim Evaluation 

pg Nilg control lung 

pg Ni/g lung 

pg Nillung 

Absolute lung wt (g) 

5 5 5 

0.219 f 0.015b - 

C 

- 

- 

0.184 f 0.009 - 

- 

- 

15 * 0.5** 

10 f 0.2** 

3 f 0.1** 

0.311 f 0.016** 

0.354 f 0.017** 

24 * 1.9** 

12 f 0.7** 

4 f 0.3** 

20 * 1.6** 

11 f 1.2* 

4 f 0.3** 

0.406 f 0.018** 

44 f 4.0** 

20 f 0.9** 

8 f 0.7** 

0.410 f 0.017** 

0.247 f 0.026 0.325 f 0.020* 

19 f 1.0** 

14 f 1.0** 

5 f 0.2** 

0.358 * 0.012** - 

13 f 0.4** 

10 * 0.6** 

3 f 0.1** - 

- 

0.178 f 0.011 

30 f 4.9** 

15 * 1.8** 

5 f 0.9** 

0.346 f 0.019** 

61 f 7.4** 

26 f 2.2** 

11 f 1.3** 

0.414 f 0.020** - 

- 

- 

a Mean f standard error; statistical tests were performed on unrounded data. 

** P<O.Ol 

* Significantly different (P10.05) from the control group by Williams’ test (lung weight) or DUM’S or Shirley’s test (lung burden parameters) 

evaluation. 

Results were below 0.0037 pg Ni (the limit of detection) for the 7-mOnth interim evaluation and below 0.0034 pg Ni for the 15-month interim 

n=4 
88 Nickel Subsulfide, NTP TR 453 

GENETIC TOXICOLOGY 

mutagenicity noted in the presence of 10 % rat liver 

TA100, with the most consistent demonstration of 

activity were observed with and without S9 in 

strain TA100. Varied indications of mutagenic 

be equivocal overall due to the response seen in 

(Table El). Results of these tests were considered to 

mutations in five strains of Salmonella typhimurium 

Nickel subsulfide was tested for induction of gene 

(Table E2). 

chromatic erythrocytes in peripheral blood samples 

increase in the frequency of micronucleated normofor 13 weeks. Nickel subsulfide did not induce an 

cytes of male and female mice exposed by inhalation 

induction of micronuclei in normochromatic erythrowithout S9. Nickel subsulfide was also tested for 

strains TA97, TA98, TA102, or TA1535, with or 

S9. No mutagenic responses were observed in 
2 years. H&E; 120~ 

of a male F344/N rat exposed to 1 mg nickel subsulfide/m3 by inhalation for lung of a male F344/N rat exposed to 0.15 mg nickel subsulfide/m3 by 

Squamous differentiation within an alveolarhronchiolar carcinoma in the lung Connective (scirrhous) tissue within an alveolarhronchiolar carcinoma in the 

PLATE 1 PLATE 2 

inhalation for 2 years. H&E; 12OX 

to 0.15 mg nickel subsulfide/m3 by inhalation for 2 years. H&E; 180 x exposed to 0.15 mg nickel subsulfide/m3 by inhalation for 2 years. Connective 

Focal alveolar epithelial hyperplasia in the lung of a male F344/N rat exposed Fibrosis and chronic active inflammation in the lung of a male F344/N rat 

PLATE 3 

phospholipid. H&E; 18OX 

tissue with slit-like spaces (S) that may have contained cholesterol or 

PLATE 4 
2 years. H&E; 215 x 

B6C3F, mouse exposed to 1.2 mg nickel subsulfide/m3 by inhalation for exposed to 1.2 mg nickel subsulfide/m3 by inhalation for 2 years. Note 

Protein and macrophages (arrow) in alveolar spaces in the lung of a male Atrophy of olfactory epithelium (A) in the nose of a male B6C3F, mouse 

PLATE 5 PLATE 6 

inflammatory exudate (arrow) within the lumen. H&E; 215 X 
89 

DISCUSSION AND CONCLUSIONS 

rats and mice exposed by inhalation. 

and carcinogenic properties of nickel subsulfide in 

studies were performed to determine the toxicologic 

ing operations (Doll et al., 1990). These NTP 

and during roasting, sintering, calcining, and smeltduring the unloading and crushing of nickel matte, 

Workplace exposure to nickel subsulfide can occur 

active inflammation. 

samples were consistent with the pulmonary chronic 

of macrophages and neutrophils within lavage 

tissue (Benson et al., 1989), and increased numbers 

identified by histopathologic analysis of the lung 

sured in lung lavage fluid) paralleled the toxicity 

The biochemical' indices for lung toxicity (as meanodes that were greater than those in the controls. 

lymphoid hyperplasia in the respiratory tract lymph 

in the lungs, atrophy of the olfactory epithelium, and 

dences of inflammation, hyperplasia, andlor fibrosis 

weights greater than those of the controls and inciindications of respiratory toxicity included lung 

deaths. In the 16-day and 13-week studies, general 

(1.8 mg nickel/m3), there were no chemical-related 

concentration was 2.5 mg nickel subsulfide/m3 

In the 13-week studies, where the highest exposure 

these deaths were attributed to respiratory toxicity. 

subsulfide/m3 died before the end of the studies; 

male and female mice exposed to 10 mg nickel 

0.44 to 7.33 mg nickel/m3)], one male rat and all 

In the 16-day studies [0.6 to 10 mg/m3 (equivalent to 

plasmic proteins, thereby causing toxicity. 

across the cell membrane and interact with cytonickel compounds because the nickel ions can diffuse 

compounds may be more toxic than the insoluble 

hydrate) being the most toxic. The soluble nickel 

most soluble nickel compound (nickel sulfate hexacompounds in water and biological fluids, with the 

toxicity reflects the relative solubility of the nickel 

subsulfide (Tables 32 and 33). The lung and nasal 

toxic and nickel oxide was less toxic than nickel 

pound studies, nickel sulfate hexahydrate was more 

In the companion 16-day and 13-week nickel comet al., 1994). 

bronchiolar epithelium (Lee et al., 1993; Costa 

damage to cytoplasmic components of the alveolar/ 

may be phagocytized and may not cause as extensive 

Alternatively, the water-insoluble nickel compounds 

was otherwise generally similar in each study. 

plasia in the paracortical region of the lymph nodes 

studies, the morphologic appearance of the hyperhexahydrate, nickel subsulfide, or cadmium oxide 

lymph nodes of animals from the nickel sulfate 

although pigment granules were not present in the 

present in the lung and respiratory tract lymph nodes; 

studies. Nickel oxide pigment granules were also 

tract lymph nodes of rats and mice from all of these 

without inflammation was present in the respiratory 

oxide (NTP, 1995). Lymphoid hyperplasia with or 

gallium oxide (NTP, unpublished data), and cadmium 

oxide, gallium arsenide (NTP, unpublished data), 

particles including nickel sulfate hexahydrate, nickel 

subsulfide was similar to that observed with other 

rats and mice after 13 weeks of exposure to nickel 

The spectrum of inflammatory lesions in the lungs of 

ology or vaginal cytology occurred in rats or mice. 

cant effects of nickel subsulfide on sperm morphimmune response (Haley et al., 1990). No signifiwas no evidence for suppression of the humoral 

exposed to 2.5 mg/m3 for 13 weeks, although there 

crease in cell-mediated immunity occurred in mice 

In nickel subsulfide immunotoxicity studies, a depresented in Table 34. 

2-year nickel studies and their nickel equivalents are 

exposure concentrations for the 16-day, 13-week, and 

tions in the 13-week studies. The nickel compound 

lesions in the lung at the higher exposure concentraweights and incidences and severity of inflammatory 

1.2 mg/m3 for mice because of increased lung 

studies were limited to 1 mg/m3 for rats and 

The highest exposure concentrations for these 2-year 

exposure-related effects on survival. Body weights 

In the 2-year nickel subsulfide studies, there were no 
90 Nickel Subsulfide, NTP TR 453 

olfactory epithelium. 

hyperplasia, and/or fibrosis, and atrophy of the 

the lungs of rats and mice including inflammation, 

the controls, and increased incidences of lesions in 

pared to controls), lung weights greater than those of 

respiratory stress (labored breathing patterns comry toxicity was manifested by clinical findings of 

0.6 and 1.2 mg/m3 male and female mice. Respiratowere reduced in 1 mg/m3 male and female rats and in 

range for this neoplasm in NTP inhalation studies. 

bined) in females exceeded the historical control 

alveolar/bronchiolar adenoma or carcinoma (comcarcinoma (combined) in females. The incidence of 

alveolar/bronchiolar carcinoma and adenoma or 

adenoma or carcinoma (combined) in males and of 

ces of alveolar/bronchiolar adenoma, carcinoma, and 

there were exposure-related increases in the incidenevidence of a carcinogenic response in rats because 

male and female rats. This was considered clear 

Nickel subsulfide exposure caused lung neoplasms in 

oxide). 

the nickel compound retained in the lungs (nickel 

sulfide) gave a stronger carcinogenic response than 

more rapidly cleared from the lungs (nickel subtions of these studies, the nickel compound that was 

eventual carcinogenic response, and under the condiThe type of nickel compound is important in the 

does not predict or parallel the lung tumor response. 

of nickel in the lung (as measured in these studies), 

there was 3 to 7 pg nickel/g lung. Thus, the amount 

15 months; in the 2-year nickel subsulfide study, 

was approximately 300 to 1,100 pg nickellg lung at 

over time. In the 2-year study of nickel oxide, there 

between the amount of nickel deposited and cleared 

and this measurement represents the difference 

various time points in the 2-year studies (Table 32), 

The level of nickel in the lung was measured at 

1,000 pg/g lung, the levels approached steady state. 

levels increased in the nickel oxide studies to 

below 30 pg nickel/g lung, and while lung nickel 

load; nickel subsulfide levels at 15 months remained 

subsulfide. There is no evidence for particle over2.5 mg/m3 nickel oxide and 0.15 and 1 mg/m3 nickel 

occurred at exposure concentrations of 1.25 and 

studies were chemically related; these responses 

served in the nickel oxide and nickel subsulfide 

The pulmonary and lung neoplasm responses obrelated to accumulation of nontoxic particles. 

250 mg/m3, suggesting that the response may be 

response was observed only at an exposure level of 

studies in rats (Lee et al., 1985), a lung neoplasm 

2 years. Similarly, in the titanium dioxide inhalation 

25 mg talc/g lung was recorded at 18 months and 

at this exposure concentration, approximately 

talc studies (NTP, 1993) was seen at 18 mg/m3; and 

genic response in the rat lung reported in the NTP 

lung neoplasm response. For example, the carcinoconcentrations (10 mg/m3 or greater) to produce a 

talc, or chromium dioxide) required higher aerosol 

relatively nontoxic particles (i.e., titanium dioxide, 

In contrast, rat carcinogenicity studies with other 

molecular changes involved in the response. 

pathologic nature of lung neoplasms and of the 

Comparisons could also be made of the histo1988) are required to produce similar effects in rats. 

1986) and titanium dioxide (250 mg/m3; Lee et al., 

trations of antimony trioxide (45 mg/m3; Groth et al., 

effects in the lungs of rats. Higher exposure concen- 

(18 mg/m3; NTP, 1993) produced carcinogenic 

Mauderly et al., 1987; Mauderly, 1994) and talc 

exposure levels to diesel exhaust (3.5 to 7 mg/m3; 

produced neoplastic responses in rats. Intermediate 

nickel (NTP, 1996a,b) or cadmium (NTP, 1995) 

For example, relatively low levels of exposure to 

various chemicals at various levels of concentration. 

neoplasms observed after inhalation exposure to 

Comparisons could be made of the types of lung 

than rats exposed to the same compound. Five of 

susceptible to proliferative and fibrotic lung lesions 

all three studies were similar in that mice were less 

neoplasms) between rats and mice were not marked, 

olar epithelial hyperplasia and alveolar/bronchiolar 

difference in the proliferative responses (focal alveof spontaneously occurring neoplasms. Although the 

greater incidence than the controls and were typical 

each of the three nickel compounds occurred with no 

to nickel sulfate hexahydrate and of mice exposed to 

The alveolar/bronchiolar neoplasms in rats exposed 

control mice were similar for all three compounds. 

were similar for all three compounds; lung lesions in 

severity of spontaneous lung lesions in control rats 

sulfate hexahydrate. Incidence, appearance, and 

exposure to nickel subsulfide, nickel oxide, or nickel 

lung pathology in rats and mice after 2 years of 

Some generalities can be made about the comparative 
Discussion and Conclusions 91 

particulates such as talc (NTP, 1993). 

but has been observed in rats exposed to inhaled 

spontaneous alveolar/bronchiolar neoplasms in rats, 

squamous differentiation is not characteristic of 

marked squamous differentiation. Such proliferative 

adenomas in rats exposed to nickel subsulfide had 

carcinomas and two f the alveolarlbronchiolar 

to nickel oxide and four of the alveolar/bronchiolar 

the alveolar/bronchiolar carcinomas in rats exposed 

only in rats and mice exposed to nickel oxide. 

in the lungs and bronchial lymph nodes was observed 

exposed to nickel oxide. Exposure-related pigment 

in rats exposed to nickel subsulfide than in rats 

filling of air spaces were somewhat more prominent 

tive alveolar epithelial proliferation, and collapse or 

fibrosis. Foci of necrotic cellular debris, regeneramacrophages in alveoli and multifocally extensive 

protein accumulations and variable numbers offoamy 

sulfide/m3 and 2.5 mg nickel oxide/m3 had abundant 

The lungs of rats exposed to 1 mg nickel subparenchymal damage secondary to inflammation. 

nickel subsulfide and nickel oxide had significant 

sulfate hexahydrate. The lungs of rats exposed to 

more severe than those in rats exposed to nickel 

rats exposed to nickel subsulfide or nickel oxide were 

lesions and severity of inflammation in the lungs of 

The increases in the incidences of proliferative 

debris. 

nuclear cells with little evidence of necrotic cell 

and nickel subsulfide were predominantly monoinflammatory foci in mice exposed to nickel oxide 

mice exposed to nickel sulfate hexahydrate, while 

and necrotic cell debris were relatively common in 

all three studies. Inflammatory foci with neutrophils 

inflammatory cells) were similar in exposed mice in 

vessels, and multifocal intra-alveolar aggregates of 

macrophages, mononuclear inflammatory cells around 

inflammatory reaction (intra-alveolar protein and 

nickel compound studies. The components of the 

the lungs of exposed mice were similar in all three 

the nickel oxide study, the nonneoplastic lesions in 

With the exception of the pigment obierved only in 

exposure to talc (NTP, 1993). In the talc study, it 

similar to those reported after chronic inhalation 

exposed to nickel subsulfide and nickel oxide were 

phage hyperplasia, and fibrosis of the lung in rats 

The inflammatory alterations of proteinosis, macroinflammatory cells in the lungs. 

pulmonary function, and cytokines released by 

increased pulmonary vascular resistance or impaired 

increased demand for catecholamines to overcome 

for adrenal medullary proliferative lesions include an 

might be related. Factors that could act as stimuli 

ations in the lungs suggests that these two findings 

which treated rats have marked inflammatory altersynthesizing cells of the adrenal medulla in studies in 

ganglia cells. Proliferation of the catecholaminefrom the same embryonic population as sympathetic 

epinephrine and norepinephrine, and are derived 

of the adrenal medulla secrete the catecholamines, 

studies with F344 rats, especially in males. The cells 

relatively common spontaneous neoplasms in chronic 

and pheochromocytoma. Pheochromocytomas are 

creased incidences of adrenal medullary hyperplasia 

subsulfide and nickel oxide, had significantly inlike rats in the current 2-year studies of nickel 

pulmonary function. Rats in the lifetime talc study, 

was concluded that these lesions resulted in impaired 

compounds tested. 

of rats or mice exposed to any of the three nickel 

incidences of proliferative lesions in the nasal cavity 

was no evidence of a chemical-related increase in the 

either rats or mice. Furthermore, after 2 years, there 

there was no evidence for nasal sinus neoplasms in 

nickel oxide study. In the nickel subsulfide studies, 

hexahydrate, the nasal mucosa was not affected in the 

2-year studies of nickel subsulfide and nickel sulfate 

epithelium of rats and mice in the 13-week and 

the atrophic changes were present in the olfactory 

the target site for chemical-related toxicity. Although 

rather than the respiratory or squamous mucosa, was 

present rodent studies, the olfactory epithelium, 

cell carcinoma (Sunderman et al., 1989). In the 

classified as anaplastic, undifferentiated, or squamous 

these sinonasal neoplasms in humans have been 

attributed to nickel exposure. The preponderance of 

Nasopharyngeal carcinoma in humans ' has been 

benign or malignant pheochromocytoma in male and 

female rats in the talc study (NTP, 1993) and of 

(combined) of the adrenal medulla in male and 

incidences of benign or malignant pheochromocytoma 

cytoma in female rats. There were also increased 

chromocytomas in male rats and benign pheochromoincreased incidences of benign and malignant pheoIn this 2-year rat study, there were exposure-related 
92 Nickel Subsulfide, NTP TR 453 

(NTP, 1988) and reserpine (NCI, 1982). 

(NTP, 1990) and orally to 2-mercaptobenzothiazole 

reported in rats exposed by inhalation to bromoethane 

incidences of pheochromocytoma have also been 

(Lee et al., 1988). Chemical-related increases in the 

trisulfide (Groth et al., 1986) and titanium dioxide 

or to other particulates including antimony trioxide or 

observed in rats exposed to nickel sulfate hexahydrate 

to nickel oxide. However, similar increases were not 

of benign pheochromocytoma in female rats exposed 

lung tumors in Syrian hamsters (Muhle et al., 1992). 

tracheally instilled nickel subsulfide did not induce 

344 rats (Yarita and Nettesheim, 1978), while intraWistar rats (Pott et al., 1987) and in female Fisher 

subsulfide induced malignant lung tumors in female 

1975). Multiple intratracheal instillations of nickel 

nickel subsulfide inhalation study (Ottolenghi et al., 

in 1 mg/m3 rats was observed in the one previous 

chemical. A chemical-related increase in lung tumors 

demonstrated the carcinogenic potential of this 

causes tumors after local injection (Table 3) and 

Other studies have shown that nickel subsulfide 

by inhalation. 

response in the lung after exposure to certain metals 

more susceptible than the mouse to a carcinogenic 

compounds such as cadmium, suggest that the rat is 

and from carcinogenicity studies of other metal 

1990). Results from these nickel subsulfide studies, 

tumor response in strain A/J mice (McNeill et al., 

evidence of an exposure-related increase in lung 

intratracheally or intraperitoneally, provided no 

carcinogenesis of nickel subsulfide, administered 

of nickel metal powder. A study of the pulmonary 

neoplasms in mice after a single intrapleural injection 

Hueper (1955) did not observe chemical-related 

lived longer than 12 months. In earlier studies, 

15 mg nickel/m3, although only three of 12 mice 

bronchial mucosa in female C57B1 mice exposed to 

Hueper (1958) observed no abnormalities of the 

have not shown evidence of carcinogenicity in mice. 

Previous studies of nickel pulmonary carcinogenesis 

gene patterns in in vitro studies (Haugen et al., 1989; 

lead to specific alterations in oncogene or suppressor 

and DNA (Huang et al., 1994). This damage may 

cells, which could cause oxidative damage to proteins 

produces a high level of oxidants in the nuclei of 

Recent studies have shown that nickel subsulfide 

lung after nickel subsulfide exposures. 

elucidate the molecular changes that occur in the rat 

genetic alterations, and further studies should help 

nickel subsulfide carcinogenesis includes specific 

These studies suggest that the mechanism involved in 

radicals (Shi et al., 1994; Tajmir-Riahi et al., 1994). 

molecule to enhance the generation of genotoxic free 

the ability of the metal and sulfide portions of the 

genic properties of nickel subsulfide may be due to 

Recent studies have also suggested that the carcinooxide or nickel sulfate (Patierno et al., 1993). 

tracheal epithelial cells more readily than nickel 

et al., 1994). Nickel subsulfide transforms rat 

Higinbotham et al., 1992; Maehle et al., 1992; Lin 

risk. 

possible to separate the types of exposure and cancer 

areas of sulfidic nickel exposure, and it was not 

soluble nickel levels were also at their highest in the 

of 2 to 8 mg nickel/m3. Oxidic nickel levels and 

and lung cancer at estimated exposure concentrations 

strong association between exposure to sulfidic nickel 

from the Sudbury sinter plant (Copper Cliff) show a 

risk of lung neoplasms (Doll et al., 1990). Studies 

workers in nickel industries may lead to an increased 

epidemiology findings that show that exposure of 

lung after nickel subsulfide exposure agree with 

The findings of the carcinogenic response in the rat 

(8/24) as well as those from the roasting and smelting 

curred in workers from the electrolysis department 

lung. This study also found that lung cancer ocanalysis did not detect sulfides (e.g., Ni,S,) in the 

an earlier study using energy dispersive X-ray 

nickel in the lung was probably nickel oxide because 

Anderson and Svenes (1989) felt that the retained 

concentration was independent of smoking habits. 

(25 cases) who died of other causes. The lung nickel 

concentrations at auopsy as nickel workers 

with lung cancer (14 cases) had the same lung nickel 

and Escobedo, 1976). Workers who were diagnosed 

approximately 20% "wet" lung weight (Henderson 

people, 0.76 k 0.39 pg/g. Dry lung represents 

34 k 48 pg/g; and for lung tissue from unexposed 

weight; for workers in electrolysis departments, 

operations was 330 k 380 pg nickel/g dry lung 

concentration for workers in roasting and smelting 

(Andersen and Svenes, 1989). The average nickel 

workers showed increased lung nickel levels 

Lung tissue specimens from 39 nickel refinery 
Discussion and Conclusions 93 

trolysis department had lower lung nickel levels. 

operations (6/15) even though those from the elecsubsulfide. 

TLV in rats in the current 2-year study of nickel 

rats and mice. Lung neoplasms occurred below the 

for nasal toxicity was approximately 0.3 mg/m3 for 

lung toxicity in mice was 0.15 mg/m3. The NOAEL 

0.15 mg/m3 (0.1 1 mg nickel/m3). The NOAEL for 

occurred even at the lowest exposure concentration of 

not reached for rats; exposure-related lung lesions 

adverse-effect level (NOAEL) for lung toxicity was 

concentrations below the TLV. A no-observedfound in the respiratory tract of rodents at exposure 

13-week studies, chemical-related toxic lesions were 

insoluble nickel compounds is 1 mg/m3, and in these 

The current threshold limit value (TLV) for waterCONCLUSIONS 

based on increased incidences of alveolar/bronchiolar 

activity* of nickel subsulfide in male F344/N rats 

studies, there was clear evidence of carcinogenic 

Under the conditions of these 2-year inhalation 

1.2 mg/m3. 

in male or female B6C3F, mice exposed to 0.6 or 

evidence of carcinogenic activity of nickel subsulfide 

chromocytoma of the adrenal medulla. There was no 

bined) and an increased incidence of benign pheoalveolar/bronchiolar adenoma or carcinoma (comincidences of alveolar/bronchiolar carcinoma and 

subsulfide in female F344/N rats based on increased 

was clear evidence of carcinogenic activity of nickel 

pheochromocytoma of the adrenal medulla. There 

malignant, and benign or malignant (combined) 

(combined) and on increased incidences of benign, 

adenoma, carcinoma, and adenoma or carcinoma 

the olfactory epithelium in the nose. 

fibrosis in the lung and inflammation and atrophy of 

inflammation, bronchialization, hyperplasia, and 

subsulfide by inhalation for 2 years resulted in 

Exposure of male and female mice to nickel 

and hyperplasia in the adrenal medulla (females). 

and atrophy of the olfactory epithelium in the nose; 

hyperplasia, and fibrosis in the lung; inflammation 

by inhalation for 2 years resulted in inflammation, 

Exposure of male and female rats to nickel subsulfide 

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

comments and the public discussion on this report appears on page 13. 
Selected Results in the 16-Day Inhalation Studies of Nickel Sulfate Hexahydrate, Nickel Subsulfide, and Nickel Oxidea 

TABLE 32 

(mg Ni/m3) 

Dose mg/m-’ 

(0.7) (1.4) (3.1) (6.1) (12.2) 

0 3.5 7 15 30 60 0 0.6 1.2 2.5 5 10 0 1.2 2.5 5 10 30 

Nickel Sulfate Hexahydrate Nickel Subsulfide Nickel Oxide 

(0.44) (0.88) (1.83) (3.65) (7.33) (0.9) (2.0) (3.9) (7.9) (23.6) 

Male Rats 

Survival 55 5553 555554 55555 5 

Absolute Lung Weightsb 

(Relative to Controls) 

Final Mean Body Weights - 72% 60% 56% 55% 45% - 109% 105% 92% 72% 52% - 99% 101% 99% 99% 96% 

0.98 1.44** 1.45**1.40* 1.40* 1.62** 1.13 1.41 1.60* 1.59* 1.82**1.54** 1.06 1.00 1.06 0.96 1.20* 1.36** 

Female mts 

Survival 55 5540 555555 55555 5 

(Relative to Controls) 

Final Mean Body Weights - 82% 71% 68% 63% - - 99% 97% 91% 78% 57% - 103% 103% 104% 101% 99% 

Absolute Lung Weights 0.76 1.28* 1.28* 1.32* 1.40**1.52** 0.82 1.12**1.12**1.36**1.42**1.25** 0.78 0.86 0.90 0.82 1.04** 1.12** 

(continued) 
Selected Results in the 16-Day Inhalation Studies of Nickel Sulfate Hexahydrate, Nickel Subsulfide, and Nickel Oxide (continued) 

TABLE 32 

Y 

(mg Nilm-’) 

Dose mg/m’ 0 3.5 7 15 30 60 0 0.6 1.2 2.5 5 10 0 1.2 2.5 5 10 30 

Nickel Sulfate Hexahydrate Nickel Subsulfide Nickel Oxide 

(0.7) (1.4) (3.1) (6.1) (12.2) (0.44) (0.88) (1.83) (3.65) (7.33) (0.9) (2.0) (3.9) (7.9) (23.6) 

2 

E 

a 

Male Mice 

C 

er 

(Relative to Controls) 

Final Mean Body Weights 

Survival 55 0000 454550 55545 5 

e 

v) 

- 95% - - - - - 99% 90% 92% 86% - - 100% 100% 98% 102% 94% 

Absolute Lung Weights 0.20 0.24 0.40**0.36**0.36**0.38** 0.22 0.20 0.22 80.31**0.38** 0.20 0.16 0.20 0.13** 0.20 

Female Mice 

Survival 55 0000 455550 55555 5 

Final Mean Body Weights 

(Relative to Controls) - 96% - - - - - 106% 104% 101% 99% - - 100% 96% 100% 95% 95% 

Absolute Lung Weights 0.16 0.22 0.36**0.36**0.38**0.40** 0.20 0.21 0.22 70.36* 0.25 0.16 0.16 0.14 0.18 20.20 

a Survival data indicate number of animals surviving. Five animals initially in group. Final mean body weights are not presented for groups with 100% mortality. 

** P10.01 

* Significantly different (P10.05) from the control by Williams’ or Dunnett’s test 

Organ weights are given in grams. 
Selected Results in the 13-Week Inhalation Studies of Nickel Sulfate Hexahydrate, Nickel Subsulfide, and Nickel Oxidea 

TABLE 33 

u+ 

\9 

(mg Ni/m’) 

Dose rng/rn’ 

(0.03) (0.06) (0.11) (0.22) (0.44) 

Nickel Sulfate Hexahydrate 

0 0.12 0.25 0.5 1 2 0 0.15 0.3 0.6 1.2 2.5 0 0.6 1.2 2.5 5 10 

Nickel Subsulfide Nickel Oxide 

(0.11) (0.22) (0.44) (0.88) (1.83) (0.4) (0.9) (2.0) (3.9) (7.9) 

Final Mean Body Weights 

Survival 

Male Rats 

(Relative to Controls) 

Alveolar Macrophage, 

Nonneoplastic Lung Lesions 

Absolute Lung Weights 

Hyperplasia 

(Severity) 

(Severity) 

(Severity) 

(Severity) 

(Severity)b 

Pigment 

Interstitial Infiltrate 

Inflammation, Granulomatous 

Inflammation, Chronic Active 

Nonneoplastic Nasal Lesions 

Atrophy, Olfactory Epithelium 

10 10 10 10 10 9 

- 99% 103% 96% 102% 95% 

1.35 1.25 1.51* 1.64**2.14**2.22** 

0 10 10 10 10 9 

00 02108 

00 0000 

(1.0) (1.5) (1.3) 

(1.0) (1.0) (1.0) (2.4) (3.6) 

10 15109 

(1 .O) (1.0) (1.0) (1.0) (1.1) 

00 0000 

00 01109 

10 10 10 10 10 10 

- 100% 95% 96% 99% 93% 

1.33 1.74**1.83**2.30**2.63**2.42** 

000000 

0 2 9 10 10 10 

0 10 10 10 10 10 

(1.0) (1.3) (1.8) (2.9) (3.7) 

(1.1) (1.5) (1.6) (3.4) (3.8) 

000000 

0011098 

(1.0) (1.9) (2.1) (1.2) 

0 0 15 1010 

10 10 10 9 10 10 

- 103% 104% 99% 102% 100% 

1.18 1.35**1.47**1.70**1.91** 2.47** 

06799 

001210 

00003 

000210 

0 10 10 9 10 

(1.0) (1.7) 

(1.0) (1.0) (1.0) (1.5) 

(2.0) 

(1.0) (1.0) (1.4) 

(1.0) (1.0) (1.0) (1.0) 

00000 0 

(continued) 
Selected Results in the 13-Week Inhalation Studies of Nickel- Sulfate Hexahydrate, Nickel Subsulfide, and Nickel Oxide (continued) 

TABLE 33 

(0.03) (0.06) (0.11) (0.22) (0.44) 

Nickel Sulfate Hexahvdrate 

0 0.12 0.25 0.5 1 2 0 0.15 0.3 0.6 1.2 2.5 0 0.6 1.2 2.5 5 10 

Nickel Subsulfide Nickel Oxide 0 

(mg Nim’) 

Dose mg/m’ g. E 

(0.11) (0.22) (0.44) (0.88) (1.83) (0.4) (0.9)- (2.0) (3.9) (7.9) 3 

a 

Y 

P) 

Final Mean Body Weights 

Survival 

Female Rats 

(Relative to Controls) 

2 

3 

10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 

R 

i;’ 

E 

0, 

- 96% 98% 98% 101% 95% - 101% 104% 101% 100% 99% - 101% 101% 98% 98% 100% 

Absolute Lung Weights 1.02 1.02 1.16**1.34**1.72**1.72** 1.01 1.29**1.39**1.82**1.85**1.81** 0.98 1.03 1.13* 1.55**1.61** 2.11** 

Alveolar Macrophage, 

Nonneoplastic Lung Lesions 

Hyperplasia 

(Severity) 

(Severity) 

(Severity) 

(Severity) 

(Severity) 

Pigment 

Interstitial Infiltrate 

Inflammation, Granulomatous 

Inflammation, Chronic Active 

0 8 10 10 10 10 

00 0 4 10 10 

00 0000 

(1.0) (1.3) (1.0) 

(1.0) (1.0) (1.1) (2.2) (3.6) 

00 0 6 10 10 

00 0000 

(1.0) (1.0) (1.0) 

000000 

0 3 9 10 10 10 

0 10 10 10 10 10 

(1.0) (1.0) (1.9) (2.6) (3.8) 

(1.0) (1.7) (1.8) (2.9) (3.8) 

000000 

0029105 

(1.0) (1.7) (2.4) (1.6) 

0000 

0001 

0 10 8 10 

(1 .O) 

(1.0) (1.0) (1.0) 

0048 

0002 

(1.0) (1.0) 

(1 .O) 

10 10 

(1.4) (2.2) 

(1.0) (1.2) 

8 10 

(1.2) 8

10 10 

(2.1) 0

44 

(1.3) 27

7 7 

NOMeOplaStiC Nasal Lesions 

Atrophy, Olfactory Epithelium 00 1 2 10 10 0008910 00000 0 

(continued) 
Selected Results in the 13-Week Inhalation Studies of Nickel Sulfate Hexahydrate, Nickel Subsulfide, and Nickel Oxide (continued) 

TABLE 33 

(0.03) (0.06) (0.11) (0.22) (0.44) 

Nickel Sulfate Hexahvdrate 

0 0.12 0.25 0.5 1 2 0 0.15 0.3 0.6 1.2 2.5 0 0.6 1.2 2.5 5 10 

Nickel Subsulfide Nickel Oxide 

(mg Ni/m3) 

Dose mg/m' 

(0.11) (0.22) (0.44) (0.88) (1.83) (0.4) (0.9) (2.0) (3.9) (7.9) 

Male Mice 

Survival 6 8' 10 10 10 8 10 10 8 9 10 10 10 10 10 9 

(Relative to Controls) 

Final Mean Body Weights 

- 105% 100% 104% 104% 102% - 102% 106% 103% 101% 97% - 101% 99% 97% 98% 97% 

Absolute Lung Weights 0.20 0.20 0.20 0.21 0.25**0.31** 0.19 0.20 0.22 0.21 0.23* 0.28** 0.21 0.22 0.21 0.21 0.24 0.29** 

Nonneoplastic Lung Lesions 

Pigment 

Interstitial Infiltrate 

Inflammation, Granulomatous 

Inflammation, Chronic Active 

Fibrosis, Focal 

Alyeolar Macrophage, Hyperplasia 

(Severity) 

(Severity) 

(Severity) 

(Severity) 

(Severity) 

(Severity) 

0 0 0 10 10 10 

00 00210 

00 0022 

00 0000 

(1.0) (1.0) (1.0) 

(1.0) (1.5) 

(1.5) (2.0) 

00 0028 

00 0000 

(1.0) (1.0) 

000000 

000057 

0000510 

0088910 

(1.2) (1.6) 

(1.6) (2.1) 

(1.0) (1.0) (2.0) (2.2) 

000000 

010232 

(1 .O) (1.0) (1.2) (1.5) 

00000 

0 10 10 10 10 

(1.0) (1.0) (1.0) (1.0) 

00000 

00000 

0 10 10 10 10 

00013 

(1.0) (1.0) (1.0) (1.0) 

(1.0) (1.0) 

0 

(1.1) 

9 

Nonneoplastic Nasal Lesions 

Atrophy, Olfactory Epithelium 00 00010 0005510 00000 0 

(continued) 

E 

e 

wl 

g 

E 

" 
Selected Results in the 13-Week Inhalation Studies of Nickel Sulfate Hexahydrate, Nickel Subsulfide, and Nickel Oxide (continued) 

TABLE 33 

E g. 

g 

(0.03) (0.06) (0.11) (0.22) (0.44) 

Nickel Sulfate Hexahvdrate 

0 0.12 0.25 0.5 1 2 0 0.15 0.3 0.6 1.2 2.5 0 0.6 1.2 2.5 5 10 

Nickel Subsulfide Nickel Oxide 0 

(mg Nilm’) 

Dose mg/m3 

(0.11) (0.22) (0.44) (0.88) (1.83) (0.4) (0.9) (2.0) (3.9) (7.9) J 

a 

D 

P) 

Female Mice 2 

h 

J 

Final Mean Body Weights 

Survival 7 10 10 10 10 10 10 8 10 9 10 8 9 10 7 10 10 9 

.- 

2 

g. F 

(Relative to Controls) - 105% 104% 105% 103% 97% - 101% 100% 101% 101% 99% 

0.20 0.20 0.20 0.20 0.22 0.27** 0.19 0.18 0.20 0.21 0.26**0.29** 0.20 0.20 0.19 0.21 0.22 0.27** 

- 97% 100% 96% 94% 97% 

Absolute Lung Weights 

Nonneoplastic Lung Lesions 

Pigment 

Interstitial Infiltrate 

Inflammation, Granulomatous 

Inflammation, Chronic Active 

Fibrosis, Focal 

Alveolar Macrophage, Hyperplasia 

(Severity) 

(Severity) 

(Severity) 

(Severity) 

(Severity) 

(Severity) 

00 010 

00 00 

(1 .O) 

000 

004 

(1 .O) 

0 

9 

(1.0) (1.0) 

10 

0 00 

(1.1) (1.0) 

10 9 

00 00 000 0 00 0 0 

(1.0) 

10 9 

(1.1) (1.1) 

68 

(1 .O) 

0 1 

(1.0) (1.1) 

1 3 

00 00 000 0 00 0 0 

0 

8 

(1.3) 

000 

023 

(1.0) (1.0) 

4 

(1.0) 

0 

1 

0 

(1.0) 

0 

9 

(1.4) 

0 

8 

(1.7) 

0 

Nonneoplastic Nasal Lesions 

Atrophy, Olfactory Epithelium 00 0005 0001610 00000 0 

a Survival data indicate number of animals surviving. Ten animals initially in group. Final mean body weights are not presented for groups with 100% mortality. 

** PSO.01 

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

Nine animals initially in group. 

Average severity grade of lesions in affected animals: l=minimal, 2=mild, 3=rnoderate, 4=rnarked 
100 Nickel Subsulfide, NTP TR 453 

of Nickel Sulfate Hexahydrate, Nickel Subsulfide, and Nickel Oxidea 

Comparison of Exposure Concentrations in the lCDay, 13-Week, and 2-Year Studies 

TABLE 34 

Amount of Compound Amount of Nickel 

16-Day Studies 

Nickel Sulfate 

Hexahydrate (22.3% Ni) 

Nickel Subsulfide (73.3% Ni) 

Nickel Oxide (78.6% Ni) 

13-Week Studies 

Nickel Sulfate 

Hexahydrate (22.3% Ni) 

Nickel Subsulfide (73.3% Ni) 

Nickel Oxide (78.6% Ni) 

0, 3.5, 7, 15, 30, 60 

0, 0.6, 1.2, 2.5, 5, 10 

0, 1.2, 2.5, 5, 10, 30 

0, 0.12. 0.25, 0.5, 1, 2 

0, 0.15, 0.3, 0.6, 1.2, 2.5 

0, 0.6, 1.2, 2.5, 5, 10 

Nickel Sulfate 

2-Year Studies 

Hexahydrate (22.3% Ni) 

Mice 

Rats 

0, 0.25, 0.5, 1 

0, 0.12. 0.25, 0.5 

Nickel Subsulfide (73.3% Ni) 

Mice 

Rats 

0, 0.6, 1.2 

0, 0.15, 1 

Nickel Oxide (78.6% Ni) 

Mice 

Rats 

0, 1.25, 2.5, 5 

0, 0.62. 1.25, 2.5 

0, 0.7, 1.4, 3.1, 6.1, 12.2 

0, 0.44, 0.88, 1.83, 3.65, 7.33 

0, 0.9, 2.0, 3.9, 7.9, 23.6 

0, 0.4, 0.9, 2.0, 3.9, 7.9 

0, 0.11, 0.22. 0.44, 0.88, 1.83 

0, 0.03. 0.06, 0.11. 0.22. 0.44 

0, 0.06. 0.11. 0.22 

0, 0.03. 0.06, 0.11 

0, 0.44. 0.88 

0, 0.11, 0.73 

0, 1.0, 2.0, 3.9 

0, 0.5, 1.0, 2.0 

a Amounts of nickel and nickel compounds are expressed in mg/mJ. Occupational exposure limits in the United States: 1 mg Nilm’ for 

nickel metals, 0.1 mg Ni/m3 for soluble nickel compounds. 
Selected Results in the 2-Year Inhalation Studies of Nickel Sulfate Hexahydrate, Nickel Subsulfide, and Nickel Oxidea 

TABLE 35 

U 

Dose mg/m' 0 0.12 0.25 0.5 

Nickel Sulfate Hexahydrate (22.3% Ni) 

0 0.15 1 

Nickel Subsulfide (73.3% Ni) Nickel Oxide (78.6% Ni) 

0 0.62 1.25 2.5 

E 

I: 

v1 

(mg NU& (0.03) (0.06) (0.11) (0.11) (0.73) (0.5) (1.0) (2.0) 

a 

3 

w 

Final Mean Body Weights 

Survival 

Male Rats 

(Relative to Controls) 

16/54 16/55 

- 99 % 

Absolute Lung Weights 

7-Month Interim Evaluation 1.67 1.62 

15-Month Interim Evaluation 2.12 2.48 

Alveolar/bronchiolar Proliferative Lesions and Neoplasms 

Adenoma or Carcinoma (Combined) 2b 0 

Carcinoma 

Adenoma 0 0 

Hyperplasia, Focal or Atypical 3 2 

Alveolar Epithelial 

2b 0 

Adrenal Medulla Proliferative Lesions and Neoplasms 

Hyperplasia 28 20 

Benign Pheochromocytoma 16 16 

Malignant Pheochromocytoma 0 3 

Benign or Malignant Pheochromocytoma 16 19 

18/55 

101 % 

2.50 

1.65 

1 

1 

0 

3 

13 

2 

12 

18 

Carcinogenic Activity 

(continued) 

No evidence 

21/55 

98 % 

3.00** 

1.89 

3 

1 

2 

2 

12 

1 

11 

26 

13/53 2 1 /53 18/53 

- 98 % 85 % 

1.87 2.38** 3.48** 

2.27 3.31** 6.84** 

2 6 11** 

0 3 6* 

0 3 7* 

0 6* 11** 

26 22 10 

13 30** 37** 

0 2 11** 

14 30** 42** 

Clear evidence 

14/54 15/53 15/53 12/52 

- 100% 95% 93% 

1.72 1.85 2.43** 2.59** 

2.20 2.15 3.30** 4.09** 

0 2 5* 3 

0 1 3 2 

3 2 

6' 4' 

lb 0 

Ib 1 

25 21 26 24 

27 24 26 32 

0 0 1 6* 

27 24 27 35** 

Some evidence 
Selected Results in the 2-Year Inhalation Studies of Nickel Sulfate Hexahydrate, Nickel Subsulfide, and Nickel Oxide (continued) 

TABLE 35 

c 

w 

Dose mg/m3 

Nickel Sulfate Hexahydrate (22.3% Ni) Nickel Subsulfide (73.3% Ni) 

0 0.12 0.25 0.5 0 0.15 1 0 0.62 1.25 2.5 

Nickel Oxide (78.6% Ni) 

(mg Ni/& (0.03) (0.06) (0.11) (0.11) (0.73) (0.5) (1.0) (2.0) 

Female Rats 

Survival 22/52 17/53 28/53 29/54 25/53 25/53 28/52 21/53 26/53 20153 26/54 

Final Meari Body Weights 

(Relative to Controls) - 97% 97% 94% - 96 % 78 x - ' 96% 92% 90% 

Absolute Lung Weights 

15-Month Interim Evaluation 

7-Month Interim Evaluation 1.25 1.22 1.22 1.45* 

1.37 1.57 1.49 1.82** 

1.31 1.75** 2.59** 

1.52 2.52** 4.14** 

1.14 1.31* 1.65** 1.78** 

1.56 1.79 2.41** 3.02** 

Alveoiatlbronchiolar Proliferative Lesions and Neoplasms 

Alveolar Epithelial 

Hyperplasia, Focal or Atypical 5 3 7 9 

Adenoma 0 0 0 1 

Carcinoma 0 0 0 0 

Adenoma or Carcinoma (Combined) 0 0 0 1 

Adrenal Medulla Proliferative Lesions and Neoplasms 

Hyperplasia 6 4 8 8 

Benign Pheochromocytoma 2 4 2 3 

Malignant Pheochromocytoma 0 0 0 0 

Benign or Malignant Pheochromocytoma 2 4 2 3 

2 6b.d 9* 

0 4 

2 5 5 

2 10* 11** 2 1 6 6 

1 0 1 4 

0 0 5* 1 

1 0 gd 5d 

lb 

5 11 16** 

2 7 36** 

1 0 1 

3 7 36** 

8 12 14 22** 

4 7 6 18** 

0 0 0 0 

4 7 6 18** 

Carcinogenic Activity No evidence Clear evidence Some evidence 

(continued) 

3 

& 

8 
Selected Results in the 2-Year Inhalation Studies of Nickel Sulfate Hexahydrate, Nickel Subsulfide, and Nickel Oxide (continued) 

TABLE 35 

(0.44) (0.88) 

0 0.6 1.2 

Nickel Sulfate Hexahydrate (22.3% Ni) Nickel Subsulfide (73.3% Ni) 

0 1.25 2.5 5 

Nickel Oxide (78.6% Ni) 3 

(mg Nilm’) 

Dose mg/m’ 0 0.25 0.5 1 ul 

(0.06) (0.11) (0.22) (1.0) (2.0) (3.9) 

a 

Male Mice F 

(Relative to Controls) 

Final Mean Body Weights 

Survival 26/61 23/61 24/62 25/61 26/61 25/59 26/58 19/57 23/67 29/66 23/69 

e 

R 

8. 

0 

- 94% 97% 91% - 92 % 92 % - 93% 93% 93% 

Absolute Lung Weights 

15-Month Interim Evaluation 

7-Month Interim Evaluation 0.21 0.20 0.22 0.23 

0.24 0.25 0.26 0.31** 

0.24 0.27 0.34** 

0.23 0.40** 0.41** 

0.19 0.21 0.24** 0.24** 

0.23 0.25 0.31* 0.38** 

Alveolar/bronchiolar Proliferative Lesions and Neoplasms 

Alveolar Epithelial 

Adenoma or Carcinoma (Combined) 13 18 7 8 

Carcinoma 9 13 4 3 

Adenoma 5 5 3 5 

Hyperplasia, Focal 0 0 0 0 0 0 0 

6 3 2 

I 2 4 

13 5 6 

1 1 2 0 

7 5 6 11 

4 10 9 6 

9 14 15 14 

Carcinogenic Activity No evidence No evidence No evidence 

(continued) 
Selected Results in the 2-Year Inhalation Studies of Nickel Sulfate Hexahydrate, Nickel Subsulfide, and Nickel Oxide (continued) 

TABLE 35 

CI 

z 

Nickel Sulfate Hexahydrate (22.3% Ni) 

0 0.6 1.2 

Nickel Subsulfide (73.3% Ni) 

0 1.25 2.5 5 

Nickel Oxide (78.6% Ni) 

(mg Ni/m’) 

Dose mg/m’ 0 0.25 0.5 1 

(0.06) (0.11) (0.22) (0.44) (0.88)(1.0) (2.0) (3.9) 

Final Mean Body Weights 

Survival 

Female Mice 

(Relative to Controls) 

34/61 39/60 45/60 

- 91% 94% 

Absolute Lung Weights 

7-Month Interim Evaluation 0.22 0.21 0.22 

15-Month Interim Evaluation 0.24 0.24 0.28 

Alveolarlbronchiolar Proliferative Lesions and Neoplasms 

Alveolar Epithelial 

Adenoma or Carcinoma (Combined) 7 6 10 

Carcinoma 4 3 9 

Adenoma 3 3 2 

0 1 1 

No evidence 

Hyperplasia, Focal 

Carcinogenic Activity 

37/60 

88 % 

0.33** 

0.25 

2 

2 

0 

0 

36/58 

- 

0.26 

0.19 

9 

I 

3 

0 

34/59 

90% 

0.39** 

0.26* 

2 

1 

1 

0 

No evidence 

38/60 

86 % 

0.50** 

0.29** 

3 

2 

1 

0 

41/64 40/66 42/63 38/64 

- 96% 94% 90% 

0.18 0.21 0.23 0.23 

0.25 0.26 0.29 0.34** 

0 0 1 0 

2 4 10* 3 

4 11 4 5 

6 15* 12 8 

Equivocal evidence 

a Survival data indicate number of animals surviving/number initially in group. 

** P10.01 

* Significantly different (P10.05) from the control by Williams’ or Dunnett’s test (lung weights) or the logistic regression test (incidences). 

Significantly different (P < 0.05) from the Lovelace Inhalation Toxicology Research Institute historical controls [4/208 (1.9%)] 

Significantly different (P<O.O5) from the Lovelace Inhalation Toxicology Research Institute historical controls [3/210 (1.4%)] 

Includes data for squamous cell carcinoma 
Lung Burden Analyses in the 16-Day, 13-Week, and 2-Year Studies of Nickel Sulfate Hexahydrate, Nickel Subsulfide, and Nickel Oxidea 

TABLE 36 

I 

(mg Nim7 

Dose mg/m3 0 0.12 0.5 2 3.5 15 30 0 0.15 0.6 2.5 10 

Nickel Sulfate Hexahydrate (22.3% Ni) Nickel Subsulfide (73.3% Ni) 

(0.4) (0.9) (2.0) (3.9) (7.9) 

0 0.6 1.2 . 2.5 5 10 

Nickel Oxide (78.6% Ni) 

2 

rn 

(0.03) (0.06) (0.44)(0.7) (3.1) (6.1) (0.11) (0.44) (1.83) (7.33) D 

a 

j 

Female Rats 

Male Rats 

16-Day Studies 

E 

2 

- b 

8 11 9 

598 - 7 18 67- 42 108 267 

R 

5 

E 

- - 9 19 77 - 54 122 340 

Male Mice - 3 - 10 20 13 - 32 46 84 

Female Mice - 4 - 8 20 8 - 31 43 71 

Female Mice 

Male Mice 

Female Rats 

Male Rats 

13-Week Studies 

-5 7 18 

-5 7 17 

-6 

-3 11 17 

13 23 

- 80 181 524 

- 42 202 736 

(continued) 
Lung Burden Analyses in the lCDay, 13-Week, and 2-Year Studies of Nickel Sulfate Hexahydrate, Nickel Subsulfide, and Nickel Oxide (continued) 

TABLE 36 

CI 

B 

(mg Ni/m3) 

Dose mgtm’ 

(0.03) (0.06) (0.11) (0.22) (0.44) (0.11) (0.44) (0.73) (0.88) 

0 0.12 0.25 0.5 1 2 0 0.15 0.6 1 1.2 

Nickel Sulfate Hexahvdrate (22.3% Ni) Nickel Subsulfide (73.3% Ni) 

0 0.62 1.25 2.55 10 

Nickel Oxide (78.6% Ni) -I 

(0.5) (1.0) (2.0) (3.9) (7.9) 

Female Mice 

Male Mice 

Female Rats 

Male Rats 

7-Month Interim Evaluation 

Female Mice 

Male Mice 

Female Rats 

Male Rats 

15-Month ‘Interim Evaluation 

- -1 

- -1 

122 

112 

- -1 

- -2 

122 

112 

-6 

-6 

- 

- 

9 

9 

10 

10 11 

14 

-4 3 

-4 7 

- 12 

26 

20 

- 15 

- 175 388 701 

- 173 477 713 

- 

169 533 861 

162 442 1,034 

- 

- 328 746 1,116 

- 262 706 949 

- 

451 1,237 2,258 

331 959 1,798 

- 

a Values represent mean amounts of nickel (pg Ni/g lung). Lung burden groups included five to seven animals. 

Results were below the limit of detection. 
107 

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Department of the Interior, Bureau of Mines, 

Mineral Facts and Problems, 1985 Edition. U.S. 

United States Bureau of Mines (1985a). Nickel. In 

Washington, DC. 

Department of the Interior, Bureau of Mines, 

Mineral Commodity Summaries 1985. U.S. 

United States Bureau of Mines (1985b). Nickel. In 

Washington, DC. 

Department of the Interior, Bureau of Mines, 

Mineral Commodity Summaries 1991. US. 

United States Bureau of Mines (1991). Nickel. In 

Environmental Assessment, Washington, DC. 

883/012FF), pp. 1-83. Office of Health and 

Nickel and Nickel Compounds (EPA/600/ 

(USEPA) (1986). Health Assessment Document for 

United States Environmental Protection Agency 

strain A/J mice. Environ. Res. 34, 328-334. 

clearance of intratracheally administered 63Ni3S, in 

Valentine, R., and Fisher, G.L. (1984). Pulmonary 

nickel workers. Carcinogenesis 5, 1525-1527. 

aberrations in peripheral lymphocytes of retired 

( 1984). Increased incidence of chromosomal 

Waksvik, H., Boysen, M., and H~getveit, A.C. 

University Press, Oxford. 

Scientific Publications No. 53, pp. 419-438. Oxford 

C. Rosenfeld, R. Saracci, and A. Sors, Eds.), IARC 

P.C. Jacquignon, E. Mastromatteo, J.P. Rigaut, 

C. Bishop, E. Buringh, W. Davis, M. Gounar, 

(F.W. Sunderman, Jr., A. itio, A. Berlin, 

products. In Nickel in the Human Environment 

airborne nickel in producing and using primary nickel 

Warner, J.S. (1984). Occupational exposure to 

H'g. ASSOC. J. 36, 801-810. 

oxide and cigarette smoke by hamsters. Am. Znd. 

Craig, D.K. (1975): Chronic inhalation of nickel 

Wehner, A.P., Busch, R.H., Olson, R.J., and 

hamsters. Prog. Exp. Tumor Res. 24, 177-198. 

(1979). Inhalation studies with Syrian golden 

Wehner, A.P., Stuart, B.O., and Sanders, C.L. 
122 Nickel Subsulfide, NTP TR 453 

Amsterdam. Carcinogenicity of nickel subsulfide for respiratory 

Ms.), pp. 555-558. Elsevier North-Holland, Inc., Yarita, T., and Nettesheim, P. (1978). 

R. Lauwerys, M. Mercier, and M. Roberfroid, 

Toxicity and Hazard Evaluation (B. Holmstedt, 9-22. 

NiO-inhalation in Wistar rats. In Mechanisms of rhabdomyosarcomata in rats. Acta Pathol. Jpn. 30, 

Kordel, W. (1980). Subchronic effects induced by Abandowitz, H.M. (1980). Nickel sulphide-induced 

Weischer, C.H., Oldiges, H., Hochrainer, D., and Yamashiro, S., Gilman, J.P.W., Hulland, T.J., and 

tract mucosa. Cancer Res. 38, 3 140-3 145. 

earlier results with 41 additional chemicals. Environ. 

predicting rodent carcinogenicity: Confirmation of 

Evaluation of four in vitro genetic toxicity tests for 

Margolin, B.H., and Tennant, R. W. (1990). 

Zeiger, E., Haseman, J.K., Shelby, M.D., 

27, 103-1 17. 

are compared with a zero dose control. Biometrics 

between treatment means when several dose levels 

Williams, D.A. (1971). A test for differences 

dose levels with a zero dose control. Biometrics 

Williams, D.A. (1972). The comparison of several Mol. Mutagen. 16 (suppl. 18), 1-14. 

28, 5 19-53 1. 

Environmental Health Criteria 108: Nickel. World 31 1 chemicals. Environ. Mol. Mutagen. 19 

World Health Organization (WHO) (1991). mutagenicity ests: V. Results from the testing of 

and Mortelmans, K. (1992). Salmonella 

Zeiger, E., Anderson, B., Haworth, S., Lawlor, T., 

Health Organization, Geneva. (Suppl. 21), 2-141. 
123 

OF NICKEL SUBSULFIDE 

SUMMARY OF LESIONS IN MALE RATS 

APPENDIX A 

IN THE 2-YEAR INHALATION STUDY 

TABLE A1 Summary of the Incidence of Neoplasms in Male Rats 

in the 2-Year Inhalation Study of Nickel Subsulfide 

TABLE A2 Individual Animal Tumor Pathology of Male Rats 

in the 2-Year Inhalation Study of Nickel Subsulfide 

in the 2-Year Inhalation Study of Nickel Subsulfide 

TABLE A5 Summary of the Incidence of Nonneoplastic Lesions in Male Rats 

in Untreated Male F344/N Rats 

TABLE A4b Historical Incidence of Pheochromocytomas of the Adrenal Medulla 

in Untreated Male F344/N Rats 

TABLE A4a Historical Incidence of Lung Neoplasms 

in the 2-Year Inhalation Study of Nickel Subsulfide 

TABLE A3 Statistical Analysis of Primary Neoplasms in Male Rats 

....................... 

155 

154 

153 

148 

130 

125 

....................... 

........................ 

.................................... 

.................................... 

....................... 
124 Nickel Subsulfide, NTP TR 453 
Lesions in Male Rats 125 

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Inhalation Study of Nickel Subsulfidea 

TABLE A1 

0 mg/m3 0.15 mg/m3 1 mg/m3 

Early deaths 

&Month interim evalualion 

7-Month interim ekluation 

Animals initially in study 

Disposition Summary 

Terminal sacrifice 

Natural deaths 

Moribund 

Survivors 

5 

5 

63 

5 

35 

13 

5 

5 

63 

4 

28 

21 

5 

5 

63 

2 

33 

18 

Animals examined microscopically 63 63 63 

Urinary System 

Special Scenes System 

Respiratory System 

Nervous System 

Musculoskeletal System 

Integumentary System 

Hematopoitic System 

Genital System 

General Body System 

Endocrine System 

Cardiovascular System 

Alimentary System 

Systems Examined At 7 Months With No Neoplasms Observed 

Testes 

Genital System 

15-Month Interim Evaluation 

(5) 

Interstitial cell, adenoma 4 (80%) 

(5) 

1 (20%) 

(5) 

2 (40%) 

Multiple organsb 

Systemic Lesions 

Leukemia mononuclear 

(5) 

1 (20%) 

Urinary System 

Special Scenes System 

Respiratory System 

Nervous System 

Musculoskeletal System 

Integumentary System 

Hematopoitic System 

General Body System 

Endocrine System 

Cardiovascular System 

Alimentary System 

Systems Examined With No Neoplasms Observed 
126 Nickel Subsulfide, NTP TR 453 

Summay of the Incidence of Neoplasms in Male Rats in the 2-Year Inhalation Study of Nickel Subsulfide (continued) 

TABLE A1 

0 mg/mg 0.15 mg/m3 1 mg/m3 

2- Year Study 

Intestine large, rectum 

Intestine large, colon 

Alimentary System 

Intestine small, duodenum 

Fibrosarcoma, metastatic, testes 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Adenocarcinoma 

Histiocytic sarcoma 

Hepatocellular adenoma 

Mesentery 

Salivary glands 

Pharynx 

Pancreas 

Tooth 

Tongue 

Stomach, glandular 

Stomach, forestomach 

Squamous cell papilloma 

Adenoma 

Squamous cell papilloma 

Gingiva, squamous cell carcinoma 

Squamous cell papilloma 

Heart 

Cardiovascular System 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

Bilateral, pheochromocytoma benign 

Bilateral, pheochromocytoma malignant 

Pheochromocytoma benign 

Pheochromocytoma complex 

Pheochromocytoma malignant 

Carcinoma 

Adenoma 

Islets, pancreatic 

Pituitary gland 

Adenoma 

Parathyroid gland 

Pars distalis, adenoma 

C-cell, carcinoma 

C-cell, adenoma 

Thyroid gland 

1 (2%) 

12 (23%) 23 (44%) 

1 (2%) 

1 (2%) 

4 (8%) 

7 (13%) 

(53) 

(52) 

(50) 

17 (33%) 

(53) 

1 (2%) 

2 (4%) 

23 (43%) 

5 (9%) 

14 (26%) 

2 (4%) 

4 (8%) 

(53) 

(51) 

(53) 

(53) 

9 (17%) 

17 (32%) 
Lesions in Male Rats 127 

TABLE A1 

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Inhalation Study of Nickel Subsulfide (continued) 

0 mg/m3 0.15 mg/m3 1 mg/m3 

Tissue NOS 

General Body System 

&Year Smdy (continued) 

Abdominal, fibrosarcoma 

Thoracic, fibrosarcoma 

Thoracic, alveolar/bronchiolar carcinoma, 

Mediastinum, alveolar/bronchiolar carcinoma, 

metastatic, lung 

metastatic, lung 

1 (50%) 

1 (100%) 

1 (100%) 

Genital System 

Testes 

Seminal vesicle 

Prostate 

Carcinoma 

Adenoma 

Preputial gland 

Tunic, fibrosarcoma 

Interstitial cell, adenoma 

Bilateral, interstitial cell, adenoma 

(53) 

2 (4%) 

1 (2%) 

(53) 

(52) 

(53) 

11 (21%) 

28 (53%) 

(53) 

2 (4%) 

(53) 

(53) 

(53) 

15 (28%) 

30 (57%) 

(53) 

(53) 

(53) 

(52) 

1 (2%) 

20 (38%) 

15 (28%) 

Lymph node 

Bone marrow 

Hematopoietic System 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Iliac, fibrosarcoma, metastatic, testes 

Histiocytic sarcoma 

Alveoladbronchiolar carcinoma, metastatic, 

lung 

Thymus 

Spleen 

1 (2%) 

(49) 

(53) 

(49) 

(53) 

Integumentary System 

Fibroadenoma 

Adenoma 

Mammaly gland (49) 

1 (2%) 

(48) 

1 (2%) 

(43) 
128 Nickel Subsulfide, NTP TR 453 

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Inhalation Study of Nickel Subsulfide (continued) 

TABLE A1 

0 mg/m3 0.15 mg/m3 1 mg/m3 

Skin 

Integumentary System (continued) 

&Year study (continued) 

Tail, squamous cell papilloma 

Tail, fibroma 

Subcutaneous tissue, lipoma 

Subcutaneous tissue, fibrosarcoma 

Pinna, squamous cell papilloma 

Pinna, keratoacanthoma 

Nose, squamous cell papilloma 

Squamous cell papilloma 

Keratoacanthoma 

Fibroma 

Carcinoma, metastatic, preputial gland 

Basal cell carcinoma 

Basal cell adenoma 

(53) 

1 (2%) 

4 (8%) 

2 (4%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

(53) 

1 (2%) 

4 (8%) 

2 (4%) 

1 (2%) 

(53) 

1 (2%) 

1 (2%) 

1 (2%) 

3 (6%) 

1 (2%) 

Musculoskeletal System 

Maxilla, osteosarcoma 

Cranium, osteosarcoma 

Bone (53) 

1 (2%) 

(53) 

1 (2%) 

Brain 

Nervous System 

Astrocytoma benign 

Oligodendroglioma NOS 

Astrocytoma NOS 

Spinal cord 

(53) (53) (53) 

1 (2%) 

Lung 

Respiratory System 

Pheochromocytoma malignant, metastatic, 

Osteosarcoma, metastatic, bone 

Histiocytic sarcoma 

Fibrosarcoma, metastatic, testes 

Chordoma, metastatic, tissue NOS 

Carcinoma, metastatic, preputial gland 

Alveolarlbronchiolar carcinoma 

Alveolar/bronchiolar adenoma, multiple 

Alveolarlbronchiolar adenoma 

adrenal medulla 

Trachea 

Nose 

(53) (53) 

3 (6%) 

3 (6%) 

(53) 

7 (13%) 

2 (4%) 

4 (8%) 

I (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

(53) 

(53) 

(53) 

(53) 

1 (2%) 
Lesions in Male Rats 129 

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Inhalation Study of Nickel Subsulfide (continued) 

TABLE A1 

0 mg/m3 0.15 mg/m3 1 mg/mg 

None 

Special Senses System 

&Year study (continued) 

Urinary bladder 

Kidney 

Urinary System 

(53) 

(53) 

Multiple organs 

Systemic Lesions 

Mesothelioma NOS 

Leukemia mononuclear 

Histiocytic sarcoma 

(53) 

1 (2%) 

28 (53%) 

1 (2%) 

(53) 

35 (66%) 

1 (2%) 

32 (60%) 

Total animals with primary neoplasms' 

Neoplasm Summary 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

2-Year study 

2-Year study 

2-Year study 

Total primary neoplasms 

Total animals with benign neoplasms 

Total benign neoplasms 

Total animals with malignant neoplasms 

Total malignant neoplasms 

Total animals with uncertain neoplasmsTotal metastatic neoplasms 

Total animals with metastatic neoplasms 

benign or malignant 

2-Year study 

2-Year study 

Total uncertain neoplasms 

53 

3 

52 

4 

52 

1 

137 

4 

157 

1 

180 

3 

52 

1 

50 

2 

49 

4 

99 

4 

114 

1 

120 

2 

45 

1 

33 37 

59 

1 

37 41 

2 

3 

2 

4 

4 

6 

1 

9 

2 1 

2 1 

' Primary neoplasms: all neoplasms except metastatic neoplasms 

Number of animals with any tissue examined microscopically 

a Number of animals examined microscopically at the site and the number of animals with neoplasm 
130 Nickel Subsulfide, NTP TR 453 

I131792399552348469854459 

5672334555661777999012333 

2335555555555555555666666 

Number of Days on Study 

0000000000000000000000000 

Carcass ID Number 

2329508326977617808468562 

3023674413065140063021456 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Esophagus 

Alimentary System 

++A++++A+++++++++++++++++ 

I+A++++A+++++++++++++++M+ 

++A++++A+++++++++++++++++ 

......................... 

Intestine small, duodenum +++++++A+++++++++++++++++ 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Histiocytic sarcoma 

Hepatocellular adenoma 

Pancreas 

Mesentery 

Tongue 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Adenoma 

Tooth 

Squamous cell papilloma 

......................... 

++A++++A+++++++++++++++++ 

++A++++A+++++++++++++++++ 

X X 

X 

+ ......................... 

......................... 

......................... 

......................... 

+ 

Heart 

Blood vessel 

Cardiovascular System 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

......................... 

......................... 

......................... 

Bilateral, pheochromocytoma benign 

Pheochromocytoma benign 

Pheochromocytoma complex 

Parathyroid gland 

Adenoma 

Islets, pancreatic 

X 

xx 

......................... X 

++M++++++++++++++++++++++ 

Pars distalis, adenoma 

Pituitary gland 

Adenoma 

C-cell, carcinoma 

C-cell, adenoma 

Thyroid gland ++M++++++++++++++++++++++ 

xx xxxx x xxx ......................... 

xx 

xx 

Thoracic, fibrosarcoma 

Tissue NOS 

General Body System 

x 

+ 

A: Autolysis precludes examination 

+ : Tissue examined microscopically 

I: Insufficient tissue 

M: Missing tissue 

Blank: Not examined 

X: Lesion present 
Lesions in Male Rats 131 

(continued) 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Nickel Subsulfide: 0 mg/m3 

TABLE A2 

1741831691563583333666666777 

5567789990001123333333333333 

6666666667771177777717117777 

Number of Days on Study 

Carcass ID Number 

0000000000000000000000000000 Total 

6215432155324361244012366156 Tissues/ 

3991701345450790129673146531 Tumors 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Esophagus 

Alimentary System 

Histiocytic sarcoma 

Hepatocellular adenoma 

Pancreas 

Mesentery 

Tongue 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Adenoma 

Tooth 

Squamous cell papilloma 

............................ 

............................ 

............................ 

............................ 

............................ 

+++++++++++++M++++++++MI ++++ ............................ 

............................ 

X X 

............................ ., A 

............................ 

............................ 

............................ 

X 

+ 

53 

51 

51 

52 

51 

46 

51 

53 

1 

1 

4 

1 

1 

1 

53 

53 

53 

1 

53 

Heart 

Blood vessel 

Cardiovascular System 

+ ............................ 53 

1 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

Bilateral, pheochromocytoma benign 

Pheochromocytoma benign 

Pheochromocytoma complex 

Parathyroid gland 

Adenoma 

Islets, pancreatic 

Pars distalis, adenoma 

Pituitary gland 

Adenoma 

C-cell, carcinoma 

C-cell, adenoma 

Thyroid gland 

............................ 

............................ 

xx xxx xx X xx 

X 

............................ X 

M+++++++++++++++I +++++++++++ 

X 

............................ 

X X X X xxx ............................ X 

X X 

X X 2 

4 

52 

20 

53 

1 

50 

2 

53 

1 

12 

1 

53 

53 

Thoracic, fibrosarcoma 

Tissue NOS 

General Body System 

+ 

1 

2 
132 Nickel Subsulfide, NTP TR 453 

(continued) 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Nickel Subsulfide: 0 mg/m3 

TABLE A2 

7131792399552348469854459 

5672334555667777999012333 

2335555555555555555666666 

2329508326977617808468562 

3023674413065140063021456 

0000000000000000000000000 

Number of Days on Study 

Carcass ID Number 

Preputial gland 

Penis 

Epididymis 

Coagulating gland 

Genital System 

Adenoma 

Testes 

Seminal vesicle 

Prostate 

Carcinoma 

Interstitial cell, adenoma 

Bilateral, interstitial cell, adenoma 

......................... +++ + ++++ + ++ +++++ 

+ ......................... 

X 

......................... 

++A++++++++++++++++++++++ 

......................... X 

X X xx xx X 

xx xx x X 

Bone marrow 

Hematopoietic System ......................... 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node + + 

......................... 

......................... 

+++++++++++++M+++++++++++ 

......................... 

Histiocytic sarcoma X 

Adenoma 

Mammary gland 

Integumentary System 

Thymus 

Spleen 

Keratoacanthoma 

Fibroma 

Carcinoma, metastatic, preputial gland 

Skin 

+++++++++++++++I ++M++++++ 

......................... 

X 

......................... 

I+I+++++++++++++++M++++++ 

X 

X 

X 

Tail, fibroma 

Subcutaneous tissue, lipoma 

Subcutaneous tissue, fibrosarcoma 

Pinna, squamous cell papilloma 

Pinna, keratoacanthoma 

Squamous cell papilloma X 

X 

X 

Bone 

Musculoskeletal System ......................... Cranium, osteosarcoma 

Spinal cord 

Brain 

Nervous System ......................... 

X 

+ 

Astrocytoma benign 
Lesions in Male Rats 133 

(continued) 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Nickel Subsulfide: 0 mg/m3 

TABLE A2 

1741831691563583333666666777 

5567789990001123333333333333 

6666666667777777777777777777 

Number of Days on Study 

Carcass ID Number 

~ ~ 

0000000000000000000000000000 Total 

~~ ~ ~ ~~ 

6215432155324361244012366156 Tissues/ 

3991701345450790729673146531 Tumors 

Penis 

Epididymis 

Coagulating gland 

Genital System 

2 

............................ 53 

++ +++++ +++ +++++++++++++ 39 

+ 

Carcinoma 

Adenoma 

Preputial gland ............................ X 

Testes 

Seminal vesicle 

Prostate 

............................ 

............................ 

............................ 

Interstitial cell, adenoma 

Bilateral, interstitial cell, adenoma xxxxxx x xx xxx xxxxxxxxx 

x xx X X 11 

28 

53 

52 

53 

2 

1 

53 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node 

Bone marrow 

Hematopoietic System 

Thymus 

Spleen 

Histiocytic sarcoma 

........................ ++++ 50 

............................ 53 

1 

++++I +I ++++++++++++++++"+++ 49 

............................ 53 

M++++++++++++++M++++++++++++ 50 

+++++++++++++++++++++++++++M 52 

5 

............................ 53 

+ ++ 

Skin 

Adenoma 

Maminary gland 

Integumentary System 

............................ 53 

1 

+++++++++++++M++++++++++++++ 49 

Tail, fibroma 

Subcutaneous tissue, lipoma 

Subcutaneous tissue, fibrosarcoma 

Pinna, squamous cell papilloma 

Pinna, keratoacanthoma 

Squamous cell papilloma 

Keratoacanthoma 

Fibroma 

Carcinoma, metastatic, preputial gland 

1 

1 

4 

2 

1 

X 

X X X 

X 

X 

1 

1 

1 

1 

X 

Bone 

Musculoskeletal System 

............................ 53 

Cranium, osteosarcoma X 1 

Spinal cord 

Brain 

Nervous System 

1 

............................ 53 

Astrocytoma benign 
134 Nickel Subsulfide, NTP TR 453 

TABLE A2 

(continued) 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Nickel Subsulfide: 0 mg/m3 

7131792399552348469854459 

5672334555667777999012333 

2335555555555555555666666 

2329508326977617808468562 

3023674413065140063021456 

0000000000000000000000000 

Number of Days on Study 

Carcass ID Number 

Lung 

Larynx 

Respiratory System 

X 

......................... 

+++++++++++++++I I ++++I +++ 

Osteosarcoma, metastatic, bone 

Histiocytic sarcoma 

Carcinoma, metastatic, preputial gland 

X 

Trachea 

Nose 

......................... 

......................... 

EY e 

Special Senses System 

+ + 

Urinary bladder 

Kidney 

Urinary System 

......................... 

+++++++A+++++++++++++++++ 

Multiple organs 

Systemic Lesions ~ ~~ 

Mesothelioma NOS 

Leukemia mononuclear 

Histiocytic sarcoma 

......................... X 

xx x x x xx x x A 
Lesions in Male Rats 135 

(continued) 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Nickel Subsulfide: 0 mg/m3 

TABLE A2 

~ 

1741831691563583333666666777 

5567789990001123333333333333 

6666666667777777777777777777 

0000000000000000000000000000 Total 

6215432155324361244012366156 Tissues/ 

3991101345450790729673146531 Tumors 

~ ~~ 

Number of Days on Study 

Carcass ID Number 

Lung 

Larynx 

Respiratory System 

++++++++++++++++++++++I +++++ 49 

............................ 53 

Carcinoma, metastatic, preputial gland 1 

Histiocytic sarcoma 1 

Osteosarcoma, metastatic, bone X 1 

............................ 53 

Trachea ............................ 53 

Nose 

EY e 

Special Senses System 

+ + 4 

Urinary bladder 

Kidney 

Urinary System 

............................ 52 

............................ 53 

Multiple organs 

Systemic Lesions 

Mesothelioma NOS 

Leukemia mononuclear 

Histiocytic sarcoma 

1 

xxxxx xxxx x xx xxx xx 28 

1 

............................ 53 
136 Nickel Subsulfide, NTP TR 453 

8589239044599183348899201 

9534566799999002223333456 

3455555555555666666666666 

Number of Days on Study 

Carcass ID Number 

2840433887259071672407741 

6877667060745955674968980 

1111111212111111111111112 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Esophagus 

Alimentary System 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Adenocarcinoma 

Pancreas 

Mesentery 

Tooth 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Adenoma 

Hepatocellular adenoma 

......................... 

+++++++++++++A++A+++++++A 

+++++++M+MM++++++++M+++++ 

+++++++++++++A+++++++++++ 

......................... 

......................... 

+++++++++++++A+++++++++++ 

+++++++++++++A+++++++++++ 

X 

+ ......................... 

+ 

......................... 

......................... 

++++++++++++++M++++++++++ 

Heart 

Cardiovascular System 

......................... 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

X 

++.+++++++M+++++++++++++++ 

......................... 

Bilateral, pheochromocytoma benign 

Pheochromocytoma benign 

Pheochromocytoma malignant 

Carcinoma 

Adenoma 

Islets, pancreatic 

Pars distalis, adenoma 

Pituitary gland 

Adenoma 

Parathyroid gland 

C-cell, carcinoma 

C-cell, adenoma 

Thyroid gland 

Tissue NOS 

General Body System 

xx 

X 

x xx X 

X X 

......................... 

X 

++++M++M++++++++++++++++M 

......................... 

+M+++++++++++++++++++++++ 

xx x X X X X 

X 

+ 

Mediastinum, alveolar/bronchiolar 

carcinoma, metastatic, lung 

Thoracic, alveoladbronchiolar 

carcinoma, metastatic, lung X 

X 
Lesions in Male Rats 137 

(continued) 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Nickel Subsulfide: 0.15 mg/m3 

TABLE A2 

0503187333333333666666666777 

9901112333333333333333333333 

6677717717777777777777777777 

Number of Days on Study 

Carcass ID Number 

1111211111111222111111112112 Total 

4459044445699000556678890691 Tissues/ 

8349591465912369265790132150 Tumors 

Esophagus 

Alimentary System 

............................ 53 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon A+++++++++++++++++++++++++++ 51 

A+++++++++++++++M+++++++MM++ 45 

A+++++++++++++++++++++++++++ 49 

A+++++++++++++++++++++++++++ 52 

Adenocarcinoma X 1 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Pancreas 

Mesentery 

Tooth 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Adenoma 

Hepatocellular adenoma 

............................ 

A+++++++++++++++++++++++++++ 

A+++++++++++++++++++++++++++ 

............................ X 

............................ 

............................ 

............................ 

53 

51 

51 

1 

53 

53 

52 

1 

53 

1 

1 

Heart 

Cardiovascular System 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

............................ 53 

............................ 53 

............................ 52 

Bilateral, pheochromocytoma benign 

Pheochromocytoma benign 

Pheochromocytoma malignant 

Carcinoma 

Adenoma 

Islets, pancreatic 

Pars distalis, adenoma 

Pituitary gland 

Adenoma 

Parathyroid gland 

C-cell, carcinoma 

C-cell, adenoma 

Thyroid gland 

xx xxx 

X 

1 

2 

............................ 53 

xx xxx xx 17 

............................ 52 

1 

............................ 50 

1 

X 4 

............................ 53 

X X 7 

xx x x xxx 23 

2 

X X 

X X X 

X 

X 

X X X 

X 

X 

Tissue NOS 

General Body System 

1 

Mediastinum, alveolar/bronchiolar 

carcinoma, metastatic, lung 

Thoracic, alveolar/bronchiolar 

carcinoma, metastatic, lung 1 

1 
138 Nickel Subsulfide, NTP TR 453 

TABLE A2 

(continued) 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Nickel Subsulfide: 0.15 mg/m3 

8589239044599183348899201 

9534566799999002223333456 

3455555555555666666666666 

Number of Days on Study 

2840433887259071672407741 

6877667060745955674968980 

1111111212111111111111112 

Carcass ID Number 

Preputial gland 

Epididymis 

Coagulating gland 

Genital System 

Testes 

Seminal vesicle 

Prostate 

Adenoma 

++++++ +++++++ +++ ++++ 

......................... 

......................... 

X 

......................... 

......................... 

......................... 

xxxxxxxxxxxx x xx 

Interstitial cell, adenoma 

Bilateral, interstitial cell, adenoma 

xx X X 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node 

Bone marrow 

Hematopoietic System 

metastatic, lung 

Alveolar/bronchiolar carcinoma, 

Thymus 

Spleen 

Mesothelioma NOS, metastatic 

........................ 

......................... + ++ ++++ + +++ 

+++++++M+++++++++++++++++ 

......................... 

+++M+++++++++++++++++++++ 

X ......................... 

++++++++M++++++++++++++++ 

Skin 

Mammary gland 

Integumentary System 

Fibroadenoma 

Tail, squamous cell papilloma 

Keratoacanthoma 

Fibroma 

Basal cell carcinoma 

I++M++++++++++++M++++++++ 

A 

......................... 

X 

X 

Bone 

Musculoskeletal System 

......................... 

Nervous System 

Respiratory System 

Spinal cord 

Brain 

Lung 

Larynx 

......................... Oligodendroglioma NOS X 

+ 

......................... 

......................... 

Alveolar/bronchiolar carcinoma 

Alveolar/bronchiolar adenoma X 

X X 

Trachea 

Nose 

......................... 

......................... 
Lesions in Male Rats 139 

(continued) 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Nickel Subsulfide: 0.15 mg/m3 

TABLE A2 

0503787333333333666666666777 

99011123333333333.33333333333 

6677777777777777777777777777 

Number of Days on Study 

Carcass ID Number 

1111211111111222111111112112 Total 

4459044445699000556678890691 Tissues/ 

8349591465912369265790132150 Tumors 

~ ~~ 

Preputial gland 

Epididymis 

Coagulating gland 

Genital System 

Testes 

Seminal vesicle 

Prostate 

Adenoma 

Interstitial cell, adenoma 

Bilateral, interstitial cell, adenoma 

............................ 

............................ 

++++++++++++ +++++++++++++++ 

A 

............................ 

............................ 

............................ 

x x xx xxxxxx xx xx x 

xxx xx X xx x xx 

53 

53 

47 

15 

30 

53 

53 

53 

2 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node 

Bone marrow 

Hematopoietic System 

metastatic, lung 

Alveolar/bronchiolar carcinoma, 

Thymus 

Spleen 

Mesothelioma NOS, metastatic 

............................ 

+++++++++++++++M++++++++++++ 

............................ 

+ +++ + 

+++++++++++++++M++++++++++++ 

+++++++++++++MM++++++M++++++ 

............................ X 

+M++++M+++++++++++++++M+++++ 

49 

53 

51 

51 

16 

52 

49 

1 

53 

1 

Skin 

Mammary gland 

Integumentary System 

1 

4 

2 

1 

............................ 53 

1 

++++++++++++++++++++++M++++M 48 

Fibroadenoma 

Tail, squamous cell papilloma 

Keratoacanthoma 

Fibroma 

Basal cell carcinoma X 

X 

X xx 

X 

Bone 

Musculoskeletal System 

+++++++++++++++M++++++++++++ 52 

Spinal cord 

Brain 

Nervous System 

1 

1 

............................ 53 

Oligodendroglioma NOS 

Lung 

Larynx 

Respiratory System 

............................ 53 

............................ 53 

3 

3 

............................ 53 

............................ 53 

Alveoladbronchiolar carcinoma 

Alveolar/bronchiolar adenoma xx 

X 

Trachea 

Nose 
140 Nickel Subsulfide, NTP TR 453 

(continued) 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Nickel Subsulfide: 0.15 mg/m3 

TABLE A2 

8589239044599183348899201 

9534566799999002223333456 

3455555555555666666666666 

2840433887259071672407741 

6877667060745955674968980 

1111111212111111111111112 

Number of Days on Study 

Carcass ID Number 

EY e 

Special Senses System 

++ + 

Urinary bladder 

Urethra 

Kidney 

Urinary System 

......................... + ......................... 

Multiple organs 

Systemic Lesions 

Mesothelioma NOS 

Leukemia mononuclear X xx xxxx xx xxxxxxxx ......................... 
Lesions in Male Rats 141 

(continued) 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Nickel Subsulfide: 0.15 mg/m3 

TABLE A2 

0503787333333333666666666177 

9901112333333333333333333333 

6677777777777771717777777717 

Number of Days on Study 

Carcass ID Number 

~ 

1111211111111222111111112112 Total 

4459044445699000556678890691 Tissues/ 

8349591465912369265790132150 Tumors 

EY e 

Special Senses System 

+ 4 

Urinary bladder 

Urethra 

Kidney 

Urinary System 

............................ 53 

1 

............................ 53 

Multiple organs 

Systemic Lesions 

Mesothelioma NOS 

Leukemia mononuclear 

1 

x xxxxxxxx X 32 

............................ 53 

x x xxx 

X 
142 Nickel Subsulfide, NTP TR 453 

8497290447840451139238033 

0202556667189992222444566 

4455555555555556666666666 

7402000801682975352534047 

4110904352148831201288202 

3333233333332233333322333 

Number of Days on Study 

Carcass ID Number 

Intestine large, rectum 

Intestine large, colon 

Esophagus 

Alimentary System 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Pharynx 

Pancreas 

Mesentery 

Tooth 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Fibrosarcoma, metastatic, testes 

Hepatocellular adenoma 

Squamous cell papilloma 

Squamous cell papilloma 

Gingiva, squamous cell carcinoma 

++++++++M++++++++++++++++ 

......................... 

......................... 

......................... 

+++++++++++A+++++++++++++ 

+++++++++++A+++++++++++++ 

......................... 

+++++++++++M+++++++++++++ 

X 

......................... 

......................... 

......................... 

......................... 

X 

+ 

Heart 

Blood vessel 

Cardiovascular System 

......................... 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

Bilateral, pheochromocytoma 

Pheochromocytoma benign 

Pheochromocytoma malignant 

Bilateral, pheochromocytoma benign 

Carcinoma 

Adenoma 

Islets, pancreatic 

Thyroid gland 

Pituitary gland 

Parathyroid gland 

malignant 

C-cell, adenoma 

Pars distalis, adenoma 

......................... 

......................... 

X xxx X X 

X 

......................... xx x xx xx 

X X 

......................... 

M++++++++++++++++++++++++ 

X 

xx x xx x xx x x ......................... X 

Tissue NOS 

General Body System 

Abdominal, fibrosarcoma X 

+ 
Lesions in Male Rats 143 

TABLE A2 

(continued) 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Nickel Subsulfide: 1 mg/m3 

8333716361333333336666666777 

7000011223333333333333333333 

6777777777777777777777777777 

Number of Days on Study 

2223323233233333332233333333 Total 

9994383944900011129901334233 Tissues/ 

7589380621478913891937596846 Tumors 

Carcass ID Number 

Intestine large, rectum 

Intestine large, colon 

Esophagus 

Alimentary System 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Pharynx 

Pancreas 

Mesentery 

Tooth 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Fibrosarcoma, metastatic, testes 

Hepatocellular adenoma 

Squamous cell papilloma 

Squamous cell papilloma 

Gingiva, squamous cell carcinoma 

............................ 

............................ 

............................ 

............................ 

............................ 

............................ 

............................ 

............................ 

X xx 

............................ + 

X 

+ 

............................ 

............................ 

A 

............................ 

52 

53 

53 

53 

52 

52 

53 

52 

1 

1 

1 

53 

1 

53 

53 

1 

1 

53 

1 

3 

Heart 

Blood vessel 

Cardiovascular System 

+ ............................ 53 

1 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

Bilateral, pheochromocytoma 

Pheochromocytoma benign 

Pheochromocytoma malignant 

Bilateral, pheochromocytoma benign 

Carcinoma 

Adenoma 

Islets, pancreatic 

Thyroid gland 

Pituitary gland 

Parathyroid gland 

malignant 

C-cell, adenoma 

Pars distalis, adenoma 

............................ 

............................ 

x xx x 

x xx x 

X X 

X xx X 

xx xx X 

............................ 

xx x x xxx x x xx xx xx 

X X 

X 

............................ 

+++++++++++++M++++++++++++++ 

xx X 

xx X xxx 

X X xx 

X 

............................ 

14 

6 

53 

53 

9 

53 

17 

53 

51 

2 

4 

53 

23 

5 

Tissue NOS 

General Body System 

Abdominal, fibrosarcoma 1 

1 
144 Nickel Subsulfide, NTP TR 453 

(continued) 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Nickel Subsulfide: 1 mg/m3 

TABLE A2 

8497290447840451139238033 

0202556667789992222444566 

4455555555555556666666666 

7402000801682975352534047 

4110904352148831201288202 

3333233333332233333322333 

Number of Days on Study 

Carcass ID Number 

Testes 

Seminal vesicle 

Prostate 

Preputial gland 

Penis 

Epididymis 

Coagulating gland 

Genital System 

......................... +++++++++++++++++++ ++++ 

......................... 

......................... 

......................... 

......................... 

xx xx x 

+ 

Tunic, fibrosarcoma 

Interstitial cell, adenoma 

Bilateral, interstitial cell, adenoma 

Lymph node 

Bone marrow 

Hematopoietic System 

Thymus 

Spleen 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

X xx 

xx x 

X 

+ + 

......................... 

X 

+ + 

Iliac, fibrosarcoma, metastatic, testes 

++++++++M+++++++M+++M++++ 

......................... 

++I++++++++++++++++++++++ 

......................... 

++++++++++++++M++++++++++ 

......................... 

~ ~~ ~ ~~ ~~ ~ ~ ~~ 

Skin 

Mammary gland 

Integumentary System 

~~ 

......................... 

......................... 

Subcutaneous tissue, lipoma 

Nose, squamous cell papilloma 

Keratoacanthoma 

Fibroma 

Basal cell adenoma X 

X 

Maxilla, osteosarcoma 

Bone 

Musculoskeletal System ......................... X 

Brain 

Nervous System 

Astrocytoma NOS 

......................... X 
Lesions in Male Rats 145 

TABLE A2 

(continued) 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Nickel Subsulfide: 1 mg/m3 

8333716361333333336666666777 

7000011223333333333333333333 

6777777777777777777777777777 

2223323233233333332233333333 Total 

99943839449000111299013342334233 Tissues/ 

7589380621478913891937596846 Tumors 

Number of Days on Study 

Carcass ID Number 

Testes 

Seminal vesicle 

Prostate 

Preputial gland 

Penis 

Epididymis 

Coagulating gland 

Genital System 

Tunic, fibrosarcoma 1 

Interstitial cell, adenoma 20 

Bilateral, interstitial cell, adenoma X x xx X X xx x xxx 15 

............................ 53 

++M+++++++++++++++++++++++++ 52 

............................ 53 

............................ 53 

1 

............................ 53 

++ +++++ ++++++++ ++++++++++ 48 

Lymph node 

Bone marrow 

Hematopoietic System 

Thymus 

Spleen 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

xx x X X xxx x xx x 

+++++++++++M++++++++++++++++ 49 

............................ 53 

+++++M++++++++++++++++++M+++ 50 

............................ 53 

+++++++++++++++++M++++M+++++ 50 

............................ 53 

1 

6 

............................ 53 

+ + 

Iliac, fibrosarcoma, metastatic, testes 

Skin 

Mammary gland 

Integumentary System 

Subcutaneous tissue, lipoma 

Nose, squamous cell papilloma 

Keratoacanthoma 

Fibroma 

Basal cell adenoma 

+++++++I M++++MMM++MM++MM+++M 43 

............................ 53 

X 

X 

X 

X 1 

1 

1 

3 

1 

X 

Maxilla, osteosarcoma 

Bone 

Musculoskeletal System 

1 

............................ 53 

Brain 

Nervous System 

1 

............................ 53 

Astrocytoma NOS 
146 Nickel Subsulfide, NTP TR 453 

8497290447840451139238033 

0202556661789992222444566 

4455555555555556666666666 

Number of Days on Study 

7402000801682975352534047 

4110904352148831201288202 

3333233333332233333322333 

Carcass ID Number 

Lung 

Larynx 

Respiratory System 

......................... 

I ......................... 

Pheochromocytoma malignant, 

Osteosarcoma, metastatic, bone 

Fibrosarcoma, metastatic, testes 

Chordoma, metastatic, tissue NOS 

Alveolar/bronchiolar carcinoma 

Alveolar/bronchiolar adenoma, multiple 

Alveolar/bronchiolar adenoma 

metastatic, adrenal medulla 

X X 

X 

X 

Trachea 

Nose 

......................... 

......................... 

EY e 

Special Senses System 

Urinaly bladder 

Kidney 

Urinary System 

Multiple organs 

Systemic Lesions 

+ 

......................... 

......................... 

xxxxx xx x xx x xxxxx xx ......................... Leukemia mononuclear 
Lesions in Male Rats 147 

(continued) 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Nickel Subsulfide: 1 mg/m’ 

TABLE A2 

8333716361333333336666666777 

7000011223333333333333333333 

6777777777777777777777777777 

2223323233233333332233333333 Total 

9994383944900011129901334233 Tissues/ 

7589380621478913891937596846 Tumors 

Number of Days on Study 

Carcass ID Number 

~ 

Lung 

Larynx 

Respiratory System ~~~ 

Pheochromocytoma malignant, 

Osteosarcoma, metastatic, bone 

Fibrosarcoma, metastatic, testes 

Chordoma, metastatic, tissue NOS 

Alveolar/bronchiolar carcinoma 

Alveolar/bronchiolar adenoma, multiple 

Alveolar/bronch~olar adenoma 

metastatic, adrenal medulla 

Trachea 

Nose 

............................ 

............................. 

X X xx 

X X 

X xx xx 

X 

X 

............................ 

++++++++++ +++++++++++++++++ 

1 

1 

1 

7 

2 

4 

53 

52 

53 

52 

1 

EY e 

Special Senses System 

+ + 3 

Urinary bladder 

Kidney 

Urinary System 

............................ 53 

............................ 53 

~~ 

Multiple organs 

Systemic Lesions 

Leukemia mononuclear 

............................ 53 

xxx xxx xx X x xx xx xxx 35 
148 Nickel Subsulfide, NTP TR 453 

of Nickel Subsulfide 

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Inhalation Study 

TABLE A3 

O mg/m3 0.15 mg/m3 1 mg/m3 

Fisher exact test 

Cochran-Armita e testd 

Logistic regression testd 

Life table testd 

First incidence (days) 

Terminal rate' 

Adjusted rateb 

Overall ratea 

Adrenal Medulla: Benign Pheochromocytoma 

P<O.OOl 

P<O.OOl 

P=O.006 

559 

4/13 (31%) 

54.4% 

13/53 (25%) 

9 

P=O.o02 

P=O.O54 

455 

16/21 (76%) 

84.6% 

30/52 (58 %) 

P < 0.001 

P=O.004 

552 

13/18 (72%) 

88.0% 

37/53 (70%) 

P<O.OOl P < 0.001 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Adrenal Medulla: Malignant Pheochromocytoma 

0/13 (0%) 

0.0% 

0153 (0%) 

e 

P<O.OOl 

P<O.OOl 

P<O.OOl 

- 

P=O.242 

P=O.309 

60 1 

0/21 (0%) 

6.9% 

2/52 (4%) 

P=O.O02 

P=O.O04 

703 

7/18 (39%) 

48.6% 

11/53 (21 %) 

P=O.243 P<O.OOl 

Fisher exact test 

Cochran-Armitage test P<O.OOl 

Logistic regression test P<O.001 

Life table test P<O.ool 

First incidence (days) 553 

Terminal rate 4/13 (31%) 

Adjusted rate 55.4 % 

Overall rate 14/53 (26%) 

Adrenal Medulla: Benign, Complex, or Malignant Pheochromocytoma 

P=O.O03 

P=O.O81 

455 

16/21 (76%) 

84.6% 

30/52 (58%) 

P<O.OOl 

P<O.ool 

552 

18/18 (100%) 

100.0% 

42/53 (79%) 

P=O.OOl P<O.OOl 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Liver: Hepatocellular Adenoma 

P=O.611 

P=O.629 

P=0.604N 

574 

1/13 (8%) 

18.5% 

4/53 (8%) 

P=0.181N 

P=O. 114N 

570 

0121 (0%) 

2.2% 

1/53 (2%) 

P=0.437N 

P=0.354N 

703 

2/18 (11%) 

14.4% 

3/53 (6%) 

P=0.181N 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: Alveolarhronchiolar Adenoma 

P=O.128 

P=O.190 

549 

0121 (0%) 

10.5% 

3/53 (6%) 

P=O.O36 

P=O.O39 

731 

5/18 (28%) 

31.6% 

6/53 (1 1 %) 

0/13 (0%) 

0.0% 

0/53 (0%) 

P=O.O22 

P=O.O29 

P=O.O33 

- 

P=O.121 P=O.O13 
Lesions in Male Rats 149 

of Nickel Subsulfide (continued) 

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Inhalation Study 

TABLE A3 

0 mg/m3 0.15 mg/m3 1 mg/m3 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Lung: Alveolar/bronchiolar Carcinoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Lung: Aheolar/bronchiolar Adenoma or Carcinoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Pancreatic Islets: Adenoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Pancreatic Islets: Adenoma or Carcinoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Pituitary Gland (Pars Distalis): Adenoma 

0/13 (0%) 

0.0% 

0/53 (0%) 

P=O.O09 

P=O.Oll 

P=O.O15 

- 

0/13 (0%) 

0.0% 

0/53 (0%) 

P=O.O02 

P=O.O03 

P=O.O05 

- 

P=O.400 

P=O.425 

P=O.468 

572 

1/13 (8%) 

9.9% 

2/53 (4%) 

P=O.189 

P=O.203 

P=O.246 

572 

1/13 (8%) 

9.9% 

2/53 (4%) 

20/53 (38%) 

P=0.380N 

P=0.388N 

P=0.300N 

52 1 

4/13 (31%) 

59.9% 

10.3% 

3/53 (6%) 

P=O.129 

P=O.171 

623 

1/21 (5%) 

P=O.121 

P=O.O20 

PzO.044 

549 

1/21 (5%) 

19.7% 

6/53 (1 1 %) 

P=O.O13 

P=O.413 

P=O.516 

690 

2/21 (10%) 

16.7 % 

4/53 (8%) 

P=O.339 

P=O.250 

P=O.367 

538 

2/21 (10%) 

18.4% 

5/53 (9%) 

P=O.219 

P=0.340N 

P=0.109N 

552 

9/21 (43%) 

54.1 % 

17/52 (33 %) 

P=0.368N 

P=O.O15 

P=O.O24 

62 1 

4/18 (22%) 

30.3% 

7/53 (13%) 

P=O.O06 

P=O.ool 

P=O.O03 

62 1 

7/18 (39%) 

48.1% 

11/53 (21 %) 

P<O.OOl 

P=O.344 

P=O.447 

559 

1/18 (6%) 

13.3% 

4/53 (8%) 

P=O.339 

P=O.147 

P=O.223 

559 

2/18 (11%) 

21.0% 

6/53 (1 1 %) 

P=O.135 

P=0.389N 

P=0.187N 

509 

4/18 (22%) 

46.2% 

17/53 (32%) 

P=0.342N 
150 Nickel Subsulfide, NTP TR 453 

of Nickel Subsulfide (continued) 

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Inhalation Study 

TABLE A3 

0 mg/m3 0.15 mg/m3 1 mg/m’ 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Preputial Gland: Adenoma or Carcinoma 

Fisher exact test 

Cochran-Amitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Skin: Fibroma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Skin: Keratoacanthoma 

P=0.109N 

P=O.llON 

P=0.096N 

553 

1/13 (8%) 

12.8% 

3/53 (6%) 

P=0.501N 

P=0.380N 

599 

0/21 (0%) 

6.4% 

2/53 (4%) 

- 

0/18 (0%) 

0.0% 

0153 (0%) 

P=0.121N 

P=0.095N 

P=0.500N P=0.121N 

P=O.563 

P=O.568 

P=O.610 

573 

0/13 (0%) 

13.2% 

3/53 (6%) 

P=0.493N 

P=0.378N 

549 

1/21 (5%) 

6.7% 

2/53 (4%) 

P=0.500N 

P=0.644N 

P=0.552N 

527 

2/18 (11%) 

12.9% 

3/53 (6%) 

P=0.661N 

5/53 (9%) 

P=0.412N 

P=0.287N 

638 

3/21 (14%) 

16.7% 

4/53 (8%) 

P=0.078N 

P=0.059N 

733 (T) 

1/18 (6%) 

5.6% 

1/53 (2%) 

P=0.090N 

P=0.072N 

P=0.067N 

574 

2/13 (15%) 

25.8% 

P=0.500N P=0.103N 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Skin: Squamous Cell Papilloma, Keratoacanthoma, or Basal Cell Adenoma or Carcinoma 

P=0.139N 

P=O.lllN 

P=0.100N 

542 

3/13 (23%) 

34.0% 

7/53 (13%) 

P=0.399N 

P=0.245N 

638 

4/21 (19%) 

24.9% 

6/53 (1 1 %) 

P=0.126N 

P=0.084N 

642 

2/18 (11%) 

13.7% 

3/53 (6%) 

P=0.500N P=0.160N 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Testes: Adenoma 

P=0.077N 

P=0.024N P=O.283 

P=0.061N P=0.222N 

52 1 455 

13/13 (100%) 20/21 (95 %) 

100.0% 

39/53 (74%) 

97.8% 

45/53 (85%) 

P=0.091N 

P=0.046N 

527 

15/18 (83%) 

91.6% 

35/53 (66%) 

P=O.115 P=0.263N 
Lesions in Male Rats 151 

of Nickel Subsulfide (continued) 

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Inhalation Study 

TABLE A3 

0 mg/m3 0.15 mg/m3 1 mg/m3 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall. rate 

Thyroid Gland (C-cell): Adenoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Thyroid Gland (C-cell): Adenoma or Carcinoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Mononuclear Cell Leukemia 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Benign Neoplasms 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Malignant Neoplasms 

P=O.O29 

P=O.O32 

P=O.O47 

594 

1/13 (8%) 

18.1% 

4/52 (8%) 

P=O.118 

P=O.133 

P=O.173 

594 

1/13 (8%) 

25.0% 

6/52 (12%) 

P=O.139 

P=O.151 

P=O.388 

539 

7/13 (54%) 

78.9% 

28/53 (53 W) 

P=0.611N 

P=0.466N 

P=0.278N 

52 1 

13/13 (100%) 

100.0% 

49/53 (92%) 

P=O.O15 

P=O.O15 

P=O.220 

361 

7/13 (54%) 

81.4% 

33/53 (62%) 

P=O.314N 

P=0.208N 

599 

1/21 (5%) 

7.0% 

2/53 (4%) 

P=0.330N 

P=0.204N 

P=0.127N 

599 

2/21 (10%) 

11.7% 

3/53 (6%) 

P=0.235N 

P=O.357 

P=0.347N 

455 

9/21 (43%) 

71.9% 

32/53 (60%) 

P=O.278 

P=O.398 

P=O.lllN 

455 

21/21 (100%) 

100.0% 

52/53 (98 %) 

P=O.181 

P=O.268 

P=0.318N 

455 

11/21 (52%) 

78.2 % 

37/53 (70%) 

P=O.269 

P=O.156 

P=O.259 

5 64 

4/18 (22%) 

34.9% 

9/53 (17%) 

P=O. 125 

P=O.358 

P=O.493 

564 

4/18 (22%) 

34.9% 

9/53 (17%) 

P=O.303 

P=O.142 

P=O.522 

408 

11/18 (61%) 

81.1% 

35/53 (66%) 

P=O. 118 

P=0.724N 

P=0.148N 

509 

18/18 (100%) 

100.0% 

50153 (94%) 

P=O.500 

P=O.O16 

P=O.386 

408 

15/18 (83%) 

93.2% 

44/53 (83 %) 

P=O.O14 
152 Nickel Subsulfide, NTP TR 453 

of Nickel Subsulfide (continued) 

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Inhalation Study 

TABLE A3 

0 mg/m3 0.15 mg/m3 1 mg/m3 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Benign or Malignant Neoplasms 

52/53 (98 %) 52/53 (98%) 53/53 (100%) 

100.0% 100.0% 100.0% 

13/13 (100%) 21/21 (100%) 18/18 (100%) 

361 455 408 

P=0.337N P=0.059N P=0.155N 

P=O.845 P=0.269N 

P=O.378 

- f 

P=0.752N P=O.500 

a Number of neoplasm-bearing animaldnumber of animals examined. Denominator is number of animals examined microscopically for adrenal gland, liver, 

(T)Terminal sacrifice 

compare directly the overall incidence rates. For all tests, a negative trend or a lower incidence in an exposure group is indicated by N. 

(directly or indirectly) the cause of death. The logistic regression test regards these lesions as nonfatal. The Cochran-Armitage and Fisher exact tests 

pairwise comparisons between the controls and that exposed group. The life table test regards neoplasms in animals dying prior to terminal kill as being 

Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding to 

Observed incidence at terminal kill 

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

lung, pancreatic islets, pituitary gland, preputial gland, testes, and thyroid gland; for other tissues, denominator is number of animals necropsied. 

Value of statistic cannot be computed. 

e Not applicable; no neoplasms in animal group 
Lesions in Male Rats 153 

Historical Incidence of Lung Neoplasms in Untreated Male F344/N Ratsa 

TABLE A4a 

Study 

Incidence in Controls 

Carcinoma 

Carcinoma Carcinoma or Squamous Cell 

Alveolar/bronchiolar 

Alveolarlbronchiolar Alveolar/bronchiolar Squamous Cell Adenoma or Carcinoma 

Adenoma 

Historical Incidence at Lovelace Inhalation Toxicology Research Institute 

Talcb 

Nickel Sulfate Hexahydrate 

Nickel Subsulfide 

Nickel Oxide 

0149 

0154 

0153 

0154 

0149 

1/54 

0153 

0154 

0149 

1 I54 

0153 

1/54 

0149 

2/54 

0153 

1/54 

Overall Historical Incidence in Inhalation Studies 

Range 

Standard deviation 

Total 

0%-10% 

3.5% 

171703 (2.4%) 

0%-2% 

1 .O% 

61703 (0.9%) 

0%-2% 

0.9% 

41703 (0.6%) 

0%-10% 

3.8% 

271703 (3.8%) 

Overall Historical Incidence in Feed Studies 

Range 

Standard deviation 

Total 

0%-6% 

2.0% 

2811,200 (2.3%) 

0%-4% 

1.2% 

1111,200 (0.9%) 011,200 (0%) 

0%-8% 

2.0% 

3911,200 (3.3%) 

Results of lifetime study 

a Data as of 17 June 1994 
154 Nickel Subsulfide, NTP TR 453 

Historical Incidence of Pheochromocytomas of the Adrenal Medulla in Untreated Male F344/N Ratsa 

TABLE A4b 

Incidence in Controls 

Study 

Malignant or NOS 

Benign Complex Malignant NOS Benign, Complex, 

Historical Incidence at Lovelace Inhalation Toxicology Research Institute 

Nickel Sulfate Hexahydrate 

Nickel Subsulfide 

Nickel Oxide 

16/54 

13/53 

27/54 

0154 

1/53 

0154 

0154 

0153 

0154 

0154 

0153 

0154 

16/54 

14/53 

27/54 

Overall Historical Incidence in Inhalation Studies 

Range 

Standard deviation 

Total 1631623 (26.2%) 21623 (0.3%) 111623 (1.8%) 71623 (1.1%) 176/623 (28.3%) 

3.9% 12.0% 

0%-14% 8%-50% 

13.1% 0.8% 2.9% 

0%-50% 0%-2% 0%-10% 

Overall Historical Incidence in Feed Studies 

Range 

Standard deviation 

Total 37911.182 (32.1%) 211.182 (0.2%) 3311.182 (2.8%) 011.182 (0%) 40011,182 (33.8%) 

11.7% 0.6% 3.2% 

0%-2% 0%-12% 

10.9% 

10%-63% 14%-63% 

a Data as of 17 June 1994; data from lifetime talc study not included because incidences of adrenal medulla pheochromocytomas increase significantly in 

aging rats, and this data would not be comparable to that in the 2-year nickel subsulfide study. 
Lesions in Male Rats 155 

of Nickel Subsulfide" 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study 

TABLE A5 

0 mg/m3 0.15 mg/m3 1 mg/m3 

Early deaths 

15-Month interim evaluation 

7-MonYh interim evaluation 

Animals initially in study 

Disposition Summary 

Terminal sacrifice 

Natural deaths 

Moribund 

Survivors 

5 

5 

63 

5 

35 

13 

5 

5 

63 

4 

28 

21 

5 

5 

63 

2 

33 

18 

Animals examined microscopically 63 63 63 

Preputial gland 

Genital System 

7-Month Interim Evaluation 

Ectasia 

(1) 

1 (100%) 

Hyperplasia, lymphoid 

Lymph node, bronchial 

Hematopoietic System 

Hyperplasia, lymphoid 

Lymph node, mediastinal (5) 

(5) 

5 (100%) 

4 (80%) 

Lung 

Respiratory System 

Interstitium, infiltration cellular 

Bronchus, hyperplasia, lymphoid 

Alveolus, proteinosis 

Alveolus, hyperplasia, macrophage 

Metaplasia, osseous 

Inflammation, chronic active 

Respiratory epithelium, hyperplasia 

Olfactory epithelium, atrophy 

Inflammation, chronic active 

Nose 

(5) 

1 (20%) 

3 (60%) 

(5) 

(5) 

5 (100%) 

1 (20%) 

3 (60%) 

5 (100%) 

1 (20%) 

5 (100%) 

(5) 

(5) 

5 (100%) 

5 (100%) 

4 (80%) 

5 (100%) 

5 (100%) 

1 (20%) 

1 (20%) 

3 (60%) 

(5) 

Urinary bladder 

Urinary System 

(3) 

Calculus, microscopic observation only 2 (67%) 

(4) 

4 (100%) 

(5) 

5 (100%) 

" Number of animals examined microscopically at the site and the number of animals with lesion 
156 Nickel Subsulfide, NTP TR 453 

of Nickel Subsulfide (continued) 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study 

TABLE A5 

0 mg/m3 0.15 mg/m3 1 mg/m3 

Special Senses System 

Nervous System 

Musculoskeletal System 

Integumentary System 

General Body System 

Endocrine System 

Cardiovascular System 

Alimentary System 

Systems Examined With No Lesions Observed 

7-Month Interim Evaluation (continued) 

Intestine large, rectum 

Alimentary System 

15-Month Interim Evaluation 

Parasite metazoan 

Intestine large, cecum 

Parasite metazoan 

Bile duct, hyperplasia 

Infiltration cellular, mononuclear cell 

Hepatodiaphragmatic nodule 

Clear cell focus 

Basophilic focus 

Liver 

1 (20%) 

(5) 

4 (80%) 

1 (20%) 

1 (20%) 

1 (20%) 

Heart 

Cardiovascular System 

Cardiomyopathy 

(5) 

1 (20%) 

Hyperplasia 

Adrenal medulla 

Endocrine System 

Pars distalis, hyperplasia 

cyst 

Pituitary gland 

Inflammation 

Preputial gland 

Genital System 

Interstitial cell, hyperplasia 

Testes 
Lesions in Male Rats 157 

of Nickel Subsulfide (continued) 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study 

TABLE A5 

O mg/m3 0.15 mg/m3 1 mg/m3 

Bone marrow 

Hematopoietic System 

15-Month Interim Evaluation (continued) 

Granuloma 

Hyperplasia, macrophage 

Hyperplasia, lymphoid 

Lymph node, bronchial 

Hyperplasia, macrophage 

Hyperplasia, lymphoid 

Lymph node, mediastinal 

Hyperplasia, lymphoid 

(5) 

(5) 

(4) 

Spleen (5) 

(5) 

4 (80%) 

5 (100%) 

(5) 

1 (20%) 

(5) 

(4) 

(5) 

1 (33%) 

4 (100%) 

4 (100%) 

1 (20%) 

(3) 

(5) 

1 (20%) 

Respiratory System 

Interstitium, infiltration cellular 

Alveolus, proteinosis 

Alveolus, hyperplasia, macrophage 

Alveolus, emphysema 

Metaplasia, osseous 

Inflammation, chronic active 

Hemorrhage, acute 

Lung 

Inflammation, chronic active 

Nose 

(5) 

3 (60%) 

1 (20%) 

2 (40%) 

1 (20%) 

(5) 

(5) 

1 (20%) 

5 (100%) 

1 (20%) 

4 (80%) 

5 (100%) 

5 (100%) 

2 (40%) 

(5) 

4 (80%) 

5 (100%) 

5 (100%) 

1 (20%) 

1 (20%) 

5 (100%) 

(5) 

Nephropathy 

Kidney 

Urinary System 

Congestion 

Calculus, microscopic observation only 

Calculus, gross observation 

Urinary bladder (5) 

(5) 

5 (100%) 

1 (20%) 

1 (20%) 

2 (40%) 

(5) 

(5) 

2 (40%) 

1 (20%) 

5 (100%) 

1 (20%) 

Special Senses System 

Nervous System 

Musculoskeletal System 

Integumentary System 

General Body System 

Systems Examined With No Lesions Observed 
158 Nickel Subsulfide, NTP TR 453 

of Nickel Subsulfide (continued) 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study 

TABLE A5 

0 mg/m3 0.15 mg/m3 1 mg/m3 

2- Year Study 

Intestine large, colon 

Alimentary System 

Parasite metazoan 

Mineralization 

Inflammation 

Parasite metazoan 

Inflammation 

Intestine large, rectum 

Parasite metazoan 

Inflammation 

Hyperplasia, lymphoid 

Intestine large, cecum 

Angiectasis 

Hyperplasia, lymphoid 

Hyperplasia, lymphoid 

Ulcer 

Hepatocyte, necrosis 

Hepatocyte, hyperplasia 

Centrilobular, atrophy 

Biliary tract, hyperplasia 

Bile duct, hyperplasia 

Thrombosis 

Pigmentation, hemosiderin 

Inflammation, focal, granulomatous 

Hepatodiaphragmatic nodule 

Eosinophilic focus 

Degeneration, fatty 

Degeneration, cystic 

Clear cell focus 

Basophilic focus 

Fat, necrosis 

Inflammation 

Salivary glands 

Hyperplasia 

Atrophy 

Infarct 

Ulcer 

Mineralization 

Hyperplasia, squamous 

Hyperkeratosis 

Diverticulum 

Stomach, forestomach 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Mesentery 

Pancreas 

(5 1) 

(51) 

3 (6%) 

4 (8%) 

1 (2%) 

1 (2%) 

3 (6%) 

6 (11%) 

9 (17%) 

11 (21%) 

1 (2%) 

27 (51%) 

(53) 

40 (75%) 

4 (8%) 

3 (6%) 

12 (23%) 

1 (2%) 

(1) 

(53) 

15 (28%) 

4 (8%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

(45) 

1 (2%) 

(49) 

1 (2%) 

(52) 

(51) 

1 (2%) 

5 (10%) 

(53) 

31 (58%) 

2 (4%) 

2 (4%) 

6 (11%) 

2 (4%) 

15 (28%) 

44 (83%) 

7 (13%) 

1 (2%) 

, 16 (30%) 

(1) 

1 (100%) 

(53) 

15 (28%) 

1 (2%) 

1 (2%) 

1 (2%) 

(52) 

(53) 

3 (6%) 

1 (2%) 

1 (2%) 

(52) 

31 (58%) 

38 (72%) 

1 (2%) 

1 (2%) 

1 (2%) 

3 (6%) 

6 (11%) 

7 (13%) 

15 (28%) 

13 (25%) 

1 (100%) 

4 (8%) 

2 (4%) 

(1) 

(53) 

25 (47%) 

5 (9%) 

6 (11%) 
Lesions in Male Rats 159 

of Nickel Subsulfide (continued) 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study 

TABLE A5 

0 mg/m3 0.15 mg/m3 1 mg/m3 

Stomach, glandular 

Albentary System (continued) 

2-YeUr %Udy (continued) 

Dysplasia 

Epithelium, mineralization 

Necrosis, focal 

(53) (53) 

1 (100%) 

1 (2%) 

1 (2%) 

1 (100%) 

1 (2%) 

2 (4%) 

Tooth (1) (1) 

(53) 

3 (6%) 

Blood vessel 

Cardiovascular System 

Mineralization 

Inflammation 

Valve, thrombosis 

Atrium, thrombosis 

Mineralization 

Inflammation 

Embolus 

Cardiomyopathy 

Heart 

1 (100%) 

(53) (53) 

7 (13%) 

1 (2%) 

2 (4%) 

1 (2%) 

36 (68%) 37 (70%) 

6 (11%) 

(53) 

36 (68%) 

1 (2%) 

4 (8%) 

1 (2%) 

Adrenal cortex 

Endocrine System 

Vacuolization cytoplasmic 

Necrosis 

Hyperplasia, focal 

Hyperplasia, diffuse 

Degeneration, cystic 

Atrophy 

Bilateral, thrombosis 

Hyperplasia 

Adrenal medulla 

Hyperplasia 

Parathyroid gland 

Hyperplasia 

Islets, pancreatic 

Pars distalis, hyperplasia 

Pars distalis, cyst 

Pars distalis, angiectasis 

Pituitary gland 

Follicular cell, hyperplasia 

Follicle, hyperplasia 

C-cell, hyperplasia 

Thyroid gland 

(53) 

2 (4%) 

3 (6%) 

1 * (2%) 

2 (4%) 

26 (49%) 

23 (43%) 

(53) 

(53) 

4 (8%) 

8 (16%) 

1 (2%) 

16 (30%) 

1 (2%) 

1 (2%) 

1 (2%) 

7 (13%) 

(50) 

(53) 

(52) 

(53) 

3 (6%) 

1 (2%) 

2 (4%) 

22 (42%) 

7 (13%) 

(52) 

(53) 

(52) 

(50) 

6 (12%) 

12 (23%) 

1 (2%) 

9 (17%) 

8 (15%) 

(53) 

(53) 

5 (9%) 

12 (23%) 

1 (2%) 

2 (4%) 

10 (19%) 

1 (2%) 

1 (2%) 

1 (2%) 

(53) 

(53) 

(51) 

(53) 

12 (23%) 

3 (6%) 

3 (6%) 

7 (13%) 

(53) 

None 

General Body System 
160 Nickel Subsulfide, NTP TR 453 

of Nickel Subsulfide (continued) 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study 

TABLE A5 

0 mg/m3 0.15 mg/m3 1 mg/m3 

Coagulating gland 

Genital System 

2-Year study (continued) 

Inflammation 

Inflammation 

Granuloma sperm 

Epididymis 

Concretion 

Penis 

Inflammation 

Hyperplasia 

Ectasia 

Preputial gland 

Inflammation 

Prostate 

Epithelium, hyperplasia 

Atrophy 

Seminal vesicle 

Interstitial cell, hyperplasia 

Germinal epithelium, degeneration 

Testes (53) 

11 (21%) 

7 (13%) 

(53) 

(47) 

3 (6%) 

3 (6%) 

(53) 

5 (9%) 

1 (2%) 

1 (2%) 

2 (4%) 

3 (6%) 

(53) 

(53) 

(53) 

13 (25%) 

13 (25%) 

1 (2%) 

(1) 

(53) 

3 (6%) 

1 (2%) 

2 (4%) 

(53) 

(52) 

1 (2%) 

2 (4%) 

(53) 

16 (30%) 

13 (25%) 

Hematopoietic System 

Myeloid cell, hyperplasia 

Necrosis 

Inflammation, suppurative 

Inflammation, focal, granulomatous 

Fibrosis 

Atrophy 

Bone marrow 

Renal, pigmentation 

Renal, angiectasis 

Pancreatic, hyperplasia, lymphoid 

Iliac, hyperplasia, lymphoid 

Lymph node 

Hyperplasia, plasma cell 

Hyperplasia, macrophage 

Hyperplasia, lymphoid 

Ectasia 

Angiectasis 

Lymph node, bronchial 

Inflammation 

Hyperplasia, plasma cell 

Hyperplasia, lymphoid 

Lymph node, mandibular 

Inflammation 

Hyperplasia, macrophage 

Hyperplasia, lymphoid 

Atrophy 

Angiectasis 

(53) (52) (53) 

1 (2%) 

2 (4%) 

4 (8%) 

1 (2%) 

1 (2%) 1 (2%) 

1 (2%) 

3 (6%) 

1 (6%) 

1 (2%) 

2 (4%) 

5 (9%) 

1 (2%) 

1 (2%) 

(5) (16) (6) 

1 (17%) 

1 (20%) 

1 (6%) 

(52) (51) (53) 

1 (2%) 

I (2%) 

1 (2%) 

5 (10%) 

1 (2%) 

2 (4%) 

4 (8%) 

1 (2%) 

5 (9%) 

1 (2%) 

1 (2%) 

14 (27%) 

29 (57%) 

1 (2%) 

10 (20%) 

7 (14%) 

1 (2%) 

28 (53%) 

34 (64%) 

4 (8%) 

3 (6%) 

(50) (51) (50) 

6 (12%) 

Lymph node, mesenteric (53) (53) (53) 

11 (21%) 

1 (2%) 

6 (11%) 
Lesions in Male Rats 161 

TABLE A5 

of Nickel Subsulfide (continued) 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study 

0 mg/m3 0.15 mg/m' 1 mg/m3 

Lymph node, mediastinal 

Hematopoietic System (continued) 

&Year Study (continued) 

Inflammation 

Hyperplasia, macrophage 

Hyperplasia, lymphoid 

Angiectasis 

Pigmentation, hemosiderin 

Infarct 

Hyperplasia, lymphoid 

Hemorrhage 

Hematopoietic cell proliferation 

Fibrosis 

Atrophy 

Degeneration 

Thymus 

Spleen 

1 (2%) 

2 (4%) 

10 (19%) 

3 (6%) 

(53) 

(50) 

17 (34%) 

(49) 

15 (31%) 

1 (2%) 

(53) 

13 (25%) 

2 (4%) 

2 (4%) 

2 (4%) 

4 (8%) 

3 (6%) 

(49) 

8 (16%) 

(53) 

12 (23%) 

1 (2%) 

1 (2%) 

10 (20%) 

3 (6%) 

(49) 

Skin 

Mammary gland 

Integumentary System 

Hyperplasia, cystic 

Tail, hyperkeratosis 

Subcutaneous tissue, inflammation 

Dermis, inflammation 

Conjunctiva, inflammation 

Cyst epithelial inclusion 

(53) 

(49) 

8 (16%) 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 1 (2%) 

1 (2%) 

Musculoskeletal System 

Mandible, necrosis 

Hyperostosis 

Fibrous osteodystrophy 

Bone (52) 

4 (8%) 

1 (2%) 

1 (2%) 

Nervous System 

Gray matter, necrosis 

Cerebrum, compression 

Cerebrum, atrophy, focal 

Necrosis 

Embolus 

cyst 

Compression 

Brain 

6 (11%) 

1 (2%) 

(53) 

4 (8%) 

(53) 

1 (2%) 

4 (8%) 

1 (2%) 

Larynx 

Respiratory System 

Inflammation 

Hyperplasia 

(49) 

3 (6%) 

4 (8%) 

(53) 

2 (4%) 

(52) 

2 (4%) 
162 Nickel Subsulfide, NTP TR 453 

of Nickel Subsulfide (continued) 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study 

TABLE A5 

0 mg/m3 0.15 mgh3 1 mg/m3 

Lung 

Respiratory System (continued) 

2-YeW study (continued) 

Interstitium, infiltration cellular 

Bronchus, hyperplasia, lymphoid 

Alveolus, proteinosis 

Alveolus, hyperplasia, macrophage 

Alveolar epithelium, hyperplasia, focal 

Mineralization 

Metaplasia, squamous 

Metaplasia, osseous 

Inflammation, chronic active 

Hemorrhage, acute 

Fibrosis 

Emphysema 

Embolus 

Olfactory epithelium, mineralization, focal 

Olfactory epithelium, atrophy 

Nasolacrimal duct, inflammation 

Thrombosis 

Inflammation, chronic active 

Inflammation, acute 

Nose 

(53) 

1 (2%) 

2 (4%) 

9 (17%) 

4 (8%) 

2 (4%) 

1 (2%) 

9 (17%) 

2 (4%) 

1 (2%) 

17 (32%) 

(53) 

3 (6%) 

12 (23%) 

1 (2%) 

2 (4%) 

(53) 

1 (2%) 

48 (91%) 

2 (4%) 

1 (2%) 

53 (100%) 

31 (58%) 

10 (19%) 

36 (68%) 

48 (91%) 

6 (11%) 

3 (6%) 

10 (19%) 

1 (2%) 

(53) 

1 (2%) 

(53) 

40 (75%) 

1 (2%) 

1 (2%) 

51 (96%) 

39 (74%) 

14 (26%) 

51 (96%) 

52 (98%) 

11 (21%) 

1 (2%) 

(52) 

3 (6%) 

9 (17%) 

3 (6%) 

4 (8%) 

18 (35%) 

EY e 

Special Senses System 

Retina, degeneration 

Iris, inflammation 

Anterior chamber, hemorrhage, chronic 

Phthisis bulbi 

Inflammation 

Cataract 

(4) 

1 (25%) 

1 (25%) 

1 (25%) 

(4) 

2 (50%) 

3 (75%) 

1 (25%) 

1 (25%) 

Kidney 

Urinary System 

Renal tubule, pigmentation 

Papilla, necrosis 

Cortex, infarct 

Nephropathy 

Mineralization 

Inflammation 

Infarct 

Hydronephrosis 

Embolus 

cyst 

Calculus, microscopic observation only 

Inflammation 

Calculus, microscopic observation only 

Calculus, gross observation 

Urinary bladder 

Urethra 

(52) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

51 (98%) 

20 (38%) 

(53) 

4 (8%) 

2 (4%) 

(53) 

2 (4%) 

53 (100%) 

(53) 

(1) 

35 (66%) 

1 (2%) 

1 (2%) 

1 (100%) 

(53) 

2 (4%) 

1 (2%) 

1 (2%) 

35 (66%) 

1 (2%) 

2 (4%) 

53 (100%) 

1 (2%) 

(53) 

3 (6%) 

2 (4%) 
163 

OF NICKEL SUBSULFIDE 

SUMMARY OF LESIONS IN FEMALE RATS 

APPENDIX B 

IN THE 2-YEAR INHALATION STUDY 

TABLE B1 Summary of the Incidence of Neoplasms in Female Rats 

in the 2-Year Inhalation Study of Nickel Subsulfide 

TABLE €52 Individual Animal Tumor Pathology of Female Rats 

in the 2-Year Inhalation Study of Nickel Subsulfide 

in the 2-Year Inhalation Study of Nickel Subsulfide 

TABLE B5 Summary of the Incidence of Nonneoplastic Lesions in Female Rats 

in Untreated Female F344/N Rats 

TABLE B4b Historical Incidence of Pheochromocytomas of the Adrenal Medulla 

in Untreated Female F344/N Rats 

TABLE B4a Historical Incidence of Lung Neoplasms 

in the 2-Year Inhalation Study of Nickel Subsulfide 

TABLE B3 Statistical Analysis of Primary Neoplasms in Female Rats 

....................... 

....................... 

....................... 

................................... 

................................... 

....................... 193 

192 

191 

186 

170 

165 
164 Nickel Subsulfide, NTP TR 453 
Lesions in Female Rats 165 

of Nickel Subsulfidea 

Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Inhalation Study 

TABLE B1 

0 mg/m3 0.15 mg/m3 1 mg/m3 

Early deaths 

15-Month interim evaluation 

7-Month interim evaluation 

Animals initially in shdy 

Disposition Summary 

Natural deaths 

Moribund 

Accidental death 

Terminal sacrifice 

Survivors 

Animals examined microscopically 

5 

5 

63 

4 

24 

25 

63 

5 

5 

63 

1 

27 

25 

63 

5 

5 

63 

1 

23 

1 

28 

63 

Urinary System 

Special Senses System 

Respiratory System 

Nervous System 

Musculoskeletal System 

Integumentary System 

Hematopoietic System 

Genital System 

General Body System 

Endocrine System 

Cardiovascular System 

Alimentary System 

Systems Examined At 7 Months With No Neoplasms Observed 

Pituitary gland 

Endocrine System 

15-Month Interim Evaluation 

(5) 

Pars distalis, adenoma 

(5) 

1 (20%) 

(5) 

2 (40%) 

Carcinoma 

Clitoral gland 

Genital System 

Polyp 

Vagina 

Sarcoma stromal 

Uterus 

Carcinoma 

Zymbal’s gland 

Special Senses System 

(1) 

1 (100%) 
166 Nickel Subsulfide, NTP TR 453 

of Nickel Subsulfide (continued) 

Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Inhalation Study 

TABLE B1 

0 mg/m3 0.15 mg/m3 1 mg/m3 

Urinary System 

Respiratory System 

Nervous System 

Musculoskeletal System 

Integumentary System 

Hematopoietic System 

General Body System 

Cardiovascular System 

Alimentary System 

Systems Examined With No Neoplasms Observed 

15-Month Interim Evaluation (continued) 

2- Year Study 

Pancreas 

Histiocytic sarcoma 

Mesentery 

Histiocytic sarcoma 

Liver 

Alimentary System 

Tooth 

Stomach, glandular 

Stomach, forestomach 

Histiocytic sarcoma 

Gingiva, squamous cell carcinoma 

Heart 

Cardiovascular System 

Schwannoma NOS 

(53) 

1 (2%) 

(53) 

2 (4%) 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

Bilateral, pheochromocytoma benign 

Pheochromocytoma benign 

Pheochromocytoma malignant 

Histiocytic sarcoma 

Carcinoma 

Adenoma 

Islets, pancreatic 

Thyroid gland 

Pituitary gland 

Parathyroid gland 

Pars distalis, adenoma 

Follicular cell, carcinoma 

C-cell, carcinoma 

C-cell, adenoma 

6 (11%) 

1 (2%) 

1 (2%) 

1 (2%) 

(53) 

(50) 

(53) 

(53) 

30 (57%) 

1 (2%) 

3 (6%) 

4 (8%) 

(53) 

(53) 

24 (45%) 

12 (23%) 

1 (2%) 

1 (2%) 

1 (2%) 

(53) 
Lesions in Female Rats 167 

TABLE B1 

of Nickel Subsulfide (continued) 

Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Inhalation Study 

Tissue NOS 

General Body System 

&Year Study (continued) 

Thoracic, squamous cell carcinoma, 

Thoracic, alveolar/bronchiolar carcinoma, 

Mediastinum, squamous cell carcinoma, 

metastatic, lung 

metastatic, lung 

metastatic, lung 

(2) 

1 (50%) 

1 (100%) 

1 (50%) 

Genital System 

Carcinoma 

Adenoma 

Clitoral gland 

Granulosa cell tumor benign 

Cystadenoma 

Ovary 

Schwannoma malignant 

Polyp stromal 

Adenoma 

Histiocytic sarcoma 

Granulosa-theca tumor benign 

Uterus 

Vagina 

1 (2%) 

(53) 

2 (4%) 

(53) 

2 (4%) 

1 (2%) 

(53) 

1 (100%) 

1 (2%) 

1 (2%) 

(1) 

Lymph node 

Bone marrow 

Hematopoietic System 

Renal, histiocytic sarcoma 

Iliac, histiocytic sarcoma 

Histiocytic sarcoma 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Histiocytic sarcoma 

Lymph node, bronchial 

Histiocytic sarcoma 

Histiocytic sarcoma 

Alveolar/bronchiolar carcinoma, metastatic, 

lung 

Thymus 

Spleen 

(49) 

(53) 

Integumentary System 

Fibroadenoma 

Carcinoma 

Adenoma 

Mammary gland (53) 

22 (42%) 

1 (2%) 

I (2%) 

(53) 

11 (21%) 

3 (6%) 

(53) 

10 (19%) 
Nickel Subsulfide, NTP TR 453 

of Nickel Subsulfide (continued) 

Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Inhalation Study 

TABLE B1 

Skin 

Integumentary System (continued) 

shdy (continued) 

Subcutaneous tissue, lipoma 

Pinna, fibrosarcoma 

Keratoacanthoma 

Histiocytic sarcoma 

Fibrosarcoma 

(53) 

1 (2%) 

(53) 

1 (2%) 

(53) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

Skeletal muscle 

Bone 

Musculoskeletal System 

(53) 

Squamous cell carcinoma, metastatic, tooth 

Squamous cell carcinoma, metastatic, tooth 

(53) 

Brain 

Nervous System 

Astrocytoma NOS 

(53) 

1 (2%) 

(53) 

Lung 

Respiratory System 

Alveolar/bronchiolar adenoma 

Adenoma 

(53) 

1 (2%) 

1 (2%) 

Squamous cell carcinoma 

Histiocytic sarcoma 

Alveolar/bronchiolar carcinoma, multiple 

Alveolar/bronchiolar carcinoma 

Nose (53) 

(53) 

5 (9%) 

1 (2%) 

(53) 

(53) 

1 (2%) 

1 (2%) 

3 (6%) 

5 (9%) 

None 

Special Senses System 

Kidney 

Urinary System 

(53) 

Nephroblastoma 

Histiocytic sarcoma 

Alveolar/bronchiolar carcinoma, metastatic, 

lung 

1 (2%) 

Urinary bladder (53) 

(53) 

1 (2%) 

1 (2%) 

(53) 

Multiple organsb 

Systemic Lesions 

Leukemia mononuclear 

Histiocytic sarcoma 

(53) 

19 (36%) 

(53) 

18 (34%) 

(53) 

22 (42%) 

1 (2%) 
Lesions in Female Rats 169 

of Nickel Subsulfide (continued) 

Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Inhalation Study 

TABLE B1 

O mg/m3 0.15 mg/m3 1 mg/m3 

Neoplasm Summary 

2-Year study 

15-Month interim evaluation 

Total animals with Primary neoplasms' 

2-Year study 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

Total animals with benign neoplasms 

2-Year study 

15-Month interim evaluation 

Total benign neoplasms 

2-Year study 

15-Month interim evaluation 

Total animals with malignant neoplasms 

Total animals with metastatic neoplasms 

2-Year study 

15-Month interim evaluation 

2-Year study 

Total metastatic neoplasms 

2-Year study 

Total animals with uncertain neoplasmsTotal primary neoplasms 

Total malignant neoplasms 

Total uncertain neoplasms 

benign or malignant 

2-Year study 

2-Year study 

48 

1 

84 

1 

37 

58 

24 

1 

25 

1 

51 

2 

100 

2 

45 

1 

64 

1 

30 

1 

33 

1 

2 

4 

3 

3 

48 

4 

109 

4 

45 

3 

I1 

3 

28 

1 

32 

1 

1 

3 

a Number of animals examined microscopically at the site and the number of animals with neoplasm 

Primary neoplasms: all neoplasms except metastatic neoplasms 

Number of animals with any tissue examined microscopically 

Lesions in Female Rats 171 

(continued) 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Nickel Subsulfide: 0 mg/m3 

TABLE B2 

7119999000000000011111112222 

1222222333333333333333333333 

7777777777777777777777777777 

Number of Days on Study 

Carcass ID Number 

0111111000000011100111110111 Total 

7230222788899902489001139111 Tissues/ 

3539279105678911025374553178 Tumors 

Tooth 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Pancreas 

Mesentery 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Esophagus 

Alimentary System 

............................ 

............................ 

............................ 

............................ 

............................ 

............................ 

+M+M++++M++++++++++M++++++I + ............................ 

............................ 

............................ 

............................ 

............................ 

+ 

53 

50 

50 

49 

49 

41 

50 

53 

53 

53 

53 

53 

1 

1 

~~~ 

Heart 

Cardiovascular System 

~~ 

Schwannoma NOS 

............................ 53 

X 1 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

............................ 53 

............................ 53 

Pheochromocytoma benign 

Pheochromocytoma malignant 

Parathyroid gland 

Carcinoma 

Islets, pancreatic 

Thyroid gland 

Pituitaly gland 

C-cell, adenoma 

Pars distalis, adenoma 

x. X 

............................ X 

............................ 

x xxxx xx X xx xx ............................ 

++++++++++++M++++++++M++++++ 

xx 

51 

24 

52 

48 

1 

53 

2 

1 

3 

None 

General Body System 

Carcinoma 

Clitoral gland 

Genital System 

Polyp stromal 

Uterus 

Granulosa cell tumor benign 

Ovaly 

2 

............................ 53 

2 

............................ 53 

1 

............................ 52 

X 

X 
172 Nickel Subsulfide, NTP TR 453 

(continued) 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Nickel Subsulfide: 0 mg/m3 

TABLE B2 

3977703834430002745577866 

6677701367714444566667791 

2334455555566666666666667 

Number of Days on Study 

Carcass ID Number 

2144937096042729340006663 

9823387189397801289021721 

0011100100100011100111011 

Thymus 

Spleen 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node 

Bone marrow 

Hematopoietic System ......................... ++ + ++ 

+M++++++++++++I ++++++++++ ......................... 

++++++++++++++++++++++M++ 

......................... 

+++++M+++I +++++++++I +M+++ 

+++++++++++++M+++++++++++ 

Integumentary System 

Fibroadenoma 

Carcinoma 

Adenoma 

Mammary gland 

Pinna, fibrosarcoma 

Skin 

......................... X 

xxx xx xX 

......................... X 

X 

Bone 

Musculoskeletal System 

~~~ ~ 

......................... 

Brain 

Nervous System 

~~~~ 

......................... 

Lung 

Larynx 

Respiratory System 

......................... 

+++++++++++I +++++++++++++ 

Alveolar/bronchiolar adenoma 

Adenoma 

Multiple organs 

Systemic Lesions 

Urinary bladder 

Nephroblastoma 

Kidney 

Urinary System 

EY e 

Special Senses System 

Trachea 

Nose 

......................... 

......................... 

X 

......................... 

.......................... 

xx 

......................... Leukemia mononuclear xx X x xxx 
Lesions in Female Rats 173 

(continued) 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Nickel Subsulfide: 0 mg/m3 

TABLE B2 

1119999000000000011111112222 

1222222333333333333333333333 

7777771717717777777717777717 

Number of Days on Study 

Carcass ID Number 

0111111000000011100111110111 Total 

7230222188899902489001139111 Tissues/ 

3539279105678911025374553178 Tumors 

~ 

Thymus 

Spleen 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node 

Bone marrow 

Hematopoietic System 

~~ 

............................ 

+++++++I ++M+++++++++++++++++ 

............................ 

+++++M+M+I +++++++"+++++I +++ 

++++++++++I +++++++++++++++++ 

++M+M++M++++++++++++I ++++++M 

+++++++++++++++++++M++++I +++ 

49 

53 

46 

52 

44 

50 

5 

53 

Mammary gland 

Integumentary System 

............................ 53 

Fibroadenoma 

Carcinoma 

Adenoma 

Pinna, fibrosarcoma 

Skin 

1 

............................ 53 

xx x 22 

1 

1 

X 

xx xx xx x 

Brain 

Nervous System 

Bone 

Musculoskeletal System 

Lung 

Larynx 

Respiratory System 

............................ 53 

............................ 53 

............................ 53 

............................ 53 

1 

X 1 

............................. 53 

............................ 52 

Alveolar/bronchiolar adenoma 

Adenoma 

X 

Trachea 

Nose 

EY e 

Special Senses System 

t 

~ 

+ 2 

Urinary bladder 

Nephroblastoma 

Kidney 

Urinary System 

............................ 53 

1 

............................ 53 

Multiple organs 

Systemic Lesions 

............................ 53 

Leukemia mononuclear xx X X xx x X 19 
174 Nickel Subsulfide, NTP TR 453 

2228604499200256029381596 

5883699900122233444578990 

4445555566666666666666667 

62785227920348927.53430917 

4223736727467174554377634 

2222222222222222222222222 

Number of Days on Study 

Carcass ID Number 

Esophagus 

Alimentary System 

......................... 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon A++++++++++++++++++++++++ 

A++++++++++++++++++++++++ 

A++++++++++++++++++++++++ 

A++++++++++++++++++++++++ 

A++++++++++++++++++++++++ 

A+++++M+++++++++++M+M++M+ 

Liver ......................... 

Tooth 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Pancreas 

......................... 

......................... 

......................... 

......................... 

+ 

Gingiva, squamous cell carcinoma X 

Heart 

Cardiovascular System 

......................... Schwannoma NOS X X 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

Bilateral, pheochromocytoma benign 

Pheochromocytoma benign 

Carcinoma 

Adenoma 

Islets, pancreatic 

Thyroid gland 

Pituitary gland 

Parathyroid gland 

Pars distalis, adenoma 

Follicular cell, carcinoma 

C-cell, carcinoma 

C-cell, adenoma 

X 

......................... 

......................... 

X 

......................... 

X 

......................... xx xxx x xxxxxxxx ......................... 

......................... 

X 

X X 

X 

Tissue NOS 

General Body System 

carcinoma, metastatic, lung 

Thoracic, squamous cell 

carcinoma, metastatic, lung 

Mediastinum, squamous cell 

++ 

X 

X 
Lesions in Female Rats 175 

(continued) 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Nickel Subsulfide: 0.15 mg/m3 

TABLE B2 

8179999999999900000111111222 

1222222222222233333333333333 

7777777777777777777777777777 

Number of Days on Study 

Carcass ID Number 

2222222222222222222222222222 Total 

7241122233355513366345568226 Tissues/ 

8052915856913810358744600367 Tumors 

Tooth 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Pancreas 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Esophagus 

Alimentary System 

Gingiva, squamous cell carcinoma 

............................ 

............................ 

............................ 

++++++++++++++++++++++++++M+ 

............................ 

............................ 

............................ 

............................ 

............................ 

M++MM+++++M+++++M++++M++M+I + ............................ 

............................. 

53 

53 

53 

52 

53 

52 

52 

52 

52 

40 

52 

53 

1 

1 

Heart 

Cardiovascular System 

~~ ~~~ 

Schwannoma NOS 2 

............................ 53 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

1 

3 

xx 4 

............................ 53 

xx xx x xxxx x X x xx x 30 

............................ 53 

M+++++++++++++I +++++++++++M 50 

1 

1 

............................ 53 

1 

6 

............................ 53 

............................ 53 

Bilateral, pheochromocytoma benign 

Pheochromocytoma benign 

Carcinoma 

Adenoma 

Islets, pancreatic 

Thyroid gland 

Pituitary gland 

Parathyroid gland 

xx X X 

X 

X 

Pars distalis, adenoma 

Follicular cell, carcinoma 

C-cell, carcinoma 

C-cell, adenoma 

X X 

X 

Tissue NOS 

General Body System 

carcinoma, metastatic, lung 

Thoracic, squamous cell 

carcinoma, metastatic, lung 

Mediastinum, squamous cell 
176 Nickel Subsulfide, NTP TR 453 

(continued) 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Nickel Subsulfide: 0.15 mg/m3 

TABLE B2 

2228604499200256029381596 

5883699900122233444578990 

4445555566666666666666667 

Number of Days on Study 

Carcass ID Number 

6278522792034892753430917 

4223736727461174554377634 

2222222222222222222222222 

Carcinoma 

Adenoma 

Clitoral gland 

Genital System 

Granulosa-theca tumor benign 

++++++++++++++++ ++++++++ 

X 

X X 

Ovary ......................... 

Uterus 

Schwannoma malignant 

Polyp stromal 

Adenoma 

Vagina 

......................... 

X 

+ 

Bone marrow 

Hematopoietic System ......................... 

Thymus 

Spleen 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node 

+++++++++++++++++++M+++++ 

......................... 

++++++++++++++I +++++++M++ 

......................... 

++++M++++++M++I ++++++++++ 

++M+++++++++++M++++++++++ 

+ + 

Integumentary System 

Fibroadenoma 

Carcinoma 

Mammary gland 

Subcutaneous tissue, lipoma 

Fibrosarcoma 

......................... X 

........................... X 

x xx 

Skin 

X 

X 

Bone 

Musculoskeletal System ......................... Squamous cell carcinoma, metastatic, 

tooth 

Skeletal muscle 

tooth 

+ 

X 

Squamous cell carcinoma, metastatic, 

X 

Nervous System 

Brain ......................... Astrocytoma NOS X 

Spinal cord + 
Lesions in Female Rats 177 

(continued) 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Nickel Subsulfide: 0.15 mg/m3 

TABLE B2 

8179999999999900000111111222 

1222222222222233333333333333 

7777777777777777777777777777 

2222222222222222222222222222 Total 

7241122233355513366345568226 Tissues1 

8052915856913810358744600367 Tumors 

Number of Days on Study 

Carcass ID Number 

Carcinoma 

Adenoma 

Clitoral gland 

Genital System 

Schwannoma malignant 

Polyp stromal 

Adenoma 

Granulosa-theca tumor benign 

Uterus 

Ovary 

Vagina 

X 

............................ 

X 

............................ X 

............................ X 

X 

1 

1 

1 

1 

53 

1 

53 

3 

2 

52 

Thymus 

Spleen 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node 

Bone marrow 

Hematopoietic System 

Integumentary System 

Fibroadenoma 

Carcinoma 

Mammary gland 

++++++++++M+++++++++++++++++ 51 

............................ 53 

+++++++M++++++++++++++++++++ 50 

............................ 53 

............................ 50 

M+++++++I +++++++++++++++++++ 49 

3 

............................ 53 

+ 

............................ 53 

X X 3 

xxx X X 11 

............................ 53 

xx 

Skin 

Subcutaneous tissue, lipoma 

Fibrosarcoma 

1 

1 

~ 

Bone 

Musculoskeletal System ~~~ ~ ____ _____ ~____ ~__________~_____ ~_____ ____ ~~ ~~ ~~ 

1 

1 

............................ 53 

Squamous cell carcinoma, metastatic, 

Skeletal muscle 

tooth 

Squamous cell carcinoma, metastatic, 

tooth 1 

Spinal cord 

Brain 

Nervous System 

Astrocytoma NOS 

1 

1 

............................ 53 
178 Nickel Subsulfide, NTP TR 453 

(continued) 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Nickel Subsulfide: 0.15 mg/m3 

TABLE B2 

2228604499200256029381596 

5883699900122233444518990 

4445555566666666666666661 

6278522792034892153430917 

4223136721461114554377634 

2222222222222222222222222 

~~ ~ 

Number of Days on Study 

Carcass ID Number 

Lung 

Larynx 

Respiratory System 

......................... 

.......................... 

Alveolar epithelium, 

Squamous cell carcinoma 

Alveolar/bronchiolar adenoma X 

X 

alveolar/bronchiolar adenoma 

Trachea 

Nose 

......................... 

......................... 

EY e 

Special Senses System 

+ + 

Urinary bladder 

Kidney 

Urinary System 

......................... 

......................... 

Multiple organs 

Systemic Lesions 

Leukemia mononuclear xx X xx ......................... X xxx 
Lesions in Female Rats 179 

(continued) 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Nickel Subsulfide: 0.15 mg/m3 

TABLE B2 

8179999999999900000111111222 

1222222222222233333333333333 

7717777717717117777777177777 

Number of Days on Study 

2222222222222222222222222222 Total 

7241122233355513366345568226 Tissues/ 

8052915856913810358744600367 Tumors 

Carcass ID Number 

Lung 

Larynx 

Respiratory System 

............................ 53 

............................ 53 

1 

5 

............................ 53 

............................ 53 

Squamous cell carcinoma 

Alveolar/bronchiolar adenoma X xx X 

~ 

Trachea 

Nose 

EY e 

Special Senses System 

~ ~~~ 

2 

~~~~ ~ ~~ ~~ ~ 

Urinary bladder 

Kidney 

Urinary System 

Multiple organs 

Systemic Lesions 

~~~ 

............................ 53 

............................ 53 

18 

............................ 53 

Leukemia mononuclear X X X xx X x xx 
180 Nickel Subsulfide, NTP TR 453 

1416832042523445113336926 

8402368990123333444670012 

2455555556666666666667777 

8505268174087621844711145 

0799711716786796505758096 

4333344343333333343333433 

Number of Days on Study 

Carcass ID Number 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Esophagus 

Alimentary System 

Pancreas 

Histiocytic sarcoma 

Mesentery 

Histiocytic sarcoma 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Histiocytic sarcoma 

......................... 

......................... 

+++++++++A+++++++++++++++ 

+++++++++A+++++++++++++++ 

+++++++++A+++++++++++++++ 

++MM+MMM+M+++++M+++M+++M+ 

......................... 

......................... 

X 

+ 

......................... X 

......................... 

......................... 

......................... 

Heart 

Blood vessel 

Cardiovascular System 

......................... 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

......................... 

......................... 

Bilateral, pheochromocytoma benign 

Pheochromocytoma benign 

Pheochromocytoma malignant 

Histiocytic sarcoma 

Parathyroid gland 

Adenoma 

Islets, pancreatic 

Thyroid gland 

Pituitary gland 

X 

xx 

X xx xx 

xx ......................... xxx 

X 

I++++++++++++++++++++++++ 

X X 

......................... 

M++++++++++++M+++++++++++ 

C-cell, adenoma 

Pars distalis, adenoma x x xx 

X X 

Tissue NOS 

General Body System 

+ 

carcinoma, metastatic, lung 

Thoracic, alveoladbronchiolar 

X 
Lesions in Female Rats 181 

(continued) 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Nickel Subsulfide: 1 mg/m3 

TABLE B2 

9999999900000000111111111122 

2222222233333333333333333333 

7717771777777777777771777717 

3333344433333344333333444433 Total 

5688801177899900567888001156 Tissues/ 

7903791934438903608269272528 Tumors 

Number of Days on Study 

Carcass ID Number 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Esophagus 

Alimentary System 

Pancreas 

Histiocytic sarcoma 

Mesentery 

Histiocytic sarcoma 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Histiocytic sarcoma 

............................ 

............................ 

............................ 

............................ 

............................ 

++++M+I MM++++++++++M++++M+++ 

............................ 

............................ 

+ 

............................ 

............................ 

............................ 

............................ 

53, 

53 

53 

52 

52 

52 

38 

53 

1 

2 

1 

53 

53 

53 

1 

53 

Heart 

Blood vessel 

Cardiovascular System 

+ ............................ 53 

1 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

Bilateral, pheochromocytoma benign 

Pheochromocytoma benign 

Pheochromocytoma malignant 

Histiocytic sarcoma 

Parathyroid gland 

Adenoma 

Islets, pancreatic 

Thyroid gland 

Pituitary gland 

C-cell, adenoma 

Pars distalis, adenoma 

............................ 53 

............................ 53 

X X X 

X 

X X 

xx x xxx xxxx x xxx xx x ............................ 

............................ X 

............................ 

++++++++++++M++++++++++++++M 

xx 

xx 

4 

52 

15 

53 

49 

1 

53 

24 

12 

1 

1 

Tissue NOS 

General Body System 

1 

carcinoma, metastatic, lung 

Thoracic, alveolar/bronchiolar 

1 
182 Nickel Subsulfide, NTP TR 453 

TABLE B2 

(continued) 

Individual Animal Tumor Pathology of Female Rats in the %Year Inhalation Study of Nickel Subsulfide: 1 mg/m3 

1416832042523445113336926 

8402368990123333444670012 

2455555556666666666667717 

8505268174087621844711145 

0799711716786796505758096 

4333344343333333343333433 

Number of Days on Study 

Carcass ID Number 

Carcinoma 

Adenoma 

Clitoral gland 

Genital System 

Polyp stromal 

Histiocytic sarcoma 

Cystadenoma 

+++++M+++++++++++++++++++ 

X 

Ovary ......................... X 

X 

Vagina 

Uterus ......................... 

+ 

X X X 

Histiocytic sarcoma 

Bone marrow 

Hematopoietic System 

Renal, histiocytic sarcoma 

Iliac, histiocytic sarcoma 

Lymph node 

Lymph node, mandibular 

Histiocytic sarcoma 

Lymph node, bronchial 

Histiocytic sarcoma 

Histiocytic sarcoma 

metastatic, lung 

Alveolar/bronchiolar carcinoma, 

Lymph node, mediastinal 

Lymph node, mesenteric 

Thymus 

Spleen 

......................... X 

+ +++ + ++ 

X 

X 

+++++++++++M+++++++++++++ 

A 

......................... 

......................... 

......................... 

X 

X 

......................... A 

+++++++++++++++++M+++++++ 

Skin 

Mammary gland 

Integumentary System 

......................... X X X 

......................... Fibroadenoma 

Pinna, fibrosarcoma 

Keratoacanthoma 

Histiocytic sarcoma 

Brain 

Nervous System 

Bone 

Musculoskeletal System 

X 

X 

X 

......................... 

......................... 
Lesions in Female Rats 183 

(continued) 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Nickel Subsulfide: 1 mg/m3 

TABLE B2 

9999999900000000111111111122 

2222222233333333333333333333 

7777777777777777777777777777 

Number of Days on Study 

Carcass ID Number 

3333344433333344333333444433 Total 

5688801177899900567888001156 Tissues/ 

7903791934438903608269272528 Tumors 

Carcinoma 

Adenoma 

Clitoral gland 

Genital System 

Polyp stromal 

Histiocytic sarcoma 

Cystadenoma 

Ovary 

Vagina 

Uterus 

3 

1 

............................ 52 

1 

1 

............................ 53 

1 

3 

............................ 53 

X 

X X 

Histiocytic sarcoma 

Bone marrow 

Hematopoietic System 

Renal, histiocytic sarcoma 

Iliac, histiocytic sarcoma 

Lymph node 

Lymph node, mandibular 

Histiocytic sarcoma 

Lymph node, bronchial 

Histiocytic sarcoma 

Histiocytic sarcoma 

metastatic, lung 

Alveolar/bronchiolar carcinoma, 

Lymph node, mediastinal 

Lymph node, mesenteric 

Thymus 

Spleen 

............................ 

+ ++ 

+++M++++M+++++++++++++++++++ 

++++++M+++++++++++++I +++++++ 

+++M+++++++MM++++++MMI ++++++ 

+++++++++++++++++++M++++++++ 

............................ 

+++++++++M++++++++++++++++++ 

1 

1 

10 

1 

53 

47 

52 

51 

1 

50 

51 

1 

53 

1 

1 

Skin 

Mammary gland 

Integumentary System 

Fibroadenoma 

Pinna, fibrosarcoma 

Keratoacanthoma 

Histiocytic sarcoma 

1 

1 

1 

............................ 53 

10 

............................ 53 

X x xx X X 

X 

Brain 

Nervous System 

Bone 

Musculoskeletal System 

............................ 53 

............................ 53 
Nickel Subsulfide, NTP TR 453 

(continued) 

Individual Animal Tumor Pathology of Female Rats in the, 2-Year Inhalation Study of Nickel Subsulfide: 1 mg/m3 

TABLE B2 

1416832042523445113336926 

8402368990123333444670012 

2455555556666666666667777 

8505268174087621844711145 

0799711716786796505758096 

4333344343333333343333433 

Number of Days on Study 

Carcass ID Number 

Lung 

Larynx 

Respiratory System 

......................... 

......................... 

Alveolar/bronchiolar carcinoma, 

Alveolar/bronchiolar carcinoma 

Alveolar/bronchiolar adenoma 

Histiocytic sarcoma 

X 

X 

multiple X 

X 

Trachea 

Nose 

......................... 

++++++++++++++++ ++++++++ 

Lacrimal gland 

EY e 

Special Senses System 

Kidney 

Urinary System 

......................... 

Histiocytic sarcoma 

metastatic, lung 

Alveolar/bronchiolar carcinoma, 

X 

X 

Urinary bladder ......................... 

Multiple organs 

Systemic Lesions 

Leukemia mononuclear 

Histiocytic sarcoma 

......................... X 

xxxx xxx X X xxx xx 
Lesions in Female Rats 185 

(continued) 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Nickel Subsulfide: 1 mg/m3 

TABLE B2 

9999999900000000111111111122 

2222222233333333333333333333 

7777777777777777777777777777 

Number of Days on Study 

Carcass ID Number 

3333344433333344333333444433 Total 

5688801177899900567888001156 Tissues/ 

7903791934438903608269272528 Tumors 

Lung 

Larynx 

Respiratory System 

Alveoladbronchiolar carcinoma, 

Alveolar/bronchiolar carcinoma 

Alveolar/bronchiolar adenoma 

Histiocytic sarcoma 

multiple 

Trachea 

Nose 

3 

X 5 

............................ 53 

+I +++++++++++++++I ++++++++++ 51 

X xx 

X X 

............................ 53 

............................ 52 

1 

1 

Lacrimal gland 

EY e 

Special Senses System 

+ 

+ 1 

1 

Kidney 

Urinary System 

............................ 53 

............................ 53 

1 

1 

Histiocytic sarcoma 

metastatic, lung 

Alveolar/bronchiolar carcinoma, 

Multiple organs 

Systemic Lesions 

Urinary bladder 

22 

1 

............................ 53 

Leukemia mononuclear 

Histiocytic sarcoma 

xx X X xxx X 
186 Nickel Subsulfide, NTP TR 453 

of Nickel Subsulfide 

Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Inhalation Study 

TABLE B3 

0 mg/m' 0.15 mg/m3 1 mg/m3 

Fisher exact test 

Cochran-Armita e testd 

Logistic regression testd 

Life table testd 

First incidence (days) 

Terminal rate' 

Adjusted rateb 

Overall ratea 

Adrenal Medulla: Benign Pheochromocytoma 

% 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Adrenal Medulla: Benign or Malignant Pheochromocytoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Clitoral Gland: Carcinoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Clitoral Gland: Adenoma or Carcinoma 

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 (includes NOS) 

P<O.OOl 

P<O.OOl 

P<O.OOl 

72 1 

1/25 (4%) 

7.6% 

2/53 (4%) 

P=O.O83 

P=O.O87 

590 

5/25 (20%) 

24.4% 

7/53 (13%) 

P<O.OOl 

P<O.OOl 

563 

22/28 (79%) 

85.5% 

36/53 (68 %) 

P=O.O80 P<O.OOl 

P<O.OOl 

P<O.OOl 

P<O.OOl 

611 

1/25 (4%) 

10.4% 

3/53 (6%) 

P=O.382 

P=O.385 

P=O.420 

514 

0/25 (0%) 

2.3% 

1/52 (2%) 

P=O.358 

P=O.363 

P=O.400 

574 

0/25 (0%) 

2.3% 

1/52 (2%) 

P=O.311 

P=O.325 

P=O.372 

729 (T) 

2/25 (8%) 

8.0% 

2/53 (4%) 

P=O.166 

P=O.170 

5 90 

5/25 (20%) 

24.4% 

7/53 (13%) 

P=O.160 

P=O.294 

P=O.324 

620 

1/25 (4%) 

9.2% 

3/52 (6%) 

P=O.309 

P=O.101 

P=O. 114 

620 

2/25 (8%) 

15.4% 

5/52 (10%) 

P=O.102 

P=O.223 

P=O.221 

594 

4/25 (16%) 

17.8% 

5/53 (9%) 

P=O.219 

P<O.OOl 

P<O.OOl 

563 

22/28 (79%) 

85.5% 

36/53 (68 %) 

P<O.OOl 

P=O.310 

P=O.340 

709 

2/28 (7%) 

10.1% 

3/52 (6%) 

P=O.309 

P=O.185 

P=O.212 

709 

3/28 (1 1 %) 

13.6% 

4/52 (8%) 

P=O.181 

P=O.230 

P=O.260 

594 

4/28 (14%) 

16.2% 

5/53 (9%) 

P=O.219 
Lesions in Female Rats 187 

of Nickel Subsulfide (continued) 

Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Inhalation Study 

TABLE B3 

0 mg/m3 0.15 mg/m3 1 mg/m3 

Fisher exact test 

Cochran-Amitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Lung: Alveolarlbronchiolar Carcinoma 

Fisher exact test 

Cochran-Armitage test P=O.O28 

Logistic regression test P=O.O30 

Life table test P=O.O42 

First incidence (days) 729 (T) 

Terminal rate 2/25 (8%) 

Adjusted rate 8.0% 

Overall rate 2/53 (4%) 

Lung: Alveolar/bronchiolar Adenoma or Carcinoma (includes NOS) 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Mammary Gland: Fibroadenoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Mammary Gland: Carcinoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Mammary Gland: Fibroadenoma or Adenoma 

0125 (0%) 

0.0% 

0/53 (0%) 

e 

P=O.O09 

P=O.OlO 

P=O.O12 

- 

P=0.037N 

P=0.034N 

P=0.030N 

377 

11/25 (44%) 

57.9% 

22/53 (42%) 

P=0.224N 

P=0.219N 

P=0.207N 

729 (T) 

1/25 (4%) 

4.0% 

1/53 (2%) 

P=0.027N 

P=O.O25N 

P=0.023N 

377 

1 1 /25 (44 % ) 

59.2% 

23/53 (43%) 

- 

012.5 (0%) 

0.0% 

0153 (0%) 

- 

- 

- 

P=O.223 

P=O.221 

594 

4/25 (16%) 

17.8% 

5/53 (9%) 

P=O.219 

P=0.016N 

P=0.027N 

594 

6/25 (24%) 

34.0% 

11/53 (21 %) 

P=0.018N 

P=O.315 

P=O.321 

594 

1/25 (4%) 

9.4% 

3/53 (6%) 

P=O.309 

P=O.OlON 

P=0.019N 

594 

6/25 (24%) 

34.0% 

11/53 (21 %) 

P=O.OllN 

P=O.O66 

P=O.O70 

635 

2/28 (7%) 

12.0% 

4/53 (8%) 

P=O.O59 

P=O.O31 

P=O.O38 

594 

6/28 (21 %) 

27.2% 

9/53 (17%) 

P=O.O26 

P=0.009N 

P=O.OlON 

594 

6/28 (21 %) 

30.0% 

10/53 (19%) 

P=O.OlON 

0128 (0%) 

0.0% 

0153 (0%) 

P=0.500N 

P=0.477N 

P=0.477N 

- 

P=0.005N 

P=0.007N 

594 

6/28 (21 %) 

30.0% 

10/53 (19%) 

P=0.006N 
188 Nickel Subsulfide, NTP TR 453 

of Nickel Subsulfide (continued) 

Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Inhalation Study 

TABLE B3 

0 mg/m3 0.15 mg/m3 1 mg/m3 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Mammary Gland: Adenoma or Carcinoma 

P=0.141N 

P=0.138N 

P=0.133N 

665 

1/25 (4%) 

6.7% 

2/53 (4%) 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Mammary Gland: Fibroadenoma, Adenoma, or Carcinoma 

P=0.013N 

P=0.012N 

P=0.012N 

377 

12/25 (48%) 

62.1 % 

24/53 (45%) 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Pituitary Gland (Pars Distalis): Adenoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Thyroid Gland (Ccell): Adenoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Thyroid Gland (C-cell): Carcinoma 

P = 0.007N 

P=0.006N 

P=O.OlON 

477 

14/25 (56%) 

67.1 % 

24/52 (46%) 

P=O.515 

P=O.524 

P=O.546 

665 

2/25 (8%) 

10.6% 

3/51 (6%) 

0125 (0%) 

0.0% 

0/51 (0%) 

P=0.356N 

P=0.359N 

P=0.334N 

- 

P=O.512 

P=O.508 

594 

1/25 (4%) 

9.4% 

3/53 (6%) 

P=O.500 

P=0.018N 

P=0.033N 

594 

6/25 (24%) 

37.8% 

13/53 (25%) 

P=0.020N 

P=O.209 

P=O.220 

482 

13/25 (52%) 

70.5% 

30/53 (57%) 

P=O.191 

P=O.519 

P=O.502 

612 

2/25 (8%) 

12.9% 

4/53 (8%) 

P=O.522 

P=O.127 

P=O.127 

538 

2/25 (8%) 

9.8 % 

3/53 (6%) 

P=O.129 

0/28 (0%) 

0.0% 

0/53 (0%) 

P=0.231N 

P=0.234N 

- 

P=0.248N 

P=0.003N 

P=0.004N 

594 

6/28 (21 %) 

30.0% 

10153 (19%) 

P=0.003N 

P=0.037N 

P=0.038N 

563 

9/28 (32%) 

42.0% 

15/53 (28 %) 

P=0.045N 

P=O.518 

P=O.520 

635 

2/28 (7%) 

12.2% 

4/52 (8%) 

P=O.511 

- 

0128 (0%) 

0.0% 

0/52 (0%) 
Lesions in Female Rats 189 

of Nickel Subsulfide (continued) 

Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Inhalation Study 

TABLE B3 

0 mg/mg 0.15 mgh’ 1 mg/m3 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Thyroid Gland (C-cell): Adenoma or Carcinoma 

P=0.524N 

P=0.517N 

P=0.482N 

665 

2/25 (8%) 

10.6% 

3/5 1 (6 %) 

P=O.175 

P=O.168 

538 

4/25 (16%) 

22.1 % 

7/53 (13%) 

P=O.518 

P=O.520 

635 

2/28 (7%) 

12.2% 

4/52 (8%) 

P=O.176 P=O.511 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Uterus: Stromal Polyp 

P=0.510N 

P=0.495N 

729 (T) 

1/25 (4%) 

4.0% 

1/53 (2%) 

P=O.442 

P=O.521 

444 

0128 (0%) 

6.5% 

3/53 (6%) 

P=O.344 

P=O.322 

P=O.369 

500 

1/25 (4%) 

6.0% 

2/53 (4%) 

P=0.500N P=O.500 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Mononuclear Cell Leukemia 

P=O.268 

P=O.252 

P=O.375 

263 

6/25 (24%) 

46.8% 

19/53 (36%) 

P=0.534N 

P=0.498N 

452 

8/25 (32%) 

48.4% 

18/53 (34 %) 

P=O.313 

P=O.437 

501 

8/28 (29%) 

50.0% 

22/53 (42%) 

P=0.500N P=O.345 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Benign Neoplasms 

P=O.118 

P=O.134 

P=O.407 

377 

18/25 (72%) 

83.1% 

37/53 (70%) 

P=O.O78 

P=O.162 

482 

24/25 (96%) 

97.8% 

45/53 (85 %) 

P=O.O65 

P=O.266 

444 

25/28 (89%) 

93.7% 

45/53 (85%) 

P=O.O52 P=O.O52 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Malignant Neoplasms 

P=O.441 

P=O.424 

P=0.537N 

263 

8/25 (32%) 

56.3% 

24/53 (45 %) 

P=O.102 

P=O.200 

452 

12/25 (48 %) 

68.7 % 

31/53 (58%) 

P=O.261 

P=O.406 

501 

12/28 (43%) 

61.9% 

28/53 (53 %) 

P=O.122 P=O.280 
190 Nickel Subsulfide, NTP TR 453 

of Nickel Subsulfide (continued) 

Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Inhalation Study 

TABLE B3 

0 mg/m3 0.15 mg/m3 1 mg/m3 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Benign or Malignant Neoplasms 

P=0.426N 

P=0.442N 

P=0.299N 

263 

20/25 (80%) 

90.6% 

48/53 (91 %) 

P=O.209 

P=O.418 

452 

24/25 (96%) 

98.1% 

51/53 (96%) 

P=0.629N 

P=0.398N 

444 

25/28 (89%) 

94.1 % 

48/53 (91 X) 

P=O.219 P=0.629N 

a Number of neoplasm-bearing animalslnumber of animals examined. Denominator is number of animals examined microscopically for adrenal gland, 

(T)Terminal sacrifice 

compare directly the overall incidence rates. For all tests, a negative trehd or a lower incidence in an exposure group is indicated by N. 

(directly or indirectly) the cause of death. The logistic regression test regards these lesions as nonfatal. The Cochran-Armitage and Fisher exact tests 

painvise comparisons between the controls and that exposed group. The life table test regards neoplasms in animals dying prior to terminal kill as being 

Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding to 

Observed incidence at terminal kill 

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

clitoral gland, lung, pituitaly gland, thyroid gland, and uterus; for other tissues, denominator is number of animals necropsied. 

e Not applicable; no neoplasms in animal group 
Lesions in Female Rats 191 

Historical Incidence of Lung Neoplasms in Untreated Female F344/N Ratsa 

TABLE B4a 

Study 

Incidence in Controls 

Carcinoma 

Carcinoma Carcinoma or Squamous Cell 

Alveolarhronchiolar 

Alveolarlbronchiolar Alveolarhronchiolar Squamous Cell Adenoma or Carcinoma 

Adenoma 

Historical Incidence at Lovelace Inhalation Toxicology Research Institute 

Talcb 

Nickel Sulfate Hexahydrate 

Nickel Subsulfide 

Nickel Oxide 

1/50 

0152 

2153 

1153 

Overall Historical Incidence in Inhalation Studies 

Range 

Standard deviation 

Total 

0%-4% 

1.5% 

71700 (1.1 %) 

0150 

0152 

0153 

0153 

0150 

0/52 

0153 

0153 

1/50 

0152 

2/53 

1/53 

01700 (0%) 01700 (0 %) 

0%-4% 

1.5% 

8/700 (1.1%) 

Overall Historical Incidence in Feed Studies 

Range 

Standard deviation 

Total 

0%-10% 

2.2% 

20/1,201 (1.7%) 

0%-2% 

0.8% 

511.201 (0.4%) 011,201 (0%) 

0%-10% 

2.2% 

25/1,201 (2.1%) 

Results of lifetime study 

a Data as of 17 June 1994 
192 Nickel Subsulfide, NTP TR 453 

Incidence in Controls 

Study 

Malignant, or NOS 

Benign Complex Malignant NOS Benign, Complex, 

Historical Incidence at Lovelace Inhalation Toxicology Research Institute 

Nickel Sulfate Hexahydrate 

Nickel Subsulfide 

Nickel Oxide 

215 1 

2/53 

4/5 1 

015 1 

0/53 

015 1 

015 1 

1/53 

015 1 

Overall Historical Incidence in Inhalation Studies 

Range 

Standard deviation 

Total 

0%-14% 

4.9% 

35/608 (5.8%) 2/608 (0.3%) 1/608 (0.2%) 

1.1 % 0.6% 

0%-4% 0%-2% 

Overall Historical Incidence in Feed Studies 

Range 

Standard deviation 

Total 

0%-2% 0%-2% 

0.6% 0.9% 

49/1,175 (4.2%) 2/1,175 (0.2%) 611,175 (0.5%) 

2.5% 

0%-8% 

015 1 

0/53 

0/5 1 

0%-2% 

0.6% 

1/608 (0.2%) 

0%-12% 

2.5% 

6/1,175 (0.5%) 

2/5 1 

3/53 

4/5 1 

2%-14% 

4.4% 

39/608 (6.4%) 

2%-12% 

2.8% 

62/1,175 (5.3%) 

a Data as of 17 June 1994; data from lifetime talc study not included because incidences of adrenal medulla pheochromocytomas increase significantly in 

aging rats, and this data would not be comparable to that in the 2-year nickel subsulfide study. 
Lesions in Female Rats 193 

of Nickel Subsulfidea 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study 

TABLE B5 

0 mg/m3 0.15 mg/m3 1 mg/m3 

Early deaths 

IS-Month interim evaluation 

7-Month interim evaluation 

Animals initially in study 

Disposition summary 

Natural deaths 

Moribund 

Accidental death 

Terminal sacrifice 

Survivors 

Animals examined microscopically 

5 

5 

63 

4 

24 

25 

63 

5 

5 

63 

1 

27 

25 

63 

5 

5 

63 

1 

23 

1 

28 

63 

Ovary 

Genital System 

7-Month Interim Evaluation 

cyst 

(1) 

1 (100%) 

Hyperplasia, lymphoid 

Lymph node, bronchial 

Hematopoietic System 

Hyperplasia, lymphoid 

Lymph node, mediastinal (4) 

(5) 

1 (25%) 

2 (40%) 

(5) 

(5) 

5 (100%) 

5 (100%) 

Lung 

Respiratory System 

Interstitium, infiltration cellular 

Bronchus, hyperplasia, lymphoid 

Alveolus, proteinosis 

Alveolus, hyperplasia, macrophage 

Metaplasia, osseous 

Inflammation, chronic active 

Olfactory epithelium, erosion, focal 

Inflammation, chronic active 

Nose 

(5) 

1 (20%) 

1 (20%) 

1 (20%) 

2 (40%) 

(5) 

(5) 

5 (100%) 

5 (100%) 

1 (20%) 

2 (40%) 

5 (100%) 

2 (40%) 

2 (40%) 

(5) 

(5) 

5 (100%) 

5 (100%) 

4 (80%) 

5 (100%) 

5 (100%) 

4 (80%) 

(5) 

Urinary System 

Special Senses System 

Nervous System 

Musculoskeletal System 

Integumentary System 

General Body System 

Endocrine System 

Cardiovascular System 

Alimentary System 

Systems Examined With No Lesions Observed 

a Number of animals examined microscopically at the site and the number of animals with lesion 
194 Nickel Subsulfide, NTP TR 453 

of Nickel Subsulfide (continued) 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study 

TABLE B5 

0 mg/m3 0.15 mg/m3 1 mg/m3 

Liver 

Alimentary System 

15-Month Interim Evaluation 

(5) 

Mixed cell focus 

Inflammation, granulomatous 

Hepatodiaphragmatic nodule 

Granuloma 

Basophilic focus 

4 (80%) 

1 (20%) 

3 (60%) 

(5) 

3 (60%) 

2 (40%) 

1 (20%) 

(5) 

1 (20%) 

5 (100%) 

1 (20%) 

5 (100%) 

Endocrine System 

Pituitary gland 

Hyperplasia 

Adrenal cortex 

Thyroid gland 

cyst 

C-cell, hyperplasia 

(5) 

1 (20%) 

(5) 

1 (20%) 

Hemorrhage, acute 

Lymph node, bronchial 

Hematopoietic System 

Hyperplasia, macrophage 

Hyperplasia, lymphoid 

Hyperplasia, macrophage 

Hyperplasia, lymphoid 

Lymph node, mediastinal 

1 (20%) 

(5) 

1 (20%) 

(5) 

2 (40%) 

1 (20%) 

1 (20%) 

(5) 

1 (20%) 2 (40%) 

1 (20%) 

4 (80%) 

2 (40%) 

(5) 

Respiratory System 

Interstitium, infiltration cellular 

Bronchus, hyperplasia, lymphoid 

Alveolus, proteinosis 

Alveolus, hyperplasia, macrophage 

Inflammation, chronic active 

Hemorrhage, acute 

Lung 

Olfactory epithelium, atrophy 

Nasolacrimal duct, hyperplasia 

Inflammation, chronic active 

Nose 

(5) 

2 (40%) 

1 (20%) 

(5) 

3 (60%) 

(5) 

5 (100%) 

5 (100%) 

5 (100%) 

3 (60%) 

4 (80%) 

(5) 

2 (40%) 

1 (20%) 

(5) 

5 (100%) 

1 (20%) 

5 (100%) 

5 (100%) 

5 (100%) 

2 (40%) 

2 (40%) 

(5) 

1 (20%) 

EY e 

Special Senses System 

Cataract 
Lesions in Female Rats 195 

of Nickel Subsulfide (continued) 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study 

TABLE B5 

0 mg/m3 0.15 mg/m3 1 mg/m3 

Kidney 

Urinary System 

15-Month Interim Evaluation (continued) 

Nephropathy 

(5) 

1 (20%) 

(5) 

2 (40%) 

(5) 

2 (40%) 

Nervous System 

Musculoskeletal System 

Integumentary System 

Genital System 

General Body System 

Cardiovascular System 

Systems Examined With No Lesions Observed 

2- Year Study 

Intestine large, cecum 

Parasite metazoan 

Intestine large, rectum 

Parasite metazoan 

Intestine large, colon 

Alimentary System 

Hyperplasia, lymphoid 

Intestine small, ileum 

Hyperplasia, lymphoid 

Intestine small, jejunum 

Parasite metazoan 

Inflammation 

Vein, thrombosis 

Hepatocyte, necrosis 

Hepatocyte, hyperplasia 

Hepatocyte, atrophy 

Centrilobular, atrophy 

Biliary tract, hyperplasia 

Bile duct, hyperplasia 

Regeneration 

Pigmentation, hemosiderin 

Mixed cell focus 

Inflammation, focal, granulomatous 

Hepatodiaphragmatic nodule 

Hematopoietic cell proliferation 

Eosinophilic focus 

Degeneration, fatty 

Degeneration, cystic 

Clear cell focus 

Basophilic focus 

Angiectasis 

Liver 

Fat, necrosis 

Necrosis 

Mesentery 

(53) 

(50) 

44 (83%) 

1 (2%) 

3 (6%) 

3 (6%) 

16 (30%) 

1 (2%) 

3 (6%) 

3 (6%) 

24 (45%) 

6 (11%) 

9 (17%) 

1 (2%) 

18 (34%) 

1 (2%) 

4 (8%) 

1 (100%) 

(40) 

2 (5%) 

(52) 

5 (10%) 

1 (2%) 

2 (4%) 

(52) 

(52) 

(53) 

1 (2%) 

1 (2%) 

22 (42%) 

3 (6%) 

1 (2%) 

2 (4%) 

12 (23%) 

4 (8%) 

1 (2%) 

46 (87%) 

13 (25%) 

1 (2%) 

1 (2%) 

5 (9%) 

40 (75%) 

13 (25%) 

2 (4%) 

1 (2%) 

5 (9%) 

11 (21%) 

2 (4%) 

14 (26%) 

2 (4%) 

I (13%) 

14 (26%) 

1 (2%) 

1 (50%) 

1 (2%) 

1 (2%) 

(2) 
Nickel Subsulfide, NTP TR 453 

TABLE B5 

of Nickel Subsulfide (continued) 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study 

0 mg/m3 0.15 mg/m3 1 mg/mJ 

Pancreas 

Alimentary System (continued) 

%Year Study (continued) 

Stomach, forestomach 

Atrophy 

Ulcer 

Inflammation 

Hyperplasia, squamous 

Peridontal tissue, inflammation 

Tooth 

(53) 

(53) 

2 (4%) 

14 (26%) 

Heart 

Blood vessel 

Cardiovascular System 

Atrium. thrombosis 

Cardiomyopathy 

Inflammation 

(53) 

2 (4%) 

25 (47%) 

(53) 

1 (2%) 

17 (32%) 

Endocrine System 

Vacuolization cytoplasmic 

Necrosis 

Hyperplasia, focal 

Hyperplasia 

Degeneration, cystic 

Adrenal cortex 

Hyperplasia 

Parathyroid gland 

Hyperplasia 

Islets, pancreatic 

Hyperplasia 

Adrenal medulla 

Pars distalis, hyperplasia 

Pars distalis, cyst 

Pars distalis, angiectasis 

Pituitary gland 

C-cell, hyperplasia 

Thyroid gland 

(53) 

1 (2%) 

11 (21%) 

(52) 

(48) 

1 (2%) 

13 (25%) 

3 (6%) 

21 (40%) 

15 (29%) 

(51) 

(53) 

11 (21%) 

11 (21%) 

2 (4%) 

1 (2%) 

11 (21%) 

3 (6%) 

11 (21%) 

5 (9%) 

3 (6%) 

(50) 

(53) 

(53) 

(53) 

(53) 

(53) 

19 (36%) 

6 (11%) 

2 (4%) 

5 (9%) 

1 (2%) 

16 (30%) 

(53) 

(49) 

(53) 

(53) 

11 (21%) 

14 (26%) 

16 (30%) 

3 (6%) 

1 (2%) 

(52) 

None 

General Body System 

Clitoral gland 

Genital System 

Inflammation 

Hyperplasia, cystic 

(52) 

1 (2%) 

5 (10%) 
Lesions in Female Rats 197 

of Nickel Subsulfide (continued) 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study 

TABLE B5 

0 mg/m3 0.15 mg/m3 1 mg/m3 

Ovary 

Genital System (cbntinued) 

&Year Study (continued) 

Inflammation 

cyst 

Atrophy 

Endometrium, hyperplasia, cystic 

Inflammation 

cyst 

Uterus 

(53) 

5 (9%) 

(53) 

3 (6%) 

1 (2%) 

1 (2%) 

(53) 

2 (4%) 

6 (11%) 

1 (2%) 

(53) 

1 (2%) 

1 (2%) 

Hematopoietic System 

Myeloid cell, hyperplasia 

Erythroid cell, hyperplasia 

Necrosis 

Inflammation, focal, granulomatous 

Fibrosis 

Atrophy 

Bone marrow 

Renal, hyperplasia, lymphoid 

Pancreatic, hyperplasia, macrophage 

Pancreatic, hyperplasia, lymphoid 

Inflammation 

Hyperplasia, plasma cell 

Hyperplasia, macrophage 

Hyperplasia, lymphoid 

Hyperplasia, plasma cell 

Hyperplasia, lymphoid 

Lymph node, mandibular 

Hyperplasia, macrophage 

Hyperplasia, lymphoid 

Atrophy 

Lymph node, mesenteric 

Hyperplasia, macrophage 

Hyperplasia, lymphoid 

Lymph node, mediastinal 

Pigmentation, hemosiderin 

Hyperplasia, lymphoid 

Hemorrhage 

Hematopoietic cell proliferation 

Fibrosis 

Atrophy 

Degeneration 

Lymph node 

Lymph node, bronchial 

Spleen 

Thymus 

(53) 

4 (8%) 

2 (4%) 

2 (4%) 

11 (22%) 

1 (20%) 

(50) 

1 (2%) 

3 (6%) 

(46) 

2 (4%) 

7 (13%) 

1 (2%) 

1 (2%) 

2 (4%) 

(49) 

(53) 

1 (33%) 

2 (4%) 

1 (2%) 

(3) 

(49) 

16 (33%) 

36 (73%) 

(50) 

5 (9%) 

1 (2%) 

6 (12%) 

5 (10%). 

(53) 

(50) 

11 (22%) 

3 (6%) 

1 (2%) 

4 (8%) 

(53) 

1 (2%) 

12 (23%) 

(51) 

1 (2%) 

1 (2%) 

3 (6%) 

(50) 

1 (2%) 

2 (4%) 

24 (48%) 

36 (72%) 

4 (8%) 

1 (2%) 

(52) 

(51) 

5 (11%) 

20 (43%) 

3 (6%) 

6 (12%) 

(47) 

(53) 

3 (6%) 

2 (4%) 

2 (4%) 

(51) 
198 Nickel Subsulfide, NTP TR 453 

of Nickel Subsulfide (continued) 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study 

TABLE B5 

0 mg/m3 0.15 mg/m3 1 mg/m3 

Mammary gland 

Integumentary System 

&Year Study (continued) 

Hyperplasia, cystic 

Fibrosis 

Tail, inflammation 

Tail, hyperkeratosis 

Ulcer 

Inflammation 

Hyperkeratosis 

Skin 

(53) 

1 (2%) 

1 (2%) 

11 (21%) 

(53) 

(53) 

14 (26%) 

1 (2%) 

(53) 

1 (2%) 

1 (2%) 

(53) 

16 (30%) 

(53) 

1 (2%) 

1 (2%) 

Bone 

Musculoskeletal System 

Cranium, hyperostosis 

Hyperostosis 

(53) 

5 (9%) 

(53) 

3 (6%) 

(53) 

3 (6%) 

7 (13%) 

Brain 

Nervous System 

Gray matter, necrosis 

Cerebrum, compression 

Hydrocephalus 

Hemorrhage 

cyst 

Degeneration, secondaxy wallerian 

Spinal cord 

(53) 

5 (9%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

10 (19%) 

(53) 

1 (2%) 

5 (9%) 

2 (4%) 

Larynx 

Respiratory System 

Inflammation 

Hyperplasia, squamous 

Lung 

Interstitium, infiltration cellular 

Bronchus, hyperplasia, lymphoid 

Alveolus, proteinosis 

Alveolus, hyperplasia, macrophage 

Alveolar epithelium, hyperplasia, focal 

Metaplasia, squamous 

Metaplasia, osseous 

Inflammation, chronic active 

Fibrosis 

Emphysema 

cyst 

7 (13%) 

1 (2%) 

2 (4%) 

8 (15%) 

2 (4%) 

28 (53%) 

(53) 

2 (4%) 

(53) 

51 (96%) 

50 (94%) 

1 (2%) 

36 (68%) 

15 (28%) 

49 (92%) 

51 (96%) 

10 (19%) 

(51) 

3 (6%) 

51 (96%) 

44 (83%) 

1 (2%) 

1 (2%) 

(53) 

43 (81%) 

18 (34%) 

53 (100%) 

52 (98%) 

11 (21%) 

3 (6%) 
Lesions in Female Rats 199 

Qf Nickel Subsulfide (continued) 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study 

TABLE B5 

Nose 

Respiratory System (continued) 

2-Yeur Study (continued) 

(53) 

Olfactory epithelium, metaplasia 

Olfactory epithelium, atrophy 

Nasolacrimal duct, inflammation 

Thrombosis 

Inflammation, chronic active 

1 (2%) 

2 (4%) 

6 (11%) 

(53) 

9 (17%) 

1 (2%) 

2 (4%) 

(52) 

2 (4%) 

1 (2%) 

20 (38%) 

1 (2%) 

16 (31%) 

EY e 

Special Senses System 

Metaplasia, squamous 

Retina, degeneration 

Cataract 

Lacrimal gland 

(2) 

2 (100%) 

2 (100%) 

(2) 

1 (50%) 

2 (100%) 

(1) 

1 (100%) 

(1) 

1 (100%) 

Urinary System 

Renal tubule, pigmentation 

Nephropathy 

Hydronephrosis 

cyst 

Kidney (53) 

40 (75%) 

51 (96%) 

1 (2%) 

43 (81%) 

52 (98%) 

43 (81%) 

50 (94%) 

1 (2%) 
200 Nickel Subsulfide, NTP TR 453 
201 

OF NICKEL SUBSULFIDE 

SUMMARY OF LESIONS IN MALE MICE 

APPENDIX C 

IN THE 2-YEAR INHALATION STUDY 

TABLE C1 Summary of the Incidence of Neoplasms in Male Mice 

in the 2-Year Inhalation Study of Nickel Subsulfide 

TABLE C2 Individual Animal Tumor Pathology of Male Mice 

in the 2-Year Inhalation Study of Nickel Subsulfide 

in the 2-Year Inhalation Study of Nickel Subsulfide 

TABLE C5 Summary of the Incidence of Nonneoplastic Lesions in Male Mice 

in Untreated Male B6C3F, Mice 

TABLE C4 Historical Incidence of Alveolar/bronchiolar Neoplasms 

in the 2-Year Inhalation Study of Nickel Subsulfide 

TABLE C3 Statistical Analysis of Primary Neoplasms in Male Mice 

....................... 

235 

234 

231 

207 

202 

....................... 

....................... 

................................... 

....................... 
Nickel Subsulfide, NTP TR 453 

Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Inhalation Study of Nickel Subsulfidea 

TABLE C1 

Early deaths 

I5-Month interim evalua&nb 

7-Month interim evaZualionb 

Animals initially in study 

Disposition Summary 

Terminal sacrifice 

Died last week of study 

Natural deaths 

Moribund 

Survivors 

Animals examined microscopically 

10 

9 

80 

8 

27 

26 

71 

10 

10 

80 

9 

26 

25 

70 

10 

10 

80 

9 

25 

25 

1 

70 

Urinary System 

Special Senses System 

Respiratory System 

Nervous System 

Musculoskeletal System 

Integumentary System 

Hematopoietic System 

Genital System 

General Body System 

Endocrine System 

Cardiovascular System 

Alimentary System 

Systems Examined At 7 Months With No Neoplasms Observed 

Liver 

Alimentary System 

15-Month Interim Evaluation 

(5) 

Hepatocellular adenoma 

Hepatocellular carcinoma 

1 (20%) 

(5) 

2 (40%) 

(5) 

1 (20%) 

1 (20%) 

Urinary System 

Special Senses System 

Respiratory System 

Nervous System 

Musculoskeletal System 

Integumentary System 

Hematopoietic System 

Genital System 

General Body System 

Endocrine System 

Cardiovascular System 

Systems Examined With No Neoplasms Observed 
Lesions in Male Mice 203 

Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Inhalation Study of Nickel Subsulfide (continued) 

TABLE C1 

O mg/m3 0.6 mg/m3 1.2 mg/m3 

Gallbladder 

Alimentary System 

2- Year Study 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Adenoma 

Fibrous histiocytoma, metastatic, liver 

Hepatocellular carcinoma, metastatic, liver 

Hepatocholangiocarcinoma 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple 

Hepatocellular carcinoma 

Hepatoblastoma 

Hemangiosarcoma 

Hemangioma 

Fibrous histiocytoma 

Fibrous histiocytoma, metastatic, liver 

Fibrous histiocytoma, metastatic. liver 

Salivaly glands 

Pancreas 

Mesentery 

Fibrous histiocytoma, metastatic, liver 

Adenocarcinoma 

Stomach, glandular 

Odontoma 

Tooth 

(54) 

(54) 

(54) 

(59) 

(55) 

1 (2%) 

12 (20%) 

1 (2%) 

1 (2%) 

11 (19%) 

3 (5%) 

(57) 

(59) 

(58) 

1 (2%) 

(4) 

2 (50%) 

Heart 

Cardiovascular System 

(61) 

Alveolarlbronchiolar carcinoma, metastatic, 

lung 1 (2%) 

Adrenal cortex 

Endocrine System 

Pheochromocytoma complex 

Fibrous histiocytoma, metastatic, liver 

Capsule, adenoma 

Fibrous histiocytoma, metastatic, liver 

Pituitaly gland 

Adenoma 

Islets, pancreatic 

Follicular cell, adenoma 

Thyroid gland 

Pars distalis, adenoma 

Adrenal medulla 

(61) 

4 (7%) 

(59) 

(59) 

1 (2%) 

1 (2%) 

(56) 
204 Nickel Subsulfide, NTP TR 453 

Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Inhalation Study of Nickel Subsulfide (continued) 

TABLE C1 

0 mg/m3 0.6 mg/m3 1.2 mg/m3 

Tissue NOS 

General Body System 

2-YeUr study (continued) 

(1) 

Thoracic, alveolar/bronchiolar carcinoma, 

Thoracic, alveoladbronchiolar carcinoma, 

Mediastinum, alveolar/bronchiolar carcinoma, 

metastatic, lung 

metastatic 

metastatic 

1 (100%) 

(1) 

1 (100%) 

1 (100%) 

Epididymis 

Coagulating gland 

Genital System 

Interstitium, granular cell tumor NOS 

Leiomyoma 

Fibrous histiocytoma, metastatic, liver 

Fibrous histiocytoma 

Interstitial cell, adenoma 

Testes 

Fibrous histiocytoma, metastatic, liver 

Seminal vesicle 

(61) 

1 (2%) 

1 (2%) 

(61) 

(61) 

1 (2%) 

1 (2%) 

1 (2%) 

(59) 

Lymph node 

Hematopoietic System 

Pancreatic, fibrous histiocytoma, metastatic, 

Axillary, fibrosarcoma, metastatic, skin 

liver 

Lymph node, bronchial 

Adenocarcinoma, metastatic, stomach, 

Hepatocellular carcinoma, metastatic 

Alveolar/bronchiolar carcinoma, metastatic, lung 

Hemangiosarcoma 

Hemangioma 

Fibrous histiocytoma 

glandular 

Lymph node, mesenteric 

Spleen 

Lymph node, mediastinal 

Alveolarlbronchiolar carcinoma, metastatic, 

Hemangiosarcoma 

Hemangioma 

lung 

Thymus 

(18) 

1 (6%) 

1 (2%) 

(48) 

1 (2%) 

1 (2%) 

(55) 

1 (2%) 

1 (2%) 

1 (2%) 
Lesions in Male Mice 205 

Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Inhalation Study of Nickel Subsulfide (continued) 

TABLE C1 

0 mg/m3 0.6 mg/m3 1.2 mg/rn3 

Skin 

Integumentary System 

&Year Study (continued) 

Tail, hemangiosarcoma 

Prepuce, fibrous histiocytoma 

Pinna, hemangiosarcoma 

Fibrous histiocytoma 

Fibrosarcoma 

(61) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

None 

Musculoskeletal System 

None 

Nervous System 

Lung 

Respiratory System 

(61 ) 

Pheochromocytoma complex, metastatic, 

Hepatocholangiocarcinoma, metastatic, liver 1 (2%) 

Hepatocellular carcinoma, metastatic, liver 2 (3%) 

Hepatoblastoma, metastatic;liver 

Fibrous histiocytoma, metastatic, liver 1 (2%) 

Alveolar/bronchiolar carcinoma 7 (11%) 

Alveolar/bronchiolar adenoma, two 1 (2%) 

Alveolar/bronchiolar adenoma 5 (8%) 

adrenal medulla 1 (2%) 

(59) 

3 (5%) 

2 (3%) 

6 (10%) 

(58) 

2 (3%) 

4 (7%) 

2 (3%) 

1 (2%) 

Harderian gland 

Special Senses System 

Adenoma 

(1) 

1 (100%) 

Kidney 

Urinary System 

Hepatoblastoma, metastatic, liver 

Fibrous histiocytoma, metastatic, liver 

Leiomyosarcoma 

Pelvis, hemangiosarcoma 

Hepatocellular carcinoma, metastatic, liver 

1 (2%) 

1 (2%) 

Urinary bladder (61) 

1 (2%) 

1 (2%) 

Multiple organs' 

Systemic Lesions 

Lymphoma malignant 

(61) 

1 (2%) 

(60) 

1 (2%) 
206 Nickel Subsulfide, NTP TR 453 

Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Inhalation Study of Nickel Subsulfide (continued) 

TABLE C1 

~ota~ animals with primary neoplasmsd 

Neoplasm Summary 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

2-Year study 

2-Year study 

2-Year study 

Total primary neoplasms 

Total animals with benign neoplasms 

Total benign neoplasms 

Total animals with malignant neoplasms 

Total malignant neoplasms 

Total animals with uncertain neoplasmsTotal metastatic neoplasms 

Total animals with metastatic neoplasms 

Total uncertain neoplasms 

benign or malignant 

2-Year study 

2-Year study 

40 

1 

56 

1 

27 

1 

30 

1 

21 

26 

7 

20 

31 

2 

40 

2 

15 

2 

16 

2 

23 

24 

6 

6 

35 

1 

46 

2 

19 

1 

23 

1 

20 

1 

22 

1 

4 

10 

1 

1 

Primary neoplasms: all neoplasms except metastatic neoplasms 

Number of animals with any tissue examined microscopically 

Five animals in each exposure group were histopathologically examined at the interim evaluations. 

a Number of animals examined microscopically at the site and the number of animals with neoplasm 
Lesions in Male Mice 207 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0 mg/m3 

TABLE C2 

Number of Days on Study 

7236430986642271445689150 

6110368902345556888888902 

0234444455555555555555566 

Carcass ID Number 

2695636177648045207366159 

4462671234204610027073634 

0000000000000000000000000 

Gallbladder 

Esophagus 

Alimentary System 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Fibrous histiocytoma, metastatic, liver 

Adenoma 

Hepatocholangiocarcinoma 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma 

Fibrous histiocytoma 

Fibrous histiocytoma, metastatic, liver 

Fibrous histiocytoma, metastatic, liver 

Salivary glands 

Pancreas 

Mesentery 

Tooth 

Stomach, glandular 

Stomach, forestomach 

Fibrous histiocytoma, metastatic, liver 

+++AA++++++++++++++++++++ 

......................... 

X 

A++AA++++++++++++++++++++ 

+++AA++++++++++++++++++++ 

+++AA+++MM+++M+++M+M+MM++ 

A++AA++++++++++++++++++M+ 

......................... 

A++AA++++++++++++++++++++ 

A++AA++++++++++++++++++++ 

A 

xxx X X X 

X 

X 

X xx 

X 

X 

+ 

......................... A 

......................... 

......................... 

......................... 

X 

+ 

Heart 

Cardiovascular System 

Metastatic, lung 

Alveolarlbronchiolar carcinoma, 

......................... 

X 

Adrenal cortex 

Endocrine System 

Pheochromocytoma complex 

Fibrous histiocytoma, metastatic, liver 

Capsule, adenoma 

Fibrous histiocytoma, metastatic, liver 

Parathyroid gland 

Adenoma 

Islets, pancreatic 

Thyroid gland 

Pituitary gland 

Adrenal medulla 

Follicular cell, adenoma 

......................... X 

+++++++++++++++++++M+++++ 

X 

......................... 

......................... 

......................... 

++++++M++++++++++++++I+++ 

X 

A: Autolysis precludes examination 

+ : Tissue examined microscopically 

I: Insufficient tissue 

M: Missing tissue 

Blank: Not examined 

X: Lesion present 
208 Nickel Subsulfide, NTP TR 453 

(continued) 

Individual Animal Tumor Pathology of Male Mce in the 2-Year Inhalation Study of Nickel Subsulfide: 0 mg/m3 

TABLE C2 

Number of Days on Study 

~~~~ 

1129111439777777770000000 

66666666771~77777777777777 

~~~ 

3467888911333333334444444 

Carcass ID Number 

0908093924112824141925974 

8340126552133366770011123 

0000000000000000000000000 

Gallbladder 

Esophagus 

Alimentary System 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

++++++++AA+++++++++++++++ 

+++++++++++++I+++++++++++ 

Fibrous histiocytoma, metastatic, liver 

++++++++AA+++++++++++++++ 

++++++++AA+++++++++++++++ 

++++++++AA+++++++++++++++ 

+++M++++++M+++M++++++++++ 

Adenoma 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Hepatocholangiocarcinoma 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma 

Fibrous histiocytoma 

Mesentery 

......................... 

++++++++AA++++++++M++++++ 

++++++++AA+++++++++++++++ 

xx 

X xx 

X 

xxx 

Fibrous histiocytoma, metastatic, liver 

Fibrous histiocytoma, metastatic, liver 

Pancreas 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Tooth 

......................... 

......................... 

......................... 

......................... 

Fibrous histiocytoma, metastatic, liver 

+ 

Heart 

Cardiovascular System 

Alveolar/bronchiolar carcinoma, 

++++++++++++++++++++++++t 

metastatic, lung 

Adrenal cortex 

Endocrine System 

......................... 

Pheochromocytoma complex 

Fibrous histiocytoma, metastatic, liver 

Capsule, adenoma 

Fibrous histiocytoma, metastatic, liver 

X X 

Adrenal medulla ......................... 

X 

Islets, pancreatic .......................... 

Thyroid gland 

Pituitary gland 

Parathyroid gland 

Adenoma 

Follicular cell, adenoma 

A 

......................... 

+++++++++++++++++M+++++++ 

+++MM+++++++++M++++++++++ 

X X 

\ 
Lesions in Male Mice 209 

(continued) 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0 mg/m3 

TABLE C2 

Number of Days on Study 

00011111111 

44444444444 

77777777777 

Carcass ID Number 

03012367789 

55712355677 

00000000000 

Tumors 

Tissues/ 

Total 

Gallbladder 

Esophagus 

Alimentary System 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Fibrous histiocytoma, metastatic, liver 

Adenoma 

Hepatocholangiocarcinoma 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma 

Fibrous histiocytoma 

Fibrous histiocytoma, metastatic, liver 

Fibrous histiocytoma, metastatic, liver 

Salivaly glands 

Pancreas 

Mesentely 

Tooth 

Stomach, glandular 

Stomach, forestomach 

Fibrous histiocytoma, metastatic, liver 

+++-, +++++++ 

+++++++++++ 

+++++++++++ 

+++++++++++ 

++++M++M+MM 

+++++++++++ 

+++++++++++ 

+++++++++++ 

++++++-I++++ 

X 

X X X 

X 

+++++++++++ 

+++++++++++ 

+++++++++++ 

+++++++++++ 

++ 4 

1 

61 

61 

61 

1 

61 

1 

1 

1 

2 

11 

11 

1 

61 

55 

56 

1 

56 

57 

45 

55 

1 

57 

60 

Heart 

Cardiovascular System 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 

+++++++++++ 

1 

61 

Adrenal cortex 

Endocrine System 

Pheochromocytoma complex 

Fibrous histiocytoma, metastatic, liver 

Capsule, adenoma 

Fibrous histiocytoma, metastatic, liver 

Parathyroid gland 

Adenoma 

Islets, pancreatic 

Thyroid gland 

Pituitaly gland 

Adrenal medulla 

Follicular cell, adenoma 

+++++++++++ 

+++++++++++ 

+++++++++++ 

+++++++++++ 

++++++M++++ 

+++++++++++ 

X 

2 

1 

61 

1 

1 

60 

4 

61 

58 

56 

1 

61 
210 Nickel Subsulfide, NTP TR 453 

(continued) 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0 mg/m3 

TABLE C2 

1236430986642211445689150 

6110368902345556888888902 

0234444455555555555555566 

2695636111648045201366159 

4462611234204610021013634 

0000000000000000000000000 

Number of Days on Study 

Carcass ID Number 

Tissue NOS 

General Body System 

carcinoma, metastatic, lung 

Thoracic, alveolar/bronchiolar 

+ 

X 

Epididymis 

Genital System 

......................... 

Fibrous histiocytoma, metastatic, liver 

Fibrous histiocytoma 

X 

Seminal vesicle 

Prostate 

Preputial gland 

Penis 

Testes 

++ + +++ + 

......................... 

......................... 

......................... + 

Interstitial cell, adenoma 

Fibrous histiocytoma, metastatic, liver X 

......................... 

Lymph node 

Bone marrow 

Hematopoietic System 

Pancreatic, fibrous histiocytoma, 

Axillary, fibrosarcoma, metastatic, skin 

metastatic, liver 

’ Lymph node, bronchial 

Alveolarlbronchiolar carcinoma, 

Lymph node, mesenteric 

Lymph node, mandibular 

metastatic, lung 

Thymus 

Spleen 

Lymph node, mediastinal 

Hemangioma 

Alveolarlbronchiolar carcinoma, 

metastatic, lung 

......................... +++ 

X 

+++ + ++ + + ++ 

X 

MI +++++++“+++++++I MM+M+M 

X 

MM++++++M+++++++++++M++++ 

+++M+++++++++++++++++M+++ 

+M++++++MM++++++M++++I++M 

......................... 

MMMMMM+MMM+MMM+MM+MMM+MMM 

A 

Skin 

Mammary gland 

Integumentary System 

Fibrosarcoma 

_____~~ ~ 

......................... 

MMMMMMMMMMMMMMMMMMMMMMMMM 

X 

Brain 

Nervous System 

Bone 

Musculoskeletal System ......................... 

......................... 
Lesions in Male Mice 211 

(continued) 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0 mg/m3 

TABLE C2 

1129111439777777770000000 

3467888911333333334444444 

6666666677777777777777777 

Number of Days on Study 

Carcass ID Number 

0908093924112824141925974 

8340126552133366770011123 

0000000000000000000000000 

Tissue NOS 

General Body System 

carcinoma, metastatic, lung 

Thoracic, alveolar/bronchiolar 

Epididymis 

Genital System 

X 

......................... 

Fibrous histiocytoma, metastatic, liver 

Fibrous histiocytoma 

Seminal vesicle 

Prostate 

Preputial gland 

Penis 

Testes 

+ ++ 

......................... 

......................... 

......................... 

Interstitial cell, adenoma 

Fibrous histiocytoma, metastatic, liver 

......................... 

Lymph node 

Bone marrow 

Hematopoietic System 

Pancreatic, fibrous histiocytoma, 

Axillary, fibrosarcoma, metastatic, skin 

metastatic, liver 

Lymph node, bronchial 

Alveolar/bronchiolar carcinoma, 

Lymph node, mesenteric 

Lymph node, mandibular 

metastatic, lung 

Thymus 

Spleen 

Lymph node, mediastinal 

Hemangioma 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 

......................... + + + 

+ +M++I ++++M++I +++M++++++ 

MM++++M++++++++++++++++++ 

++M++++++++++++++++++++++ 

X 

MMM+MM++++++M++++++++++++ 

......................... 

M+MM+MMMIMMMMMMMMM++MMMMM 

Skin 

Mammary gland 

Integumentary System 

Fibrosarcoma 

......................... 

MMMMMMMMMMMMMMMMMMMMMMMMM 

Bone 

Musculoskeletal System 

......................... 

Brain 

Nervous System 

~ ~ ~~ 

......................... 
212 Nickel Subsulfide, NTP TR 453 

(continued) 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0 mg/m3 

TABLE C2 

11111111111 

Number of Days on Study 

00011111111 

44444444444 

Carcass ID Number 

03012361189 

55112355611 

00000000000 

Tumors 

Tissues/ 

Total 

Tissue NOS 

General Body System 

carcinoma, metastatic, lung 

Thoracic, alveolar/bronchiolar 

Epididymis 

Genital System 

Fibrous histiocytoma, metastatic, liver 

Fibrous histiocytoma 

Seminal vesicle 

ProstatePreputial gland 

Penis 

Testes 

Interstitial cell, adenoma 

Fibrous histiocytoma, metastatic, liver 

+++++++++++ 

+++++++++++ 

+++++++++++ 

+++++++++++ 

+ 

+++++++++++ 

X 

61 

61 

61 

12 

1 

1 

61 

1 

61 

1 

Lymph node 

Bone marrow 

Hematopoietic System 

Pancreatic, fibrous histiocytoma, 

Axillary, fibrosarcoma, metastatic, skin 

metastatic, liver 

Lymph node, bronchial 

Alveolar/bronchiolar carcinoma, 

Lymph node, mesenteric 

Lymph node, mandibular 

metastatic, lung 

Thymus 

Spleen 

Lymph node, mediastinal 

Hemangioma 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 

+ 

+++++++++++ 

+ 

++MMM+MM++M 

+++++++++++ 

++M++++++M+ 

+M+++++++++ 

+++++++++++ 

M+MMMI MMM++ 

1 

18 

61 

40 

1 

58 

52 

1 

48 

61 

12 

1 

1 

Skin 

Mammary gland 

Integumentary System 

Fibrosarcoma 

+++++++++++ 

MMMMMMMMMMM 

1 

61 

Bone 

Musculoskeletal System 

+++++++++++ 61 

Brain 

Nervous System 

+++++++++++ 61 
Lesions in Male Mice 213 

(continued) 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0 mg/m3 

TABLE C2 

Number of Days on Study 

7236430986642271445689150 

6110368902345556888888902 

02344444555,55555555555566 

Carcass ID Number 

2695636177648045207366159 

4462671234204610027073634 

0000000000000000000000000 

Lung 

Larynx 

Respiratory System 

......................... 

......................... 

Pheochromocytoma complex, metastatic, 

Hepatocholangiocarcinoma, metastatic, liver 

Hepatocellular carcinoma, metastatic, liver 

Fibrous histiocytoma, metastatic, liver 

Alveolar/bronchiolar carcinoma 

Alveolar/bronchiolar adenoma, two 

Alveolar/bronchiolar adenoma 

X 

X 

X 

adrenal medulla 

Trachea 

Nose 

......................... 

......................... 

Adenoma 

Harderian gland 

Special Senses System 

Kidney 

Urinary System ......................... 

Pelvis, hemangiosarcoma 

Fibrous histiocytoma, metastatic, liver X 

X 

Urinary bladder 

Urethra 

Ureter ++ 

+++ ......................... Leiomyosarcoma X 

Multiple organs 

Systemic Lesions 

Lymphoma malignant 
214 Nickel Subsulfide, NTP TR 453 

(continued) 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0 mg/m3 

TABLE C2 

Number of Days on Study 

1129111439777777770000000 

3467888911333333334444444 

6666666611777777177771777 

0908093924112824141925974 

8340126552133366170011123 

0000000000000000000000000 

Carcass ID Number 

Lung 

Larynx 

Respiratory System 

......................... 

+++++++++++++I+++++++++++ 

Pheochromocytoma complex, metastatic, 

Hepatocholangiocarcinoma, metastatic, liver 

Hepatocellular carcinoma, metastatic, liver 

Fibrous histiocytoma, metastatic, liver 

Alveolarlbronchiolar carcinoma 

Alveolar/bronchiolar adenoma, two 

Alveolar/bronchiolar adenoma 

xx 

X X xx 

X 

xxx 

adrenal medulla X 

Adenoma 

Harderian gland 

Special Senses System 

Trachea 

Nose 

......................... 

......................... 

X 

+ 

Kidney 

Urinary System ......................... 

Pelvis, hemangiosarcoma 

Fibrous histiocytoma, metastatic, liver 

Urinary bladder 

Urethra 

Ureter 

+ ++ ........................... 

Multiple organs 

Systemic Lesions 

Leiomyosarcoma 

......................... Lymphoma malignant 
Lesions in Male Mice 215 

(continued) 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0 mg/m3 

TABLE C2 

Number of Days on Study 

00011111111 

44444444444 

71717717117 

Carcass ID Number 

03072367789 

55712355611 

00000000000 

Tumors 

Tissues/ 

Total 

Lung 

Larynx 

Respiratory System 

Pheochromocytoma complex, metastatic, 

Hepatocholangiocarcinoma, metastatic, liver 

Hepatocellular carcinoma, metastatic, liver 

Fibrous histiocytoma, metastatic, liver 

Alveolar/bronchiolar carcinoma 

Alveolar/bronchiolar adenoma, two 

Alveolar/bronchiolar adenoma 

adrenal medulla 

Trachea 

Nose 

+++++++++++ 

+++++++++M+ 

A 

X 

X X 

+++++++++++ 

+++++++++++ 

1 

2 

1 

7 

1 

5 

61 

59 

60 

61 

1 

Adenoma 

Harderian gland 

Special Senses System 

1 

1 

Kidney 

Urinary System 

+++++++++++ 

Pelvis, hemangiosarcoma 

Fibrous histiocytoma, metastatic, liver 

Urinary bladder 

Urethra 

Ureter 

+++++++++++ 

Leiomyosarcoma 

6 

2 

1 

1 

61 

1 

61 

Multiple organs 

Systemic Lesions 

Lymphoma malignant 

+++++++++++ 

X 1 

61 
216 Nickel Subsulfide, NTP TR 453 

0734559954315399930018028 

2364516678122344567888991 

0012344444555555555555556 

4386799230064183965976082 

8239390191081878809626216 

1221212212212111121121221 

Number of Days on Study 

- 

Carcass ID Number 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Gallbladder 

Esophagus 

Alimentary System 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple 

Hepatocellular carcinoma 

Hemangioma 

Tooth 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Pancreas 

AA+A+A+A+++++A+++++++++AA 

AA+A+A+A+++++A+++++++++AA 

AA+A+A+++++++A+++++++++++ 

AAMA+AMM+M++MA+M+M++++M++ 

A++A+++++++++A+M+++++++++ 

AA+M+A+M+++++A+++++++++AM 

+++A+++++++++++++++++++++ 

+++A+++++++++++++++++++++ 

AA+A+A+A+++++A+++++++++AA 

X 

xx 

X X X xx xx 

X X 

X 

+++A+++++++++++++++++++++ 

+++A+++++++++++++++++++++ 

+++A+++++++++++++++++++++ 

+++A+++++++++++++++++++++ 

Heart 

Cardiovascular System 

+++A+++++++++++++++++++++ 

Islets, pancreatic 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

Thyroid gland 

Pituitary gland 

Parathyroid gland 

Adenoma 

Follicular cell, adenoma 

+++A+++++++++++++++++++++ 

+++A+++++++++++++++++++++ 

+++A+++++++++++++++++++++ 

X 

"+A+++++++++++++++++++++ 

........................ 

+++A+++++++++++++++++++++ 

None 

General Body System 

Epididymis 

Genital System 

Testes 

Seminal vesicle 

Prostate 

Preputial gland 

Penis 

Leiomyoma 

+++A+++++++++++++++++++++ 

+ ++ 

+++A+++++++++++++++++++++ 

+++A+++++++++++++++++++++ 

+++A+++++++++++++++++++++ 

+M+A+++++++++++++++++++++ 

++ 
Lesions in Male Mice 217 

(continued) 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0.6 mg/m3 

TABLE C2 

0611462461777777770000000 

2367789013333333334444444 

6666666777777777777777717 

0761448189030445301625191 

8011622982779900347788012 

1222122112111122221111222 

Number of Days on Study 

Carcass ID Number 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Gallbladder 

Esophagus 

Alimentary System 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple 

Hepatocellular carcinoma 

Hemangioma 

Tooth 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Pancreas 

......................... 

+++++A+++++++++++++++++++ 

+++++A+++++++++++++++++++ 

+++++A+++++++++++++++++++ 

+++++A+++++++++++++++++++ 

++++MM+M++++++++++++M++M+ 

......................... 

++++MA+++++++++++++++++++ 

......................... 

X 

xx xx X 

xx X 

......................... 

......................... 

......................... 

......................... 

Heart 

Cardiovascular System 

......................... 

Islets, pancreatic 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

Thyroid gland 

Pituitary gland 

Parathyroid gland 

Adenoma 

Follicular cell, adenoma 

......................... 

......................... 

......................... 

......................... 

......................... 

++++++++++++++++++++++++M 

X 

Epididymis 

Genital System 

Testes 

Seminal vesicle 

Prostate 

Preputial gland 

Penis 

Leiomyoma 

......................... 

+ + ++ 

......................... 

......................... 

......................... 

......................... 
218 Nickel Subsulfide, NTP TR 453 

(continued) 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0.6 mg/m3 

TABLE C2 

Number of Days on Study 

0000111111 

4444444444 

7777117777 

2564387126 

2223661333 

2222112222 

Tumors 

Tissues/ 

Total 

Carcass ID Number 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Gallbladder 

Esophagus 

Alimentary System 

+++.+++++++ 

++++++++++ 

M++++M++M+ 

++++++++++ 

++++++++++ 

++++++++++ 

51 

54 

39 

56 

50 

59 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple 

Hepatocellular carcinoma 

Hemangioma 

Tooth 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Pancreas 

++++++++++ 

++++++++++ 

++++++++++ 

xx 

X X 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

+ 

59 

51 

51 

59 

59 

59 

59 

1 

8 

2 

15 

1 

1 

Heart 

Cardiovascular System 

++++++++++ 59 

Islets, pancreatic 

Adrenal medulla 

Adrenal cortex 

Endocrine System 

Thyroid gland 

Pituitary gland 

Parathyroid gland 

Adenoma 

Follicular cell, adenoma 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

+M++++++++ 

59 

59 

59 

59 

58 

55 

1 

1 

None 

General Body System 

Epididymis 

Genital System 

Testes 

Seminal vesicle 

Prostate 

Preputial gland 

Penis 

Leiomyoma 

++++++++++ 

X 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

59 

59 

59 

58 

9 

1 

59 
Lesions in Male Mice 219 

(continued) 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0.6 mg/m3 

TABLE C2 

Number of Days on Study 

0734559954315399930018028 

2364516678122344567888991 

0012344444555555555555556 

Carcass ID Number 

4386799230064183965976082 

8239390191081878809626216 

1221212212212111121121221 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node 

Bone marrow 

Hematopoietic System 

Spleen 

Lymph node, mediastinal 

Hemangiosarcoma 

Thymus 

Hemangiosarcoma 

M++A+++M+++++++++++++++++ 

+++A++++++++++++++++++++M 

MM+A+++++I +++++I M+I ++++++ 

+ + 

+++A+++++++++++++++++++++ 

+ + ++ + 

MMMAMMMMI ++""+MI MMM+++M 

+++A+++++++++++++++++++++ 

+++A+++++++++++M+I++M++M+ 

Skin 

Mammary gland 

Integumentary System 

Tail, hemangiosarcoma 

Prepuce, fibrous histiocytoma 

MMMAMMMMMMMMMMMMMMMMMMMMM 

+++A+++++++++++++++++++++ 

Bone 

Musculoskeletal System 

+++A+++++++++++++++++++++ 

Spinal cord 

Brain 

Nervous System 

+++A+++++++++++++++++++++ 

+ 

Lung 

Larynx 

Respiratory System 

+++A+++++++++++++++++++++ 

+++A+++++++++++++++++++++ 

Hepatocellular carcinoma, metastatic, liver 

Alveolar/bronchiolar carcinoma 

Alveolar/bronchiolar adenoma 

X X X 

Trachea 

Nose 

+++A+++++++++++++++++++++ 

+++A+++++++++++++++++++++ 

Ear 

Special Senses System 

t 

Urinary bladder 

Urethra 

Kidney 

Urinary System 

+++A+++++++++++++++++++++ 

+ +++ + 

+++A+++++++++++++++++++++ 

Multiple organs 

Systemic Lesions 

Lymphoma malignant 

+++ ..................... 
220 Nickel Subsulfide, NTP TR 453 

Carcass ID Number 

~ 

0761448189030445301625191 

8011622982779900347788012 

1222122112111122221111222 

~~ ~ 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node 

Bone marrow 

Hematopoietic System 

......................... 

++++++++++++++++++++++++t 

++++M+++++M+++++++++++++I 

......................... ++ + + 

Spleen 

Lymph node, mediastinal 

Hemangiosarcoma 

Thymus 

Hemangiosarcoma 

X 

++++++++++I +M+++++++++I ++ 

++++++M++++++++++++++++++ 

MM+++M+MMMI I I I I ++MI I1 +I ++ 

X 

Skin 

Mammary gland 

Integumentary System 

Tail, hemangiosarcoma 

Prepuce, fibrous histiocytoma 

+++++++++++++++++++I+++++ 

MM+MMMMMMMMMMMM+MMMMMMMMM 

X 

X 

Bone 

Musculoskeletal System 

......................... 

Spinal cord 

Brain 

Nervous System 

......................... 

Lung 

Larynx 

Respiratory System 

......................... 

......................... 

Hepatocellular carcinoma, metastatic, liver 

Alveolar/bronchiolar carcinoma 

Alveolar/bronchiolar adenoma X X X 

xx 

Trachea 

Nose 

......................... 

......................... 

~ ____~ ~~~ _____ - 

Ear 

Special Senses System 

Urinary bladder 

Urethra 

Kidney 

Urinary System 

____ ____~~~ 

......................... 

+ 

......................... + + 

Multiple organs 

Systemic Lesions 

Lymphoma malignant 'X ......................... 
Lesions in Male Mice 221 

(continued) 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0.6 mg/m3 

TABLE C2 

Number of Days on Study 

0000111111 

4444444444 

7777777777 

Carcass ID Number 

2564387126 

2223661333 

2222112222 

Tumors 

Tissues1 

Total 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node 

Bone marrow 

Hematopoietic System 

Spleen 

Lymph node, mediastinal 

Hemangiosarcoma 

Thymus 

Hemangiosarcoma 

++++++++++ 

++++++++++ 

MM+++M++++ 

++++++++++ 

++++++++++ 

+MI I M+I M+M 

++++++++M+ 

57 

52 

53 

11 

59 

58 

18 

1 

51 

1 

Skin 

Mammary gland 

Integumentary System 

Tail, hemangiosarcoma 

Prepuce, fibrous histiocytoma 

++++++I +++ 

MMMMMMMMMM 

1 

1 

57 

2 

Bone 

Musculoskeletal System 

Spinal cord 

Brain 

Nervous System 

++++++++++ 59 

++++++++++ 

1 

59 

Lung 

Larynx 

Respiratory System 

++++++++++ 

I M++M+++++ 

Hepatocellular carcinoma, metastatic, liver 

Alveolarlbronchiolar carcinoma 

Alveolar/bronchiolar adenoma 

xx 

X 

Trachea 

Nose 

++++++++++ 

++++++++++ 

59 

59 

6 

2 

3 

59 

56 

Ear 

Special Senses System 

1 

Urinary bladder 

Urethra 

Kidney 

Urinary System 

++++++++++ 

+++++++,+++ 59 

8 

59 

Multiple organs 

Systemic Lesions 

Lymphoma malignant 

++++++++++ 

1 

59 
222 Nickel Subsulfide, NTP TR 453 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 1.2 mg/m3 

TABLE C2 

Number of Days on Study 

8405451409395429577624111 

I453789255668233800244666 

1123333444444555566666666 

Carcass ID Number 

9369328832660465911032355 

9358265642474789535867568 

3333333333333333333333333 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Gallbladder 

Esophagus 

Alimentary System 

Liver 

++A+++A+++++++A++++++++++ 

++A+++A+++++++A++++++++++ 

++A+++A+A+++++A++++++++++ 

++A+++A+A++++++++++++++++ 

++A++MMM+++++++++++++++++ 

++A++++++++++++++++++++++ 

++A+++A+A+++++A++++++++++ 

......................... 

Hepatocellular carcinoma, metastatic, liver 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma 

Hepatoblastoma 

Hemangiosarcoma X 

......................... 

X xxxxx x 

X x xx 

++A++++++++++++++++++++++ 

++A++++++++++++++++++++++ 

......................... 

++A++++++++++++++++++++++ 

X 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Pancreas 

Odontoma 

Tooth 

Adenocarcinoma 

X 

+ 

Heart 

Cardiovascular System ......................... 

Adrenal cortex 

Endocrine System 

Pituitary gland 

Parathyroid gland 

Islets, pancreatic 

Adrenal medulla 

Capsule, adenoma 

Thyroid gland 

Follicular cell, adenoma 

Pars distalis, adenoma 

++++M++++++++++++++++++++ 

+++++++M+++++++++++++++++ 

MM+++M+M+++M++++++++++++M 

++A++++++++++++++++++++++ 

++M+M++++++++++++++++++++ 

X 

......................... 

Tissue NOS 

General Body System 

Thoracic, alveolar/bronchiolar 

Mediastinum, alveolar/bronchiolar 

carcinoma, metastatic 

+ 

X 

carcinoma, metastatic X 
Lesions in Male Mice 223 

(continued) 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 1.2 mg/m3 

TABLE C2 

~ ~~ ~~ ~ 

Number of Days on Study 

Carcass ID Number 

I198592466789440834716057 

3779434936222345'788899024 

3333333333333333333333433 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Gallbladder 

Esophagus 

Alimentary System 

+++A+A+++++++++++++++++++ 

+++A+A+++M+++++++++++++++ 

+++A+A+++++++++++++++++++ 

+++A+A+++++++++++++++++++ 

M+MA+M++++++++++++++M+M++ 

+++A+A+++++++++++++++++++ 

+++A+A+++++++++++++++++++ 

......................... 

Hepatocellular carcinoma, metastatic, liver 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma 

Hepatoblastoma 

Hemangiosarcoma 

Liver 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Pancreas 

Odontoma 

Tooth 

Adenocarcinoma 

X 

+++A+++++++++++++++++++++ 

xx xx X 

X 

xxx X X 

+++A+A+++++++++++++++++++ 

+++A+A+++'++++++++++++++++ 

+++A+++++++++++++++++++++ 

+++A+++++++++++++++++++++ 

X X 

X 

+ 

Heart 

Cardiovascular System ......................... 

Adrenal cortex 

Endocrine System 

Pituitary gland 

Parathyroid gland 

Islets, pancreatic 

Adrenal medulla 

Capsule, adenoma 

Thyroid gland 

Follicular cell, adenoma 

Pars distalis, adenoma 

+++A+++++++++++++++++++++ 

+++A+I+++++++++++++++MM++ 

++MA+++++++++++++M+++++++ 

+++A+++++++++++++++++++++ 

M++A+++++++++++++++++++++ 

+++A+++++++++++++++++++++ 

X 

Tissue NOS 

General Body System 

Thoracic, alveolar/bronchiolar 

Mediastinum, alveolar/bronchiolar 

carcinoma, metastatic 

carcinoma, metastatic 
224 Nickel Subsulfide, NTP TR 453 

(continued) 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 1.2 mg/m3 

TABLE C2 

Number of Days on Study 

0000000111 

4444444444 

7777777777 

Carcass ID Number 

2101237408 

5678899234 

3333333333 

Tumors 

Tissues/ 

Total 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Gallbladder 

Esophagus 

Alimentary System 

Liver 

Hepatocellular carcinoma, metastatic, 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma 

Hepatoblastoma 

Hemangiosarcoma 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Pancreas 

Odontoma 

Tooth 

Adenocarcinoma 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

liver 

++++++++++ 

X X 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

X 

++ 

55 

54 

54 

55 

50 

57 

54 

60 

2 

4 

1 

57 

57 

59 

58 

3 

11 

12 

1 

1 

59 

1 

Heart 

Cardiovascular System 

++++++++++ 60 

Adrenal cortex 

Endocrine System 

Pituitary gland 

Parathyroid gland 

Islets, pancreatic 

Adrenal medulla 

Capsule, adenoma 

Thyroid gland 

Follicular cell, adenoma 

Pars distalis, adenoma 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

X 

++++++++++ 

1 

59 

1 

55 

51 

58 

56 

1 

58 

Tissue NOS 

General Body System 

1 

Thoracic, alveolar/bronchiolar 

Mediastinum, alveolar/bronchiolar 

carcinoma, metastatic 

carcinoma, metastatic 

1 

1 
Lesions in Male Mice 225 

(continued) 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 1.2 mg/m3 

TABLE C2 

Number of Days on Study 

8405451409395429577624111 

1453189255668233800244666 

1123333444444555566666666 

Carcass ID Number 

9369328832660465911032355 

9358265642474789535867568 

3333333333333333333333333 

Coagulating gland 

Genital System 

Testes 

Seminal vesicle 

Prostate 

Preputial gland 

Penis 

Epididymis 

Interstitium, granular cell tumor NOS 

......................... + ++++ + 

......................... 

......................... 

......................... 

......................... 

Lymph node, bronchial 

Lymph node 

Bone marrow 

Blood 

Hematopoietic System 

Lymph node, mesenteric 

Lymph node, mandibular 

Hepatocellular carcinoma, metastatic 

A 

......................... + +++ ++ ++ + 

M+++I ++++I +++++++++I +++++ 

++A+++++++++++++++++M++++ 

++++++M++++++++++++M+++++ 

Hemangioma 

Fibrous histiocytoma 

Adenocarcinoma, metastatic, stomach, glandular 

Lymph node, mediastinal 

Integumentary System 

Thymus 

Spleen 

Skin 

Mammary gland 

++A++++++++++++++++M+++++ 

MI MMMAAMMI MM++++M+MI MMMMI 

Hemangioma 

+M+M++++IM+++M+M+M++M++++ 

......................... 

MMMMMMMMMMMMMMMMMMMMMMMMM 

Pinna, hemangiosarcoma 

Fibrous histiocytoma 

Bone 

Musculoskeletal System 

......................... 

Spinal cord 

Brain 

Nervous System 

+ 

......................... 

Lung 

Larynx 

Respiratory System 

~~ 

Hepatocellular carcinoma, metastatic, liver 

Hepatoblastoma, metastatic, liver 

Alveolar/bronchiolar carcinoma 

Alveolar/bronchiolar adenoma 

Trachea 

Nose 

++A++++++++++++.++++++++++ 

......................... 

X 

+++++++++++++++M+++++++++ 

......................... 
Nickel Subsulfide, NTP TR 453 

TABLE C2 

(continued) 

Individual Animal Tumor Pathology of Male Mice in the %Year Inhalation Study of Nickel Subsulfide: 1.2 mg/m3 

5566551364177777177111700 

6667901113333333333333344 

6666677777777777777777777 

7198592466189440834716051 

3779434936222345788899024 

3333333333333333333333433 

Number of Days on Study 

Carcass ID Number 

Genital System 

Coagulating gland 

Prostate 

Preputial gland 

Penis 

Epididymis 

Interstitium, granular cell tumor NOS X 

+ 

+++A+++++++++++++++++++++ 

......................... 

+ 

+++A+++++++++++++++++++++ 

+ 

Testes 

Seminal vesicle 

Lymph node, bronchial 

Lymph node 

Bone marrow 

Blood 

Hematopoietic System 

Lymph node, mandibular 

Fibrous histiocytoma 

Adenocarcinoma, metastatic, stomach, glandular 

Lymph node, mesenteric 

+++A+++++++++++++++++++++ 

+++A+++++++++++++++++++++ 

+++A+++++++++++++++++++++ 

+++A+++++++++++++++++++++ 

+ + 

Hepatocellular carcinoma, metastatic X 

++MA+++++++++++++++++++++ 

+++A++M++++++++++++++++++ 

X 

X 

Hemangioma 

Thymus 

Spleen 

Lymph node, mediastinal 

Hemangioma 

X 

+++A+++++++++++++++++++++ 

++MA+++M++I +MI I MI ++I I I MMI 

MM+A++M+++MI +"+I ++M+++M+ 

Skin 

Mammary gland 

Integumentary System 

MMMMMMMMMMMMMMMMMMMMMMMMM 

+++A+++++++++++++++++++++ 

Pinna, hemangiosarcoma 

Fibrous histiocytoma X 

X 

Bone 

Musculoskeletal System 

+++A+++++++++++++++++++++ 

Spinal cord 

Brain 

Nervous System 

+++A+++++++++++++++++++++ 

Lung 

Larynx 

Respiratory System 

+++A+++++++++++++++++++++ 

+++A+++++++++++++++++++++ 

Hepatocellular carcinoma, metastatic, liver 

Hepatoblastoma, metastatic, liver 

Alveolar/bronchiolar carcinoma 

Alveolarlbronchiolar adenoma X X 

X 

X X 

X 

Trachea 

Nose 

+++M+++++++++++++++++++++ 

+++A+++++++++++++++++++++ 
Lesions in Male Mice 227 

(continued) 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 1.2 mg/m3 

TABLE C2 

Number of Days on Study 

0000000111 

4444444444 

1111111111 

Carcass ID Number 

2101231408 

5618899234 

3333333333 

Tumors 

Tissues/ 

Total 

Coagulating gland 

Genital System 

Testes 

Seminal vesicle 

Prostate 

Preputial gland 

Penis 

Epididymis 

Interstitium, granular cell tumor NOS 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

59 

59 

59 

60 

8 

59 

1 

1 

Lymph node, bronchial 

Lymph node 

Bone marrow 

Blood 

Hematopoietic System 

Lymph node, mesenteric 

Lymph node, mandibular 

++++++++++ 

54 

11 

59 

55 

56 

1 

+++++++M++ 

Hepatocellular carcinoma, metastatic 

+M++++++++ 

++++++++++ 

Hemangioma 

Fibrous histiocytoma 

Adenocarcinoma, metastatic, stomach, glandular 

Thymus 

Spleen 

Lymph node, mediastinal 

++++++++++ 

MI I ++I I MI M 

Hemangioma X 

+*++++++++ 

1 

1 

1 

41 

1 

57 

17 

Skin 

Mammary gland 

Integumentary System 

++++++++++ 

MMMMMMMMMM 

1 

1 

59 

Pinna, hemangiosarcoma 

Fibrous histiocytoma 

Bone 

Musculoskeletal System 

++++++++++ 59 

Spinal cord 

Brain 

Nervous System 

++++++++++ 

1 

59 

Lung 

Larynx 

Respiratory System 

++++++++++ 

++++++++++ 

Hepatocellular carcinoma, metastatic, liver 

Hepatoblastoma, metastatic, liver 

Alveolarlbronchiolar carcinoma 

Alveolar/bronchiolar adenoma 

X X 

Trachea 

Nose 

++++++++++ 

++++++++++ 

58 

59 

2 

1 

4 

2 

58 

59 

- 
228 Nickel Subsulfide, NTP TR 453 

(continued) 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 1.2 mg/m3 

TABLE C2 

8405451409395429577624111 

1453789255668233800244666 

1123333444444555566666666 

Number of Days on Study 

Carcass ID Number 

9369328832660465911032355 

9358265642474789535867568 

3333333333333333333333333 

None 

Special Senses System 

Kidney 

Urinary System 

......................... 

Hepatocellular carcinoma, metastatic, liver 

Hepatoblastoma, metastatic, liver 

Urinary bladder 

Urethra ++ + ++ 

++A++++++++++++++++++++++ 

+ 

Multiple organs 

Systemic Lesions 

......................... 
Lesions in Male Mice 229 

(continued) 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 1.2 mg/m3 

TABLE C2 

Number of Days on Study 

55665513647177177117171~0 

6667901113333333333333344 

6666677771177777777777777 

Carcass ID Number 

7198592466789440834716057 

3119434936222345188899024 

3333333333333333333333433 

None 

Special Senses System 

Kidney 

Urinary System 

+++A+++++++++++++++++++++ 

Hepatocellular carcinoma, metastatic, liver 

Hepatoblastoma, metastatic, liver 

X 

X 

Urinary bladder 

Urethra 

+++A+++++++++++++++++++++ 

++ +++ 

Multiple organs 

Systemic Lesions ~~ ~~ ~ 

......................... 
230 Nickel Subsulfide, NTP TR 453 

(continued) 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 1.2 mg/m3 

TABLE C2 

0000000111 

4444444444 

7771711111 

Number of Days on Study 

Carcass ID Number 

2101237408 

5678899234 

3333333333 

Tumors 

Tissues/ 

Total 

None 

Special Senses System 

Kidney 

Urinary System 

Hepatoblastoma, metastatic, liver 

++++++++++ 

Hepatocellular carcinoma, metastatic, liver 

Urinary bladder 

Urethra 

++++++++++ 58 

11 

1 

1 

59 

Multiple organs 

Systemic Lesions 

++++++++++ 60 
Lesions in Male Mice 231 

Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Inhalation Study of Nickel Subsulfide 

TABLE C3 

O mg/m3 0.6 mg/m3 1.2 mg/m3 

Fisher exact test 

Cochran-Armita e testd 

Logistic regression testd 

Life table testd 

First incidence (days) 

Terminal rate' 

Adjusted rateb 

Overall ratea 

Liver: Hepatocellular Adenoma 

% P=O.419 

P=O.410 

P=O.450 

508 

7/26 (27 %) 

37.4% 

13/61 (21%) 

P=0.278N 

P=0.293N 

525 

3/25 (12%) 

25.6% 

9/59 (15 %) 

P=O.454 

P=O.492 

485 

8/25 (32%) 

41.7% 

14/59 (24%) 

P=0.268N P=O.461 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Liver: Hepatocellular Carcinoma 

P=O.418 

P=O.242 

P=O.442 

434 

3/26 (12%) 

27.0% 

11/61 (18%) 

P=O.123 

P=O.137 

415 

5/25 (20%) 

40.4% 

17/59 (29%) 

P=O.272 

P=O.492 

450 

1/25 (4%) 

28.2% 

12/59 (20%) 

P=O.119 P=O.464 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Liver: Hepatocellular Adenoma or Carcinoma 

P=O.405 

P=O.222 

P=O.449 

434 

10126 (38%) 

56.6% 

24/61 (39%) 

P=0.489N 

P=0.507N 

415 

6/25 (24%) 

49.6% 

22/59 (37%) 

P=O.257 

P=O.487 

450 

9/25 (36%) 

58.0% 

25/59 (42%) 

P=0.483N P=O.440 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Lung: Alveolarlbronchiolar Adenoma 

P=0.131N 

P=0.144N 

734 

1/25 (4%) 

7.7% 

2/58 (3%) 

P=0.103N 

P=0.088N 

P=0.097N 

64 1 

3/26 (12%) 

19.8% 

6/61 (10%) 

P=0.261N 

P=0.269N 

66 1 

2/25 (8%) 

10.8% 

3/59 (5%) 

P=0.262N P=0.153N 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Lung: Alveolar/bronchiolar Carcinoma 

P=0.211N 

P=0.184N 

P=0.200N 

552 

5/26 (19%) 

23.7% 

7/61 (11%) 

P=0.088N 

P=0.092N 

737 (T) 

2/25 (8%) 

8.0% 

2/59 (3 %) 

P=0.261N 

P=0.279N 

66 1 

3/25 (12%) 

14.3% 

4/58 (7%) 

P=0.090N P=0.294N 
232 Nickel Subsulfide, NTP TR 453 

Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Inhalation Study of Nickel Subsulfide (continued) 

TABLE C3 

0 mg/m3 0.6 mg/m3 1.2 mg/m3 

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 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Thyroid Gland (Follicular Cell): Adenoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Hemangiosarcoma 

P=0.051N 

P=0.035N 

P=0.045N 

552 

8/26 (31 %) 

40.7% 

13/61 (21 %) 5/59 (8%) 

P=0.058N 

P=0.076N 

66 1 

4/25 (1 6 %) 

21.4% 

6/58 (10%) 

P=0.083N 

P=0.038N 

P=0.046N 

66 1 

4/25 (16%) 

18.5% 

P=O.O42N 

P=0.109N 

P=O.llON 

P=0.106N 

313 

2/26 (8 96) 

12.4% 

4/61 (7%) 

P=0.192N 

P=0.195N 

704 

0125 (0%) 

3.6% 

1/59 (2%) 

P=0.193N 

P=0.192N 

P=0.188N 

737 (T) 

1/25 (4%) 

4.0% 

1/59 (2%) 

P=0.193N 

P=O.393 

P=O.392 

P=O.390 

480 

0/26 (0%) 

1.8% 

1/61 (2%) 

P=O.292 

P=O.291 

67 1 

2/25 (8%) 

10.9% 

3/60 (5 %) 

P=O.494 

P=O.493 

66 1 

1/25 (4%) 

6.5% 

2/60 (3 %) 

P=O.303 P=O.494 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Hemangioma or Hemangiosarcoma 

P=O.268 

P=O.271 

P=O.268 

480 

1/26 (4%) 

5.6% 

2/61 (3%) 

P=O.317 

P=O.323 

67 1 

2/25 (8%) 

14.2% 

4/60 (7%) 

P=O.328 

P=O.329 

661 

2/25 (8%) 

13.6% 

4/60 (7%) 

P=O.332 P=O.332 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Benign Neoplasms 

P=0.060N 

P=0.060N 

P=0.076N 

313 

16/26 (62%) 

72.2% 

28/61 (46%) 

P=0.025N 

P=0.045N 

475 

6/25 (24%) 

42.8% 

16/60 (27 %) 

P=0.077N 

P=0.092N 

144 

10125 (40%) 

53.1% 

19/60 (32%) 

P=0.022N P=0.078N 
Lesions in Male Mice 233 

TABLE C3 

Statistical Analysis. of Primary Neoplasms in Male Mice in the 2-Year Inhalation Study of Nickel Subsulfide (continued) 

O mg/m3 0.6 mg/m3 1.2 mg/m3 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Malignant Neoplasms 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Benign or Malignant Neoplasms 

P=O.511 

P=O.348 

P=O.510 

434 

8/26 (3 1 %) 

49.1 % 

21/61 (34%) 

P=0.182N 

P=0.193N 

P=0.255N 

313 

19/26 (73%) 

84.3% 

41/61 (67%) 

P=O.384 

P=O.369 

415 

9/25 (36%) 

55.2% 

23/60 (38%) 

P=O.398 

P=O.lOON 

P=0.201N 

415 

11/25 (44%) 

68.0% 

32/60 (53 %) 

P=0.085N 

P=O.375 

P=O.551 

450 

7/25 (28%) 

50.6% 

21/60 (35%) 

P=O.549 

P=0.221N 

P=0.272N 

144 

15/25 (60%) 

76.6% 

35/60 (58 %) 

P=0.206N 

a Number of neoplasm-bearing animaldnumber of animals examined. Denominator is number of animals examined microscopically for liver, lung, and 

(T)Terminal sacrifice 

Observed incidence at terminal kill 

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

thyroid gland; for other tissues, denominator is number of animals necropsied. 

compare directly the overall incidence rates. For all tests, a negative trend or a lower incidence in an exposure group is indicated by N. 

(directly or indirectly) the cause of death. The logistic regression test regards these lesions as nonfatal. The Cochran-Armitage and Fisher exact tests 

pairwise comparisons between the controls and that exposed group. The life table test regards neoplasms in animals dying prior to terminal kill as being 

* Beneath he control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding to 
234 Nickel Subsulfide, NTP TR 453 

Historical Incidence of Alveolar/bronchiolar Neoplasms in Untreated Male B6C3F, Micea 

TABLE C4 

Study Carcinoma 

Incidence in Controls 

Adenoma Adenoma or Carcinoma 

Historical Incidence at Lovelace Inhalation Toxicology Research Institute 

Talc 

Nickel Sulfate Hexahydrate 

Nickel Subsulfide 

Nickel Oxide 

6/45 

5/61 

6/61 

7/57 

Overall Historical Incidence in Inhalation Studies 

Range 

Standard deviation 

Total 

6%-36% 

7.0% 

141/952’(14.8%) 

Overall Historical Incidence in Feed Studies 

Range 

Standard deviation 

Total 

4%-28% 

6.4% 

194/1,319 (14.7%) 

7/45 

9/61 

7/61 

4/57 

0%-16% 

5.7% 

75/952 (7.9%) 

12/45 

13/61 

13/61 

9/57 

10%-42% 

8.0% 

205/952 (21.5%) 

0%-14% 

3.9% 

6411,319 (4.9%) 

4%-32% 

7.6% 

249/1,319 (18.9%) 

a Data as of 17 June 1994 
Lesions in Male Mice 235 

of Nickel Subsulfidea 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study 

TABLE C5 

0 mg/m3 0.6 mg/m3 1.2 mg/m3 

Early deaths 

IS-Month interim evaluationb 

7-Month interim evaluationb 

Animals initially in study 

Disposition Summary 

Terminal sacrifice 

Died last week of study 

Natural deaths 

Moribund 

Survivors 

Animals examined microscopically 

10 

9 

80 

8 

27 

71 

26 

10 

10 

80 

9 

26 

70 

25 

10 

10 

80 

9 

25 

70 

25 

1 

7-Month Interim Evaluation 

Lymph node 

Hematopoietic System 

Hyperplasia 

Lymph node, mediastinal 

Hyperplasia, macrophage 

Hyperplasia, lymphoid 

Lumbar, pigmentation 

Lumbar, hyperplasia, lymphoid 

Lymph node, bronchial 

(1) 

1 (100%) 

1 (100%) 

(3) 

(1) 

1 (100%) 

1 (100%) 

(4) 

1 (50%) 

2 (50%) 

2 (50%) 

(2) 

(5) 

5 (100%) 

3 (60%) 

(3) 

Lung 

Respiratory System 

Interstitium, infiltration cellular 

Alveolus, proteinosis 

Alveolus, hyperplasia, macrophage 

Inflammation, chronic active 

Olfactory epithelium, respiratory epithelium, 

Olfactory epithelium, atrophy 

Nose 

degeneration 

(5) 

1 (20%) 

(5) 

5 (100%) 

5 (100%) 

5 (100%) 

3 (60%) 

1 (20%) 

(5) 

(5) 

3 (60%) 

5 (100%) 

5 (100%) 

5 (100%) 

4 (80%) 

(5) 

Urinary bladder 

Urinary System 

Calculus, microscopic observation only 

a Number of animals examined microscopically at the site and the number of animals with lesion 

Five animals in each exposure group were histopathologically examined at the interim evaluations. 
Nickel Subsulfide, NTP TR 453 

of Nickel Subsulfide (continued) 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study 

TABLE C5 

0 mg/m3 0.6 mg/mJ 1.2 mg/m3 

Special Senses System 

Nervous System 

Musculoskeletal System 

Integumentary System 

Genital System 

General Body System 

Endocrine System 

Cardiovascular System 

Alimentary System 

Systems Examihed With No Lesions Observed 

7-Month Interim Evaluation (continued) 

Intestine large, cecum 

Alimentary System 

15-Month Interim Evaluation 

Liver 

Basophilic focus 

Hyperplasia, focal, lymphoid 

Hyperplasia, focal 

Stomach, forestomach 

Mixed cell focus 

Hyperplasia, focal 

Stomach, glandular 

Endocrine System 

Thyroid gland 

Hypertrophy 

Adrenal cortex 

Follicular cell, hyperplasia 

(5) 

1 (20%) 

Preputial gland 

Genital System 

Inflammation, chronic 

Ectasia 

(5) 

1 (20%) 

4 (80%) 

(5) 

1 (20%) 

5 (100%) 

(5) 

1 (20%) 

3 (60%) 

Hyperplasia, lymphoid 

Lymph node, inguinal 

Hematopoietic System 

Hyperplasia, macrophage 

Hyperplasia, lymphoid 

Lymph node, bronchial 

Hyperplasia, lymphoid 

Lymph node, mandibular 

(3) 

(1) 

3 (100%) 

3 (100%) 

1 (100%) 

(3) 

(5) 

1 (25%) 

4 (80%) 

5 (100%) 

(4) 
Lesions in Male Mice 237 

of Nickel Subsulfide (continued) 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study 

TABLE C5 

0 mg/m3 0.6 mg/m3 1.2 mg/m3 

Lymph node, mesenteric 

Hematopoietic System (continued) 

15-Month Interim Evaluation (continued) 

Hematopoietic cell proliferation 

Hyperplasia, lymphoid 

Hemorrhage, chronic 

(5) 

1 (20%) 

1 (20%) 

Spleen (5) (5) 

1 (20%) 

Skin 

Integumentary System 

Subcutaneous tissue, abscess 

Ulcer 

(5) 

1 (20%) 

(5) 

1 (20%) 

Bone 

Musculoskeletal System 

Femur, hyperostosis 

(5) 

1 (20%) 

Lung 

Respiratory System 

Interstitium, infiltration cellular 

Bronchus, hyperplasia, lymphoid 

Alveolus, proteinosis 

Alveolus, hyperplasia, macrophage 

Bronchialization 

Inflammation, chronic active 

Inflammation, focal 

Olfactory epithelium, atrophy 

Trachea 

Nose 

2 (40%) 

(5) 

3 (60%) 

(5) 

4 (80%) 

1 (20%) 

5 (100%) 

5 (100%) 

4 (80%) 

5 (100%) 

3 (60%) 

(5 ) 

(5) 

(5) 

5 (100%) 

2 (40%) 

5 (100%) 

5 (100%) 

5 (100%) 

5 (100%) 

4 (80%) 

(5) 

(5 1 

Urinary System 

Nephropathy 

Hydronephrosis 

Kidney 

Calculus, microscopic observation only 

Calculus, microscopic observation only 

(5) 

Urinary bladder 

Urethra 

(5) 

(2) 

1 (20%) 

2 (100%) 

(5) 

1 (20%) 

Special Senses System 

Nervous System 

General Body System 

Cardiovascular System 

Systems Examined With No Lesions Observed 
Nickel Subsulfide, NTP TR 453 

TABLE C5 

of Nickel Subsulfide (continued) 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study 

0 mg/m3 0.6 mglm3 1.2 mg/m3 

Gallbladder 

Alimentary System 

2- Year Study 

Intestine large, cecum 

Lymphoid tissue, hyperplasia 

Epithelium, hyperplasia 

Angiectasis 

Peyer’s patch, hyperplasia, lymphoid 

Lymphoid tissue, hyperplasia, lymphoid 

Sinusoid, cyst 

Bile duct, hyperplasia 

Regeneration 

Pigmentation, hemosiderin 

Necrosis 

Mixed cell focus 

Inflammation 

Infiltration cellular, lymphocyte 

Infarct 

Hepatodiaphragmatic nodule 

Eosinophilic focus 

Degeneration 

Basophilic focus 

Atrophy 

Acinw, hyperplasia 

Acinus, atrophy 

Infiltration cellular, lymphocyte 

Stomach, glandular 

Inflammation 

Stomach, forestomach 

Inflammation 

Developmental malformation 

Inflammation 

Degeneration 

Liver 

Intestine small, ileum 

Pancreas 

Tooth 

(50) 

1 (2%) 

1 (2%) 

(54) 

(51) 

(59) 

2 (3%) 

2 (3%) 

2 (3%) 

1 (2%) 

1 (2%) 

1 (2%) 

3 (5%) 

1 (2%) 

1 (2%) 

1 (2%) 

9 (15%) 

(59) 

1 (2%) 

1 (2%) 

(59) 

(59) 

1 (2%) 

2 (3%) 

(1) 

1 (100%) 

(55) 

(54) 

1 (2%) 

3 (5%) 

3 (5%) 

1 (2%) 

2 (3%) 

(59) 

(55) 

2 (3%) 

1 (2%) 

6 (10%) 

1 (2%) 

1 (2%) 

(58) 

(57) 

(57) 

1 (2%) 

(4) 

1 (25%) 

2 (50%) 

Heart 

Cardiovascular System 

Cardiomyopathy 

Angiectasis 

(61) 

1 (2%) 

(59) 

2 (3%) 

(60) 

1 (2%) 
Lesions in Male Mice 239 

of Nickel Subsulfide (continued) 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study 

TABLE C5 

O mg/m3 0.6 mg/m3 1.2 mg/m3 

Adrenai cortex 

Endocrine System 

&Year Study (continued) 

Necrosis 

Hypertrophy, focal 

Hyperplasia, focal 

Hyperplasia 

Degeneration 

Cyst 

Pituitary gland 

Hyperplasia 

Islets, pancreatic 

Follicular cell, hyperplasia 

Thyroid gland 

Pars distalis, hyperplasia 

(61) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

2 (3%) 

1 (2%) 

1 (2%) 

1 (2%) 

(59) 

(58) 

(59) 

25 (42%) 

4 (7%) 

(59) 

(55) 

1 (2%) 

24 (41%) 

None 

General Body System 

Penis 

Genital System 

Preputial gland 

Inflammation 

Concretion 

Inflammation, chronic 

Hyperplasia 

Ectasia 

Atrophy 

Prostate 

Hyperplasia 

Seminal vesicle 

Inflammation 

Germinal epithelium, atrophy 

Atrophy 

Testes 

(12) 

5 (42%) 

4 (33%) 

24 (39%) 

2 (3%) 

28 (44%) 

3 (5%) 

(61 ) 

(61) 

14 (24%) 

(59) 

(59) 

(59) 

1 (2%) 

2 (3%) 

(8) 

6 (75%) 

1 (13%) 

(60) 

1 (2%) 

42 (70%) 

2 (3%) 

7 (12%) 

(59) 

(59) 

(59) 

2 (3%) 

Hematopoietic System 

Myeloid cell, hyperplasia 

Erythroid cell, hyperplasia 

Thrombosis 

Bone marrow (61) 

23 (38%) 

2 (3%) 

(59) 

3 (5%) 

11 (19%) 

(59) 

1 (2%) 

1 (2%) 

15 (25%) 
Nickel Subsulfide, NTF' TR 453 

of Nickel Subsulfide (continued) 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study 

TABLE C5 

0 mg/m3 0.6 mg/m3 1.2 mg/m3 

Lymph node 

Hematopoietic System (continued) 

2-Yk'Ur shdy (continued) 

Renal, hyperplasia, plasma cell 

Renal, hyperplasia, lymphoid 

Pancreatic, hyperplasia, lymphoid 

Lumbar, pigmentation 

Lumbar, hyperplasia, plasma cell 

Inguinal, pigmentation 

Inguinal, inflammation 

Inguinal, hyperplasia, plasma cell 

Inguinal, hyperplasia, lymphoid 

Iliac, pigmentation 

Iliac, inflammation 

Iliac, hyperplasia, plasma cell 

Iliac, hyperplasia, lymphoid 

Hyperplasia, plasma cell 

Hyperplasia, macrophage 

Hyperplasia, lymphoid 

Hyperplasia, plasma cell 

Hyperplasia, lymphoid 

Lymph node, mandibular 

Inflammation 

Hyperplasia, macrophage 

Hyperplasia, lymphoid 

Lymph node, mesenteric 

Hyperplasia, plasma cell 

Hyperplasia, macrophage 

Hyperplasia, lymphoid 

Lymph node, mediastinal 

Inflammation 

Hyperplasia, lymphoid 

Hematopoietic cell proliferation 

Atrophy 

Degeneration 

Atrophy 

Lymph node, bronchial 

Spleen 

Thymus 

(18) 

2 (11%) 

6 (33%) 

2 (11%) 

1 (6%) 

7 (39%) 

3 (17%) 

1 (6%) 

1 (6%) 

1 (6%) 

1 (6%) 

1 (6%) 

1 (3%) 

1 (3%) 

4 (10%) 

1 (2%) 

3 (6%) 

3 (5%) 

1 (2%) 

2 (3%) 

(40) 

(52) 

(58) 

(12) 

1 (8%) 

2 (17%) 

(61) 

16 (26%) 

8 (13%) 

1 (2%) 

12 (25%) 

5 (45%) 

1 (9%) 

1 (9%) 

1 (9%) 

1 (9%) 

3 (27%) 

(11) 

3 (27%) 

3 (27%) 

6 (55%) 

1 (9%) 

1 (9%) 

1 (9%) 

1 (9%) 

(53) 

47 (89%) 

40 (75%) 

(57) 

5 (9%) 

1 (2%) 

1 (6%) 

1 (6%) 

1 (6%) 

8 (14%) 

11 (19%) 

1 (2%) 

(18) 

(58) 

(54) 

50 (93%) 

49 (91%) 

(56) 

1 (2%) 

(55) 

6 (11%) 

(17) 

1 (6%) 

1 (6%) 

(57) 

1 (2%) 

8 (14%) 

5 (9%) 

1 (2%) 

(41) 

16 (39%) 
Lesions in Male Mice 241 

of Nickel Subsulfide (continued) 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study 

TABLE C5 

0 mg/m3 0.6 mg/m3 1.2 mg/m3 

Skin 

Integumentary Sjrstem 

&Year Study (continued) 

Tail, necrosis 

Tail, inflammation 

Tail, hyperkeratosis 

Prepuce, inflammation 

Prepuce, hyperkeratosis 

Pinna, hyperkeratosis 

Parakeratosis 

Necrosis 

Inflammation 

Fibrosis 

Alopecia 

(61) 

1 (2%) 

2 (3%) 

1 (2%) 

1 (2%) 

1 (2%) 

16 (26%) 

1 (2%) 

(57) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

9 (16%) 

(59) 

2 (3%) 

1 (2%) 

1 (2%) 

13 (22%) 

1 (2%) 

Bone 

Musculoskeletal System 

Hyperostosis 

Developmental malformation 

Brain 

Nervous System 

(61) 

Medulla, demyelination 

Medulla, degeneration, focal 

Cerebellum, white matter, degeneration, focal 

Degeneration, secondary Wallerian 

1 (2%) 

1 (2%) 

Spinal cord 

(59) 

1 (2%) 

Lung 

Respiratory System 

Interstitium, infiltration cellular 

Bronchus, hyperplasia, lymphoid 

Alveolus, proteinosis 

Alveolus, hyperplasia, macrophage 

Alveolus, hyperplasia 

Alveolar epithelium, metaplasia, squamous 

Bronchialization 

Inflammation, chronic active 

Inflammation, acute 

Infiltration cellular 

Fibrosis 

Autolysis 

Olfactory epithelium, degeneration 

Olfactory epithelium, atrophy 

Inflammation, acute 

Foreign body 

Nose 

3 (5%) 

1 (2%) 

6 (10%) 

10 (16%) 

22 (36%) 

(61) 

3 (5%) 

1 (2%) 

53 (90%) 

52 (88%) 

57 (97%) 

55 (93%) 

29 (49%) 

51 (97%) 

(59) 

8 (14%) 

27 (46%) 

(58) 

16 (28%) 

1 (2%) 

54 (93%) 

53 (91%) 

2 (3%) 

55 (95%) 

14 (24%) 

57 (98%) 

58 (100%) 

1 (2%) 

1 (2%) 

6 (10%) 

55 (93%) 

3 (5%) 

1 (2%) 

(59) 
242 Nickel Subsulfide, NTP TR 453 

of Nickel Subsulfide (continued) 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study 

TABLE C5 

O mg/m3 0.6 mg/m3 1.2 mg/mg 

None 

Special Senses System 

2-y~~ Study (continued) 

Urinary System 

Renal tubule, necrosis 

Renal tubule, degeneration 

Renal tubule, atrophy 

Pelvis, inflammation 

Cortex, fibrosis 

Cortex, atrophy 

Nephropathy 

Inflammation 

Infiltration cellular, lymphocyte 

Hydronephrosis 

Kidney 

Inflammation 

Inflammation 

Concretion 

Calculus, microscopic observation only 

Inflammation 

Hyperplasia 

Crystals 

Calculus, microscopic observation only 

Calculus, gross observation 

Urinary bladder 

Urethra 

Ureter 

(61) 

9 (15%) 

5 (8%) 

7 (11%) 

8 (13%) 

5 (8%) 

(59) 

1 (2%) 

3 (5%) 

5 (8%) 

1 (2%) 

2 (3%) 

10 (17%) 

(59) 

6 (10%) 

2 (3%) 

2 (3%) 

5 (8%) 

1 (2%) 

2 (3%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (17%) 

5 (83%) 

1 (2%) 

1 (2%) 

1 (2%) 

(61) 

11 (18%) 

7 (88%) 

2 (3%) 

1 (2%) 

4 (7%) 
243 

OF NICKEL SUBSULFIDE 

SUMMARY OF LESIONS IN FEMALE MICE 

APPENDIX D 

IN THE 2-YEAR INHALATION STUDY 

TABLE D5 Summary of the Incidence of Nonneoplastic Lesions in Female Mice 

TABLE D4 Historical Incidence of Alveolarlbronchiolar Neoplasms 

TABLE D3 Statistical Analysis of Primary Neoplasms in Female Mice 

TABLE D2 Individual Animal Tumor Pathology of Female Mice 

TABLE Dl Summary of the Incidence of Neoplasms in Female Mice 

in the 2-Year Inhalation Study of Nickel Subsulfide 

in Untreated Female B6C3Fl Mice 

in the 2-Year Inhalation Study of Nickel Subsulfide 

in the 2-Year Inhalation Study of Nickel Subsulfide 

in the 2-Year Inhalation Study of Nickel Subsulfide ....................... 

285 

284 

280 

250 

244 

....................... 

....................... 

.................................. 

....................... 
244 Nickel Subsulfide, NTP TR 453 

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Inhalation Study of Nickel Subsulfidea 

TABLE Dl 

0 mg/m3 0.6 mg/m3 1.2 mg/m3 

Early deaths 

15-Month interim evaluat%onb 

7-Month interim evaluationb 

Animals initially in study 

Disposition Summary 

Terminal sacrifice 

Natural deaths 

Moribund 

Animals examined microscopically 

Missexed 

Survivors 

10 

10 

80 

8 

14 

2 

36 

68 

10 

10 

80 

11 

15 

34 

70 

10 

10 

80 

9 

13 

38 

70 

Uterus 

Genital System 

7-Month Interim Evaluation 

Polyp stromal 

(1) 

1 (100%) 

Urinary System 

Special Senses System 

Respiratory System 

Nervous System 

Musculoskeletal System 

Integumentary System 

Hematopoietic System 

General Body System 

Endocrine System 

Cardiovascular System 

Alimentary System 

Systems Examined With No Neoplasms Observed 

Liver 

Alimentary System 

15-Month Interim Evaluation 

(5) 

Hepatocellular adenoma 

Hemangiosarcoma 

Hemangioma 

1 (20%) 

1 (20%) 

(5) 

1 (20%) 

Musculoskeletal System 

Integumentary System 

Hematopoietic System 

Genital System 

General Body System 

Endocrine System 

Cardiovascular System 

Systems Examined With No Neoplasms Observed 
Lesions in Female Mice 245 

TABLE Dl 

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide (continued) 

0 mg/m3 0.6 mg/m3 1.2 mg/m3 

Urinary System 

Special Senses System 

Respiratory System 

Nervous System 

Systems Examined With No Neoplasms Observed (continued) 

15-Month Interim Evaluation (continued) 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, colon 

Gallbladder 

Alimentary System 

2- Year Study 

Adenoma 

Intestine small, jejunum 

Polyp adenomatous 

Squamous cell carcinoma, metastatic, stomach, 

Sarcoma, metastatic, skeletal muscle 

Histiocytic sarcoma, metastatic, uterus 

Histiocytic sarcoma 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple 

Hepatocellular carcinoma 

Hemangiosarcoma 

Liver 

forestomach 

Mesentery 

Squamous cell carcinoma, metastatic, stomach, 

Sarcoma, metastatic, skeletal muscle 

Histiocytic sarcoma 

Carcinoma, metastatic, islets, pancreatic 

forestomach 

Pancreas 

Oral mucosa 

Squamous cell carcinoma, metastatic, stomach, 

Sarcoma, metastatic, skeletal muscle 

Buccal, squamous cell papilloma 

Stomach, forestomach 

Salivary glands 

forestomach 

Alveolar/bronchiolar carcinoma, metastatic, 

Squamous cell papilloma 

Squamous cell carcinoma 

Stomach, glandular 

lung 
246 Nickel Subsulfide, NTP TR 453 

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide (continued) 

TABLE Dl 

0 mg/m3 0.6 mg/m3 1.2 mg/m3 

Heart 

Cardiovascular System 

2-Year study (continued) 

(58) 

Sarcoma, metastatic, skeletal muscle 

Hemangiosarcoma, metastatic, liver 

Alveolar/bronchiolar carcinoma, metastatic, 

lung 

1 (2%) 

(59) 

Endocrine System 

Squamous cell carcinoma, metastatic, stomach, 

Sarcoma, metastatic, skeletal muscle 

Hepatocellular carcinoma, metastatic, liver 

Adenoma 

Adrenal cortex 

Pheochromocytoma benign 

Adrenal medulla 

forestomach 

Carcinoma 

Adenoma 

Islets, pancreatic 

Pars distalis, adenoma 

Pituitary gland 

Follicular cell, carcinoma 

Follicular cell, adenoma 

Thyroid gland 

(58) 

1 (2%) 

1 (2%) 

1 (2%) 

General Body System 

Mediastinum, alveolar/bronchiolar carcinoma, 

Histiocytic sarcoma 

Tissue NOS 

Thoracic, alveolar/bronchiolar carcinoma, 

Mediastinum, hemangiosarcoma 

metastatic, lung 

metastatic, lung 

1 (25%) 

1 (25%) 

1 (25%) 

Genital System 

Squamous cell carcinoma, metastatic, stomach, 

Sarcoma, metastatic, skeletal muscle 

Luteoma 

Histiocytic sarcoma, metastatic, utem 

Histiocytic sarcoma 

Cystadenoma 

Ovary 

Teratoma malignant 

Teratoma benign 

forestomach 

(58) 

1 (2%) 

2 (3%) 

1 (2%) 

1 (2%) 

1 (2%) 
Lesions in Female Mice 247 

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide (continued) 

TABLE Dl 

0 mg/m3 0.6 mg/m3 

Uterus 

Genital System (continued) 

2-YeUt study (continued) 

Endometrium, adenocarcinoma 

Polyp stromal 

Histiocytic sarcoma 

Hemangioma 

Fibroma 

(60) 

1 (2%) 

1 (2%) 

2 (3%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

Bone marrow 

Hematopoietic System 

Histiocytic sarcoma, metastatic, uterus 

Histiocytic sarcoma 

Hemangiosarcoma, metastatic, tissue NOS 

Pancreatic, sarcoma, metastatic, skeletal 

Pancreatic, histiocytic sarcoma 

Iliac, sarcoma, metastatic, skeletal muscle 

Iliac, hemangiosarcoma 

muscle 

Renal, histiocytic sarcoma, metastatic. 

metastatic, stomach, forestomach 

Pancreatic, squamous cell carcinoma, 

Histiocytic sarcoma 

Alveolar/bronchiolar carcinoma, metastatic, 

Renal, sarcoma, metastatic, skeletal muscle 

Squamous cell carcinoma, metastatic, stomach, 

Histiocytic sarcoma, metastatic, uterus 

Lymph node 

uterus 

Lymph node, bronchial 

lung 

forestomach 

Lymph node, mesenteric 

Lymph node, mandibular 

Sarcoma, metastatic, skeletal muscle 

Histiocytic sarcoma, metastatic, uterus 

Hemangiosarcoma, metastatic, spleen 

Squamous cell carcinoma, metastatic, stomach, 

Histiocytic sarcoma, metastatic, uterus 

Lymph node, mediastinal 

forestomach 

Teratoma malignant, metastatic, ovary 

Histiocytic sarcoma, metastatic, uterus 

Histiocytic sarcoma 

Hemangiosarcoma, metastatic, tissue NOS 

Hemangiosarcoma 

Fibrosarcoma 

Histiocytic sarcoma 

Alveolar/bronchiolar carcinoma, metastatic, 

Spleen 

lung 

Thymus 

(60) 

1 (2%) 

1 (11%) 

1 (2%) 

(9) (12) 

1 (8%) 

1 (9%) 

1 (8%) 

1 (9%) 

(59) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

(15) (30) 

1 (7%) 

1 (4%) 

(58) (60) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

(58) 

1 (2%) 

(57) 

1 (2%) 

1 (2%) 
248 Nickel Subsulfide, NTP TR 453 

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide (continued) 

TABLE Dl 

0 mg/m3 0.6 mg/m3 1.2 mg/m3 

Mammary gland 

Integumentary System 

2- Yf?Ul. study (continued) 

Skin 

Adenocarcinoma 

Sebaceous gland, squamous cell carcinoma 

Pinna, squamous cell carcinoma 

Pinna, fibrosarcoma 

Keratoacanthoma 

Fibrosarcoma 

Fibroma 

2 (3%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

Skeletal muscle 

Bone 

Musculoskeletal System 

(1) 

(57) 

(3) 

(60) 

Hemangiosarcoma 

Alveolar/bronchiolar carcinoma, metastatic, 

Hemangiosarcoma 

lung 1 (100%) 

1 (33%) 

Sarcoma 

Hemangiosarcoma, metastatic, spleen 

1 (33%) 

1 (33%) 

1 (100%) 

Brain 

Nervous System 

Lung 

Larynx 

Respiratory System 

Epithelium, squamous cell carcinoma 

Squamous cell carcinoma, metastatic, stomach, 

Sarcoma, metastatic, skeletal muscle 

Histiocytic sarcoma 

Hepatocellular carcinoma, metastatic, liver 

Hepatocellular carcinoma, metastatic 

Alveolar/bronchiolar carcinoma 

Alveolarlbronchiolar adenoma 

Adenocarcinoma, metastatic, mammary gland 

forestomach 

Trachea 

Nose 

Hemangioma 

(58) 

(54) 

2 (3%) 

1 (2%) 

7 (12%) 

3 (5%) 

1 (2%) 

(59) 

(55) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

6 (10%) 

1 (2%) 

(59) 

(57) 

2 (3%) 

1 (2%) 

1 (2%) 

1 (2%) 

Harderian gland 

Special Senses System 

Adenoma 
Lesions in Female Mice 249 

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide (continued) 

TABLE Dl 

0 mg/m3 0.6 mg/m3 1.2 mg/m3 

Kidney 

Urinary System 

&Year ShCdy (continued) 

Alveolar/bronchiolar carcinoma, metastatic, 

Adenocarcinoma, metastatic, mammary gland 

Squamous cell carcinoma, metastatic, stomach, 

Histiocytic sarcoma, metastatic, uterus 

Histiocytic sarcoma 

Teratoma malignant, metastatic, ovary 

lung 

Urinary bladder 

forestomach 

(58) 

1 (2%) 

1 (2%) 

1 (2%) 1 (2%) 

(60) 

1 (2%) 

Multiple organs' 

Systemic Lesions 

Lymphoma malignant 

Histiocytic sarcoma 

(60) 

6 (10%) 

2 (3%) 

~otal animals with primary neoplasmsd 

Neoplasm Summary 

2-Year study 

15-Month interim evaluation 

"/-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

7-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

7-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

7-Month interim evaluation 

15-Month interim evaluation 

2-Year study 

15-Month interim evaluation 

2-Year study 

2-Year study 

2-Year study 

Total primary neoplasms 

Total animals with benign neoplasms 

Total benign neoplasms 

Total animals with malignant neoplasms 

Total malignant neoplasms 

Total metastatic neoplasms 

Total animals with metastatic neoplasms 

42 

2 

69 

2 

27 

2 

35 

2 

26 

18 

7 

34 

39 

55 

22 

25 

27 

30 

12 

37 

37 

1 

1 

49 

1 

1 

1 

20 

1 

26 

22 

1 

23 

1 

2 

7 

Primary neoplasms: all neoplasms except metastatic neoplasms 

Number of animals with any tissue examined microscopically 

Five animals in each exposure group were histopathologically examined at the interim evaluations. 

a Number of animals examined microscopically at the site and the number of animals with neoplasm 
250 Nickel Subsulfide, NTP TR 453 

80598623803594903146373 

80810356900447788999223 

233455555666666666667’77 

42586749130528459972834 

34504444852802130881198 

11111111011011111001100 

Number of Days on Study 

Carcass ID Number 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Gallbladder 

Esophagus 

Alimentary System 

A++AA++++++++++A+++++++ 

++++A+++M+M++++A+++++++ 

++++A++++++++++A+++++++ 

A+++A++++++A+++A+++++++ 

....................... 

Intestine small, duodenum A++AA++++++A+++A+++++++ 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Polyp adenomatous 

Histiocytic sarcoma, metastatic, uterus 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma 

Hemangiosarcoma 

Carcinoma, metastatic, islets, pancreatic 

Mesentery 

A++AA++++++++++A+++++++ 

....................... 

A++AA++++++++++A+++++++ 

A 

xx 

X X 

X 

xxxxx 

X 

X 

+ 

Pancreas ....................... 

Tooth 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Squamous cell papilloma 

++++A++++++++++++++++++ 

....................... 

++++A++++++++++++++++++ 

Heart 

Cardiovascular System 

....................... Hemangiosarcoma, metastatic, liver 

Endocrine System 

Adrenal medulla 

Adenoma 

Adrenal cortex 

Pheochromocytoma benign 

Parathyroid gland 

Carcinoma 

Islets, pancreatic 

Thyroid gland 

Pituitary gland 

....................... 

....................... 

....................... 

....................... X 

....................... 

+++++++M+++++++++M+++++ 

Follicular cell, carcinoma 

Follicular cell, adenoma 

Pars distalis, adenoma X 

X 

None 

General Body System 

~ ~~~ ~ 

A: Autolysis precludes examination 

+ : Tissue examined microscopically 

I: Insufficient tissue 

M: Missing tissue 

Blank: Not examined 

X: Lesion present 
Lesions in Female Mice 251 

(continued) 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0 mg/m3 

TABLE D2 

Number of Days on Study 

3333333334444444445555555 

3333333333333333333333333 

1111111111111111111111111 

~ ~~ 

Carcass ID Number 

6051021246940577118186918 

9002335558901112348991133 

0111111110011111110001111 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Gallbladder 

Esophagus 

Alimentary System 

Intestine small, ileum 

Intestine small, jejunum 

Polyp adenomatous 

+++++++t+++++++++++++++++ 

......................... 

++++++MMM++M++M+++++++++M 

......................... 

++++++++I++++++++++++++++ 

+++++++++++M+++++++++++++ 

......................... 

......................... 

Liver ......................... 

Histiocytic sarcoma, metastatic, uterus 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma 

Hemangiosarcoma 

Carcinoma, metastatic, islets, pancreatic 

X X xx 

X X 

X 

X X 

Mesentery 

Stomach, forestomach 

Salivary glands 

Pancreas 

Heart 

Cardiovascular System 

Tooth 

Stomach, glandular 

X 

+ 

......................... 

......................... 

......................... 

Squamous cell papilloma X 

......................... + 

......................... Hemangiosarcoma, metastatic, liver 

Endocrine System 

Adrenal medulla 

Adenoma 

Adrenal cortex 

Pheochromocytoma benign 

Parathyroid gland 

Carcinoma 

Islets, pancreatic 

Thyroid gland 

Pituitary gland 

Follicular cell, carcinoma 

Follicular cell, adenoma 

Pars distalis, adenoma 

......................... X 

......................... X 

......................... 

A 

......................... 

+++++++++M++++M+++++++++M 

X xx X X 

......................... X 

X 

None 

General Body System 
252 Nickel Subsulfide, NTP TR 453 

(continued) 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0 mg/m3 

TABLE D2 

7777777777 

Number of Days on Study 

5566666666 

3333333333 

9031169688 

5689002345 

1100111111 

Tumors 

Tissues/ 

Total 

Carcass ID Number 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Gallbladder 

Esophagus 

Alimentary System 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Polyp adenomatous 

Histiocytic sarcoma, metastatic, uterus 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma 

Hemangiosarcoma 

Carcinoma, metastatic, islets, pancreatic 

Mesentery 

Stomach, forestomach 

Salivaly glands 

Pancreas 

Tooth 

Stomach, glandular 

Squamous cell papilloma 

++++++++++ 

++++++++++ 

M+++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

X 

++++++++++ 

++++++++++ 

++++++++++ 

xx 

xx 

X 

+ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

+ 

53 

54 

47 

56 

53 

51 

2 

57 

1 

57 

58 

58 

1 

3 

1 

4 

9 

11 

2 

58 

54 

54 

1 

Heart 

Cardiovascular System 

++++++++++ 

Hemangiosarcoma, metastatic, liver X 1 

58 

Endocrine System 

Adrenal medulla 

Adenoma 

Adrenal cortex 

Pheochromocytoma benign 

Parathyroid gland 

Carcinoma 

Islets, pancreatic 

Thyroid gland 

Pituitary gland 

Follicular cell, carcinoma 

Follicular cell, adenoma 

Pars distalis, adenoma 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

+M++++++++ 

X 

++++++++++ 

1 

2 

58 

8 

58 

52 

1 

58 

1 

58 

1 

58 

None 

General Body System 
Lesions in Female Mice 253 

(continued) 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0 mg/m3 

TABLE D2 

Number of Days on Study 

80598623803594903146373 

80810356900441188999223 

23345555566666666666717 

42586149130528459912834 

34504444852802130881198 

11111111011011111001100 

Carcass ID Number 

Ovary 

Clitoral gland 

Genital System 

....................... 

++++++++M++++++++++M+++ 

Histiocytic sarcoma, metastatic, uterus 

Cystadenoma 

Polyp stromal 

Histiocytic sarcoma 

Hemangioma 

X 

X 

Uterus ....................... 

X 

Lymph node 

Bone marrow 

Hematopoietic System ....................... 

Renal, histiocytic sarcoma, 

Iliac, hemangiosarcoma 

Histiocytic sarcoma, metastatic, uterus 

++ 

X 

+ + 

metastatic, uterus 

X 

X 

Lymph node, mandibular 

Lymph node, bronchial 

Spleen 

Lymph node, mediastinal 

Lymph node, mesenteric 

I I +++++++I ++++++++++++M 

Histiocytic sarcoma, metastatic, uterus 

+M+++++++++M+++++++++++ 

+++++M+++++++++++++++++ 

Fibrosarcoma 

Histiocytic sarcoma, metastatic, uterus 

Histiocytic sarcoma, metastatic, uterus 

Histiocytic sarcoma, uterus 

X 

MMM+MM+MMMM++M+MMM++MMM 

X 

....................... X 

X 

Skin 

Mammary gland 

Integumentary System 

Thymus ....................... 

+++M+++++++++++++++++++ 

Pinna, fibrosarcoma 

Adenocarcinoma 

Skeletal muscle 

Bone 

Musculoskeletal System 

....................... X 

....................... + 

Alveolarlbronchiolar carcinoma, 

metastatic, lung X 

Spinal cord 

Brain 

Nervous System ........................ + 
254 Nickel Subsulfide, NTP TR 453 

(continued) 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0 mg/m3 

TABLE D2 

3333333334444444445555555 

3333333333333333333333333 

7777777177711177771717177 

Number of Days on Study 

Carcass ID Number 

6051021246940577718186918 

9002335558901112348991133 

0111111110011111110001111 

Ovary 

Clitoral gland 

Genital System 

.......................... 

......................... 

Histiocytic sarcoma, metastatic, uterus 

Cystadenoma 

Polyp stromal 

Histiocytic sarcoma 

Hemangioma 

X 

Uterus ......................... X 

X 

Lymph node 

Bone marrow 

Hematopoietic System ......................... 

Renal, histiocytic sarcoma, 

Iliac, hemangiosarcoma 

Histiocytic sarcoma, metastatic, uterus 

+ + 

metastatic, uterus 

Lymph node, mandibular 

Lymph node, bronchial 

Spleen 

Lymph node, mediastinal 

Lymph node, mesenteric 

++++++++++++I +I M+++++I +++ 

Histiocytic sarcoma, metastatic, uterus 

I MI "+""++MI M++MMMMMM++ 

+++++++++++++++M+++++++++ 

......................... 

Fibrosarcoma 

Histiocytic sarcoma, metastatic, uterus 

Histiocytic sarcoma, metastatic, uterus 

Histiocytic sarcoma, metastatic, uterus 

......................... 

Mammary gland 

Integumentary System 

Thymus ......................... 

......................... Adenocarcinoma 

Skin 

X 

......................... Pinna, fibrosarcoma 

Skeletal muscle 

Bone 

Musculoskeletal System ......................... 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 

Spinal cord 

Brain 

Nervous System ......................... 
Lesions in Female Mice 255 

(continued) 

Individual Animal Tumor Pathology of Female Mice in the 2-Year alation Study of Nickel Subsulfide: 0 mg/m3 

TABLE D2 

Number of Days on Study 

~ ~ ~~ ~ 

5566666666 

3333333333 

7777777777 

9031169688 

5689002345 

1100111111 

Tumors 

Tissues/ 

Total 

Carcass ID Number 

Ovary 

Clitoral gland 

Genital System 

++++++++++ 

+++++M++++ 

2 

58 

55 

Cystadenoma 

Histiocytic sarcoma, metastatic, uterus 

Polyp stromal 

Histiocytic sarcoma 

Hemangioma 

Uterus ++++++++++ 

X 

1 

2 

1 

58 

1 

Bone marrow 

Hematopoietic System 

+++++++M++ 51 

Renal, histiocytic sarcoma, 

Iliac, hemangiosarcoma 

Histiocytic sarcoma, metastatic, uterus 

Lymph node + 

X 

++ 

metastatic, uterus 

Lymph node, bronchial ++++++++++ 

Histiocytic sarcoma, metastatic, uterus 

Lymph node, mandibular 

Spleen 

Lymph node, mediastinal 

Lymph node, mesenteric 

Fibrosarcoma 

Histiocytic sarcoma, metastatic, uterus 

Histiocytic sarcoma, metastatic, uterus 

Histiocytic sarcoma, metastatic, uterus 

Thymus 

++++++++++ 

++++++++++ 

MMMMM+MMMM 

++++++++++ 

++++++++++ 

1 

9 

1 

58 

1 

1 

58 

1 

15 

1 

55 

57 

1 

50 

1 

Skin 

Mammary gland 

Integumentary System 

Pinna, fibrosarcoma 

Adenocarcinoma 

++++++++++ 

++++++++++ 

X 2 

58 

1 

57 

Skeletal muscle 

Bone 

Musculoskeletal System 

+++++++M++ 

1 

1 

57 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 

Spinal cord 

Brain 

Nervous System 

++++++++++ 

1 

58 
256 Nickel Subsulfide, NTP TR 453 

(continued) 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0 mg/m’ 

TABLE D2 

80598623803594903146373 

80810356900447788999223 

2334555556666666666617’1 

Number of Days on Study 

Carcass ID Number 

42586749130528459972834 

34504444852802130881198 

11111111011011111001100 

Lung 

Larynx 

Respiratory System 

....................... 

+++++I+++++++++++++++++ 

Hepatocellular carcinoma, metastatic, liver 

Hepatocellular carcinoma, metastatic 

Alveolar/bronchiolar carcinoma 

Alveolar/bronchiolar adenoma 

Adenocarcinoma, metastatic, mammary gland 

X 

X 

xx 

Trachea 

Nose 

+++++++++++++++++++M+++ 

....................... 

Harderian gland 

EY e 

Ear 

Special Senses System 

Adenoma 

~~~~ ~ ~ 

+ 

X 

+ 

+ 

Kidney 

Urinary System ....................... 

Histiocytic sarcoma, metastatic, uterus 

Alveolar/bronchiolar carcinoma, 

Adenocarcinoma, metastatic, mammary gland 

metastatic, lung X 

X 

Urinary bladder ++++A++++++++++++++++++ 

Multiple organs 

Systemic Lesions 

Lymphoma malignant 

Histiocytic sarcoma 

....................... A 

X 
Lesions in Female Mice 257 

(continued) 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0 mg/m3 

TABLE D2 

~~~~ 

Number of Days on Study 

~ ~ ~~ ~~ ~~ 

3333333334444444445555555 

3333333333333333333333333 

7777777777777777777777777 

~~ 

6051021246940577718786918 

9002335558901112348991133 

0111111110011111110001111 

Carcass ID Number 

Lung 

Larynx 

Respiratory System 

......................... 

+I+++++++++M+++++++++++++ 

Hepatocellular carcinoma, metastatic, liver 

Hepatocellular carcinoma, metastatic 

Alveolar/bronchiolar carcinoma 

Alveolar/bronchiolar adenoma 

Adenocarcinoma, metastatic, mammary gland X 

X X 

X 

X X X xx 

Trachea 

Nose 

......................... 

......................... 

Harderian gland 

EY e 

Ear 

Special Senses System 

Adenoma 

t 

Kidney 

Urinary System ......................... 

Histiocytic sarcoma, metastatic, uterus 

Alveolar/bronchiolar carcinoma, 

Adenocarcinoma, metastatic, mammary gland X 

metastatic, lung 

Multiple organs 

Systemic Lesions 

Urinary bladder ++++++++++++I++++++++++++ 

......................... 

Lymphoma malignant 

Histiocytic sarcoma 

X X X 
Nickel Subsulfide, NTP TR 453 

(continued) 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0 mg/m3 

TABLE D2 

5566666666 

3333333333 

7777777777 

9031169688 

5689002345 

1100111111 

Number of Days on Study 

Tumors 

Tissues/ 

Total 

Carcass ID Number 

Lung 

Larynx 

Respiratory System 

++++++++++ 

+M++++++++ 

58 

54 

Hepatocellular carcinoma, metastatic, liver 

Hepatocellular carcinoma, metastatic 

Alveolar/bronchiolar carcinoma 

Alveolar/bronchiolar adenoma 

Adenocarcinoma, metastatic, mammary gland 

X 

Trachea 

Nose 

++++++++++ 

++++++++++ 

2 

1 

7 

3 

1 

57 

58 

Harderian gland 

EY e 

Ear 

Special Senses System 

Adenoma 

+ 

Kidney 

Urinary System 

++++++++++ 58 

Histiocytic sarcoma, metastatic, uterus 

Alveoladbronchiolar carcinoma, 

Adenocarcinoma, metastatic, mammary gland 

metastatic, lung 

Urinary bladder ++++++++++ 

1 

56 

1 

1 

Multiple organs 

Systemic Lesions 

++++++++++ 

5 

2 

58 

Lymphoma malignant 

Histiocytic sarcoma X 

X 
Lesions in Female Mice 259 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0.6 mg/m3 

TABLE D2 

Number of Days on Study 

2663500623536223555241270 

1100223356023455555991112 

1124455555666666666661777 

Carcass ID Number 

8848137674715711414822920 

1004990451715450488507611 

3332223223232223222232233 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Gallbladder 

Esophagus 

Alimentary System 

Squamous cell carcinoma, metastatic, 

Sarcoma, metastatic, skeletal muscle 

Histiocytic sarcoma 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple 

Hepatocellular carcinoma 

Hemangiosarcoma 

stomach, forestomach 

Mesentery 

A+AA++++++++++++++A+++A++ 

A+AAA+++++++M+++A+M+++AM+ 

......................... 

......................... 

A+AAA+++++++++++A+A+++A+A 

A+AAA+++++++++++A+A+++A++ 

A+AAA+++++++++++A+A+++A++ 

A+AAA+++++++++++++A+++A++ 

A+AMA+M+++++M+++M+A+++AM+ 

X X X X 

X 

X X 

X 

X 

+ 

X 

++ 

X 

Squamous cell carcinoma, metastatic, 

Sarcoma, metastatic, skeletal muscle X 

stomach, forestomach X 

Pancreas A+A+++t+++++++++++A++++M+ 

Squamous cell carcinoma, metastatic, 

Sarcoma, metastatic, skeletal muscle X 

stomach, forestomach X 

Salivary glands ......................... 

Stomach, glandular 

Stomach, forestomach 

Alveolar/bronchiolar carcinoma, 

Squamous cell carcinoma 

metastatic, lung 

Tooth 

......................... X 

A++A+++++++++++++++++++++ 

X 

Heart 

Cardiovascular System 

......................... Alveolar/bronchiolar carcinoma, 

metastatic, lung 

Sarcoma, metastatic, skeletal muscle 

X 

X 

Adrenal cortex 

Endocrine System 

++ +++++++++++++M++++++++ 

Squamous cell carcinoma, metastatic, 

Sarcoma, metastatic, skeletal muscle X 

stomach, forestomach X 

Parathyroid gland 

Adenoma 

Islets, pancreatic 

Adrenal medulla 

A+A+++++++++++++++A++++M+ 

++ +++++++++++++M++++++++ 

X 

MM+++M++M++++++++++++++++ 
260 Nickel Subsulfide, NTP TR 453 

(continued) 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0.6 mg/m3 

TABLE D2 

8333333333344444444444555 

2333333333333333333333333 

7777777777777777777777777 

Number of Days on Study 

Carcass ID Number 

1226150583515903484939636 

6466718991144456667900577 

2222222223322222222233222 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Gallbladder 

Esophagus 

Alimentary System 

Squamous cell carcinoma, metastatic, 

Sarcoma, metastatic, skeletal muscle 

Histiocytic sarcoma 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple 

Hepatocellular carcinoma 

Hemangiosarcoma 

stomach, forestomach 

Mesentery 

Squamous cell carcinoma, metastatic, 

Sarcoma, metastatic, skeletal muscle 

stomach, forestomach 

Pancreas 

Squamous cell carcinoma, metastatic, 

Sarcoma, metastatic, skeletal muscle 

stomach, forestomach 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Alveolar/bronchiolar carcinoma, 

Squamous cell carcinoma 

metastatic, lung 

Tooth 

......................... 

......................... 

.......................... 

......................... 

......................... 

M++++++++++M+++++++++++++ 

......................... 

......................... 

......................... 

X 

X X 

X X 

X 

X 

xx 

+ 

......................... 

......................... 

......................... 

......................... 

t 

Heart 

Cardiovascular System 

Sarcoma, metastatic, skeletal muscle 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 

......................... 

Adrenal cortex 

Endocrine System 

Squamous cell carcinoma, metastatic, 

Sarcoma, metastatic, skeletal muscle 

stomach, forestomach 

Parathyroid gland 

Adenoma 

Islets, pancreatic 

Adrenal medulla 

......................... 

......................... 

......................... 

......................... 
Lesions in Female Mice 261 

(continued) 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0.6 mg/m3 

TABLE D2 

~~ 

Number of Days on Study 

~~ ~~~ 

5556666666 

3333333333 

7777777777 

3503785669 

8024678901 

2332222233 

Tumors 

Tissues/ 

Total 

Carcass ID Number 

Liver 

Intestine small, ileum 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Gallbladder 

Esophagus 

Alimentary System 

Squamous cell carcinoma, metastatic, 

Sarcoma, metastatic, skeletal muscle 

Histiocytic sarcoma 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple 

Hepatocellular carcinoma 

Hemangiosarcoma 

stomach, forestomach 

Mesentery 

Squamous cell carcinoma, metastatic, 

Sarcoma, metastatic, skeletal muscle 

stomach, forestomach 

Pancreas 

Squamous cell carcinoma, metastatic, 

Sarcoma, metastatic, skeletal muscle 

stomach, forestomach 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

Alveolar/bronchiolar carcinoma, 

Squamous cell carcinoma 

metastatic, lung 

Tooth 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

+++M++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

X X 

X X 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

60 

52 

53 

53 

54 

47 

55 

51 

60 

1 

2 

2 

8 

1 

8 

1 

1 

4 

1 

1 

56 

1 

60 

60 

1 

58 

1 

1 

1 

Heart 

Cardiovascular System 

++++++++++ 

Sarcoma, metastatic, skeletal muscle 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 

60 

1 

1 

Adrenal cortex 

Endocrine System 

Squamous cell carcinoma, metastatic, 

Sarcoma, metastatic, skeletal muscle 

stomach, forestomach 

Parathyroid gland 

Adenoma 

Islets, pancreatic 

Adrenal medulla 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++M+ 

1 

58 

56 

58 

1 

55 

1 
262 Nickel Subsulfide, NTP TR 453 

(continued) 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0.6 mg/m3 

TABLE D2 

Number of Days on Study 

2663500623536223555241270 

1100223356023455555991112 

1124455555666666666667777 

Carcass ID Number 

8848137674715711414822920 

1004990451715450488507611 

3332223223232223222232233 

Thyroid gland 

Pituitary gland 

Endocrine System (continued) 

Follicular cell, adenoma 

Pars distalis, adenoma 

+IM+++++++++++++++++M++++ 

xx ......................... X 

General Body System 

Mediastinum, alveolar/bronchiolar 

Histiocytic sarcoma 

Tissue NOS 

Thoracic, alveolar/bronchiolar 

Mediastinum, hemangiosarcoma 

carcinoma, metastatic, lung 

carcinoma, metastatic, lung 

++ + 

X 

+ 

X 

X 

X 

Ovary 

Clitoral gland 

Genital System 

A+++++++++++++++++M++++++ 

++M++++++++++++++++++++++ 

Squamous cell carcinoma, metastatic, 

Sarcoma, metastatic, skeletal muscle 

Luteoma 

Teratoma benign 

X 

stomach, forestomach 

X 

X 

Polyp stromal 

Histiocytic sarcoma 

Teratoma malignant 

UteNS 

X 

......................... 

X 

Lymph node 

Bone marrow 

Hematopoietic System ......................... 

metastatic, stomach, forestomach 

Pancreatic, squamous cell carcinoma, 

Pancreatic, histiocytic sarcoma 

Hemangiosarcoma, metastatic, tissue NOS X 

+ ++ 

X 

+++ + 

X 

Lymph node, bronchial MM++++M++++++++++++++++++ 

Alveolar/bronchiolar carcinoma, 

Squamous cell carcinoma, metastatic, 

Histiocytic sarcoma 

metastatic, lung X 

X 

stomach, forestomach X 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular ......................... 

A+AA+++++++M++++++A++++++ 

Squamous cell carcinoma, metastatic, 

Hemangiosarcoma, metastatic, spleen X 

MMMM++++M++MM++++M+++++M+ 

stomach, forestomach X 
Lesions in Female Mice 263 

(continued) 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0.6 mg/m3 

TABLE D2 

Number of Days on Study 

~~ 

a333333333344444444444555 

2333333333333333333333333 

7177117771777777777771777 

~ ~~ 

1226150583515903484939636 

6466718991144456661900517 

2222222223322222222233222 

Carcass ID Number 

Thyroid gland 

Pituitary gland 

Endocrine System (continued) 

......................... 

Follicular cell, adenoma 

Pars distalis, adenoma X X X 

......................... X X 

General Body System 

Mediastinum, alveolar/bronchiolar 

Histiocytic sarcoma 

Tissue NOS 

Thoracic, alveolar/bronchiolar 

Mediastinum, hemangiosarcoma 

carcinoma, metastatic, lung 

carcinoma, metastatic, lung 

Ovary 

Clitoral gland 

Genital System 

Squamous cell carcinoma, metastatic, 

Sarcoma, metastatic, skeletal muscle 

Luteoma 

Polyp stromal 

Histiocytic sarcoma 

Teratoma malignant 

Teratoma benign 

stomach, forestomach 

Uterus 

......................... 

......................... 

x 

......................... X 

Lymph node 

Bone marrow 

Hematopoietic System 

......................... 

metastatic, stomach, forestomach 

Pancreatic, squamous cell carcinoma, 

Pancreatic, histiocytic sarcoma 

Hemangiosarcoma, metastatic, tissue NOS 

++ + 

Squamous cell carcinoma, metastatic, 

Histiocytic sarcoma 

Metastatic, lung 

Alveolarlbronchiolar carcinoma, 

Lymph node, bronchial ......................... 

stomach, forestomach 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Squamous cell carcinoma, metastatic, 

Hemangiosarcoma, metastatic, spleen 

stomach, forestomach 

++M++++++++++++++++++++++ 

......................... 

"++I ++I M++MM+I MI +"I MMMM 
264 Nickel Subsulfide, NTP TR 453 

(continued) 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0.6 mg/m3 

TABLE D2 

5556666666 

3333333333 

7777777777 

Number of Days on Study 

3503785669 

8024678901 

2332222233 

Tumors 

Tissues/ 

Total 

Carcass ID Number 

Thyroid gland 

Pituitary gland 

Endocrine System (continued) 

++++++++++ 

4 

60 

5 

57 

Follicular cell, adenoma 

Pars distalis, adenoma 

++++++++++ 

X 

General Body System 

Mediastinum, alveolar/bronchiolar 

Histiocytic sarcoma 

Tissue NOS 

Thoracic, alveolar/bronchiolar 

Mediastinum, hemangiosarcoma 

carcinoma, metastatic, lung 

carcinoma, metastatic, lung 

1 

4 

1 

1 

1 

Ovary 

Clitoral gland 

Genital System 

Squamous cell carcinoma, metastatic, 

Sarcoma, metastatic, skeletal muscle 

Luteoma 

Polyp stromal 

Histiocytic sarcoma 

Teratoma malignant 

Teratoma benign 

++++++++++ 

++++++++++ 

1 

1 

58 

59 

stomach, forestomach 

1 

1 

1 

1 

1 

Uterus ++++++++++ 60 

Lymph node 

Bone marrow 

Hematopoietic System 

++,++++++++ 

1 

12 

1 

60 

metastatic, stomach, forestomach 

Pancreatic, squamous cell carcinoma, 

Pancreatic, histiocytic sarcoma 

Hemangiosarcoma, metastatic, tissue NOS 

+ + 

57 

1 

Alveolar/bronchiolar carcinoma, 

Lymph node, bronchial 

Squamous cell carcinoma, metastatic, 

Histiocytic sarcoma 

metastatic, lung 

++++++++++ 

1 

1 

stomach, forestomach 

54 

59 

1 

1 

28 

1 

Lymph node, mediastinal 

Lymph node, mesenteric 

Lymph node, mandibular 

Squamous cell carcinoma, metastatic, 

Hemangiosarcoma, metastatic, spleen 

stomach, forestomach 

++++++++++ 

+++++M++++ 

"++++"MI 
Lesions in Female Mice 265 

(continued) 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0.6 mg/m3 

TABLE D2 

Number of Days on Study 

2663500623536223555241270 

1100223356023455555991112 

1124455555666666666667777 

Carcass ID Number 

8848137674715711414822920 

1004990451715450488507611 

3332223223232223222232233 

Spleen 

Hematopoietic System (continued) 

......................... 

Teratoma malignant, metastatic, ovary 

Histiocytic sarcoma 

Hemangiosarcoma, metastatic, tissue NOS 

Hemangiosarcoma 

Skin 

Mammary gland 

Integumentary System 

Histiocytic sarcoma 

metastatic, lung 

Alveolar/bronchiolar carcinoma, 

X 

X 

X 

X 

Thymus A+A++++++++++M+++++++++++ 

X 

X 

M++++++++++++++++++++++++ 

Keratoacanthoma 

Adenocarcinoma X 

......................... X 

~~ 

Skeletal muscle 

Bone 

Musculoskeletal System 

~~ 

Sarcoma 

Hemangiosarcoma, metastatic, spleen 

Hemangiosarcoma 

......................... + + 

X 

X 

Brain 

Nervous System ......................... 

Lung 

Larynx 

Respiratory System 

++++++++++++++M++++++++++ 

Epithelium, squamous cell carcinoma 

Squamous cell carcinoma, metastatic, 

Sarcoma, metastatic, skeletal muscle 

Lymphoma malignant lymphocytic, metastatic 

Histiocytic sarcoma 

Hepatocellular carcinoma, metastatic, liver 

Alveoladbronchiolar carcinoma 

Alveolar/bronchiolar adenoma 

X 

++A++++++++++++++++++++++ 

X X 

X 

xx X 

X 

X 

X 

stomach, forestomach X 

Ear 

Special Senses System 

Trachea 

Nose 

++A+++M.++++++++M+++++++++ 

A++++++++++++++++++++++++ 
266 Nickel Subsulfide, NTP TR 453 

TABLE D2 

(continued) 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0.6 mg/m3 

8333333333344444444444555 

2333333333333333333333333 

1171111117177111111111111 

Number of Days on Study 

Carcass ID Number 

1226150583515903484939636 

6466118991144456661900511 

2222222223322222222233222 

Spleen 

Hematopoietic System (continued) 

......................... 

Teratoma malignant, metastatic, ovaly 

Histiocytic sarcoma 

Hemangiosarcoma, metastatic, tissue NOS 

Hemangiosarcoma 

Histiocytic sarcoma 

metastatic, lung 

Alveolarlbronchiolar carcinoma, 

Thymus ......................... 

Mammary gland 

Integumentary System 

......................... 

Keratoacanthoma 

Adenocarcinoma 

Skin 

X 

......................... 

Skeletal muscle 

Bone 

Musculoskeletal System 

......................... 

X 

+ 

Sarcoma 

Hemangiosarcoma, metastatic, spleen 

Hemangiosarcoma 

Brain 

Nervous System 

......................... 

Lung 

Larynx 

Respiratory System 

Alveolar/bronchiolar carcinoma 

Alveolar/bronchiolar adenoma 

Epithelium, squamous cell carcinoma 

+++++++++I ++++++++++++I +I 

......................... X 

Squamous cell carcinoma, metastatic, 

Sarcoma, metastatic, skeletal muscle 

Lymphoma malignant lymphocytic, metastatic 

Histiocytic sarcoma 

Hepatocellular carcinoma, metastatic, liver X 

stomach, forestomach 

Trachea 

Nose .......................... 

......................... 

Ear 

Special Senses System 

+ 
Lesions in Female Mice 267 

(continued) 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0.6 mg/m3 

TABLE D2 

5556666666 

3333333333 

1111111111 

3503185669 

8024618901 

2332222233 

Number of Days on Study 

Tumors 

Tissues/ 

Total 

Carcass ID Number 

Spleen 

Hematopoietic System (continued) 

++++++++++ 

1 

1 

1 

1 

60 

1 

1 

51 

Teratoma malignant, metastatic, ovary 

Histiocytic sarcoma 

Hemangiosarcoma, metastatic, tissue NOS 

Hemangiosarcoma 

Histiocytic sarcoma 

metastatic, lung 

Alveolar/bronchiolar carcinoma, 

Thymus ++++++++++ 

Skin 

Mammary gland 

Integumentary System 

Keratoacanthoma 

Adenocarcinoma 

++++++++++ 

X 

++++++++++ 

1 

60 

3 

59 

Skeletal muscle 

Bone 

Musculoskeletal System 

Sarcoma 

Hemangiosarcoma, metastatic, spleen 

Hemangiosarcoma 

++++++++++ 

1 

1 

1 

3 

60 

Brain 

Nervous System 

++++++++++ 60 

Lung 

Larynx 

Respiratory System 

Epithelium, squamous cell carcinoma 

Squamous cell carcinoma, metastatic, 

Sarcoma, metastatic, skeletal muscle 

Lymphoma malignant lymphocytic, metastatic 

Histiocytic sarcoma 

Hepatocellular carcinoma, metastatic, liver 

Alveolar/bronchiolar carcinoma 

Alveolar/bronchiolar adenoma 

stomach, forestomach 

Trachea 

Nose 

+I ++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

1 

1 

1 

6 

1 

1 

59 

1 

55 

51 

59 

1 

Ear 

Special Senses System 

1 
Nickel Subsulfide, NTP TR 453 

(continued) 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0.6 mg/m3 

TABLE D2 

2663500623536223555241270 

1100223356023455555991112 

1124455555666666666667777 

Number of Days on Study 

Carcass ID Number 

8848137674715711414822920 

1004990451715450488507611 

3332223223232223222232233 

Kidney 

Urinary System ......................... 

Teratoma malignant, metastatic, ovary 

Squamous cell carcinoma, metastatic, 

Alveolar/bronchiolar carcinoma, 

stomach, forestomach 

metastatic, lung X 

X 

X 

Urinary bladder A+AAA+++++++++++++A++++++ 

Multiple organs 

Systemic Lesions 

Lymphoma malignant 

Histiocytic sarcoma 

......................... X 

X X X X X 
Lesions in Female Mice 269 

(continued) 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0.6 mg/m’ 

TABLE D2 

Number of Days on Study 

8333333333344444444444555 

2333333333333333333333333 

7777777777777777777777777 

Carcass ID Number 

1226150583515903484939636 

6466778991144456667900577 

2222222223322222222233222 

~ ~ ~~ ~~ ~ ~~ ~~ ~~ ~ 

Kidney 

Urinary System 

......................... 

Teratoma malignant, metastatic, ovary 

Squamous cell carcinoma, metastatic, 

Alveolarlbronchiolar carcinoma, 

stomach, forestomach 

metastatic, lung 

Urinary bladder ......................... 

Multiple organs 

Systemic Lesions 

Lymphoma malignant 

Histiocytic sarcoma 

......................... X 

X 
270 Nickel Subsulfide, NTP TR 453 

(continued) 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 0.6 mg/m3 

TABLE D2 

Number of Days on Study 

5556666666 

3333333333 

7777171711 

Carcass ID Number 

3503785669 

8024678901 

2332222233 

Tumors 

Tissues/ 

Total 

Kidney 

Urinary System 

++++++++++ 60 

Teratoma malignant, metastatic, ovary 

Squamous cell carcinoma, metastatic, 

Alveolar/bronchiolar carcinoma, 

stomach, forestomach 

metastatic, lung 1 

55 

1 

1 

Multiple organs 

Systemic Lesions 

Urinary bladder ++++++++++ 

++++++++++ 

7 

2 

60 

Lymphoma malignant 

Histiocytic sarcoma 

X 
Lesions in Female Mice 271 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 1.2 mg/m3 

TABLE D2 

Number of Days on Study 

4905973000878023253922333 

7344906888802335780233333 

0234455555546666667777777 

Carcass ID Number 

6224190049629372659473285 

2257457347010326511137034 

4444444444444444444444444 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Gallbladder 

Esophagus 

Alimentary System 

Intestine small, ileum 

Histiocytic sarcoma 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma 

Hemangiosarcoma 

Liver 

Buccal, squamous cell papilloma 

Histiocytic sarcoma 

Adenoma 

Pancreas 

Oral mucosa 

Mesentery 

Tooth 

Stomach, glandular 

Stomach, forestomach 

Salivaly glands 

+A+++++++++AA+A++AAA+++++ 

+A+++++++++AA+A++AAA+++++ 

+A+++++++++A++A++A+A+++++ 

........................ 

+A++++++M++A++A++A+A+++++ 

+A+++M+++++AM+A++AA++++++ 

......................... 

X 

......................... 

+A+++++++++A++A++AAA+++++ 

X 

X 

X .I 

A 

X 

t 

xx 

X X 

X 

+A+++++++++A+++++++++++++ 

+A+++++++++A+++++++++++++ 

......................... 

+++++++++++A+++++++++++++ 

+ 

Adrenal cortex 

Endocrine System 

Heart 

Cardiovascular System 

......................... 

......................... 

Pheochromocytoma benign 

Sarcoma, metastatic, skeletal muscle 

Hepatocellular carcinoma, metastatic, liver X 

X 

Adrenal medulla 

Pituitary gland 

Parathyroid gland 

Islets, pancreatic 

Thyroid gland 

......................... 

......................... 

+M++M++++++++MI+++++M++++ 

+++++++++++A+++++++++++++ 

Follicular cell, adenoma 

Pars distalis, adenoma X X 

......................... 

Tissue NOS 

General Body System 

+ 
272 Nickel Subsulfide, NTP TR 453 

(continued) 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 1.2 mg/m3 

TABLE D2 

3333334444444555555555555 

3333333333333333333333333 

7777777777777777777777777 

Number of Days on Study 

Carcass ID Number 

6716188094974173822383480 

4456770223356001134445556 

4444444444444444444444444 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Gallbladder 

Esophagus 

Alimentary System 

Intestine small, ileum 

Histiocytic sarcoma 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma 

Hemangiosarcoma 

Liver 

Buccal, squamous cell papilloma 

Histiocytic sarcoma 

......................... 

......................... 

......................... 

++++M+M+MM+++++++M+M+++++ 

......................... 

......................... 

......................... 

Adenoma 

......................... 

......................... 

X 

X 

xxxx X 

Pancreas 

Oral mucosa 

Mesentery 

Tooth 

Stomach, glandular 

Stomach, forestomach 

Salivary glands 

......................... 

......................... 

......................... 

......................... 

Heart 

Cardiovascular System ......................... 

Adrenal cortex 

Endocrine System 

Pheochromocytoma benign 

Sarcoma, metastatic, skeletal muscle 

Hepatocellular carcinoma, metastatic, 

Adrenal medulla 

Pituitary gland 

Parathyroid gland 

Islets, pancreatic 

Thyroid gland 

Follicular cell, adenoma 

Pars distalis, adenoma 

......................... liver 

......................... 

......................... 

......................... 

......................... 

X 

X X X 

......................... 

Tissue NOS 

General Body System 
Lesions in Female Mice 273 

(continued) 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 1.2 mg/m3 

TABLE D2 

Number of Days on Study 

5566666666 

3333333333 

77?17????? 

Carcass ID Number 

8531560535 

6702235566 

4444444444 

Tumors 

Tissues/ 

Total 

Intestine small, jejunum 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Gallbladder 

Esophagus 

Alimentary System 

++++++++++ 

++++++++++ 

++++++++++ 

+MM+M++M++ 

++++++++++ 

++++++++++ 

++++++++++ 

53 

53 

55 

48 

54 

53 

60 

Adenoma 1 

Intestine small, ileum 

Histiocytic sarcoma 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma 

Hemangiosarcoma 

Liver 

Buccal, squamous cell papilloma 

Histiocytic sarcoma 

Pancreas 

Oral mucosa 

Mesentely 

Tooth 

Stomach, glandular 

Stomach, forestomach 

Salivaly glands 

++++++++++ 

++++++++++ 

X 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

X 

+ 

1 

58 

58 

60 

59 

1 

1 

1 

1 

2 

1 

9 

4 

1 

60 

54 

Heart 

Cardiovascular System 

+++++++ ++ 59 

Adrenal cortex 

Endocrine System 

Pheochromocytoma benign 

Sarcoma, metastatic, skeletal muscle 

Hepatocellular carcinoma, metastatic, 

Adrenal medulla 

Pituitary gland 

Parathyroid gland 

Islets, pancreatic 

Thyroid gland 

Follicular cell, adenoma 

Pars distalis, adenoma 

++++++++++ 

liver 

++++++++++ 

X 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

1 

1 

60 

3 

60 

3 

60 

55 

59 

1 

60 

Tissue NOS 

General Body System 

1 
274 Nickel Subsulfide, NTP TR 453 

(continued) 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 1.2 mg/m3 

TABLE D2 

4905913000818023253922333 

7344906888802335180233333 

0234455555566666661111111 

Number of Days on Study 

Carcass ID Number 

6224190049629312659413285 

2251451341010326511131034 

4444444444444444444444444 ~~ 

Ovary 

Clitoral gland 

Genital System 

......................... 

MA++M++++++++++M+M+M+++++ 

Histiocytic sarcoma 

Fibroma 

Histiocytic sarcoma 

Cystadenoma X 

X 

Uterus ......................... 

X 

Endometrium, adenocarcinoma 

Polyp stromal X 

X 

Bone marrow 

Blood 

Hematopoietic System 

Lymph node 

+A+++++++++++++++++++++++ 

Renal, sarcoma, metastatic, skeletal muscle 

Pancreatic, sarcoma, metastatic, 

Iliac, sarcoma, metastatic, skeletal muscle 

Histiocytic sarcoma X 

+ ++++ 

X 

skeletal muscle 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node, mediastinal 

Histiocytic sarcoma 

Hemangiosarcoma 

Spleen 

X 

X 

......................... 

+++++++++++A++M++++++++++ 

......................... 

Sarcoma, metastatic, skeletal muscle X 

......................... 

M+M+MM+M+M++MM+M+++M++MIM 

X 

Thymus 

Skin 

Mammary gland 

Integumentary System 

Sebaceous gland, squamous cell 

Pinna, squamous cell carcinoma 

Pinna, fibrosarcoma 

Fibrosarcoma 

Fibroma 

+++M+++++++M+MMMM++++++++ 

......................... 

......................... 

X 

X 

X 

X 

carcinoma X 

Sarcoma 

Skeletal muscle 

Hemangiosarcoma 

Bone 

Musculoskeletal System 

........................ 

X 

+ 

Spinal cord 

Brain 

Nervous System ......................... + 
Lesions in Female Mice 275 

(continued) 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 1.2 mg/m’ 

TABLE D2 

Number of Days on Study 

3333334444444555555555555 

3333333333333333333333333 

7777777177177777777777777 

Carcass ID Number 

6716188094974173822383480 

4456770223356001134445556 

4444444444444444444444444 

Ovary 

Clitoral gland 

Genital System 

Histiocytic sarcoma 

Cystadenoma 

Endometrium, adenocarcinoma 

Polyp stromal 

Histiocytic sarcoma 

Fibroma 

Uterus 

......................... 

+M+++++++++++++++++++++++ 

......................... X 

Bone marrow 

Blood 

Hematopoietic System 

Lymph node 

Renal, sarcoma, metastatic, skeletal muscle 

Pancreatic, sarcoma, metastatic, 

Iliac, sarcoma, metastatic, skeletal muscle 

Histiocytic sarcoma 

skeletal muscle 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node, mediastinal 

Histiocytic sarcoma 

Hemangiosarcoma 

Spleen 

Sarcoma, metastatic, skeletal muscle 

Thymus 

+ ......................... 

+ + + 

+M+++++++++++++++++++++++ 

......................... 

......................... 

......................... 

I +++M+M+I ”“+I +++I ++MM++ 

X 

......................... 

Skin 

Mammary gland 

Integumentary System 

Sebaceous gland, squamous cell 

Pinna, squamous cell carcinoma 

Pinna, fibrosarcoma 

Fibrosarcoma 

Fibroma 

carcinoma 

......................... 

......................... 

Sarcoma 

Skeletal muscle 

Hemangiosarcoma 

Bone 

Musculoskeletal System 

......................... X 

Spinal cord 

Brain 

Nervous System 

......................... 
276 Nickel Subsulfide, NTP TR 453 

(continued) 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 1.2 mg/m3 

TABLE D2 

5566666666 

3333333333 

7771777777 

Number of Days on Study 

8531560535 

6702235566 

4444444444 

Tumors 

Tissues1 

Total 

Carcass ID Number 

Ovary 

Clitoral gland 

Genital System 

++++++++++ 

++++M+++++ 

1 

60 

52 

Cystadenoma 

Endometrium, adenocarcinoma 

Histiocytic sarcoma 

Fibroma 

Histiocytic sarcoma 

Uterus 

Polyp stromal 

++++++++++ 

1 

1 

1 

1 

60 

1 

Bone marrow 

Blood 

Hematopoietic System 

Lymph node 

Renal, sarcoma, metastatic, skeletal muscle 

Pancreatic, sarcoma, metastatic, 

Iliac, sarcoma, metastatic, skeletal muscle 

Histiocytic sarcoma 

skeletal muscle 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node, bronchial 

Lymph node, mediastinal 

Histiocytic sarcoma 

Hemangiosarcoma 

Spleen 

Sarcoma, metastatic, skeletal muscle 

Thymus 

++++++++++ 

++ + 

++++++++++ 

++++++++++ 

++++++++++ 

++++++++++ 

++MMMM+M++ 

++++++++++ 

1 

11 

1 

59 

1 

54 

1 

1 

60 

30 

1 

57 

60 

59 

1 

1 

Skin 

Mammary gland 

Integumentary System 

Sebaceous gland, squamous cell 

Pinna, squamous cell carcinoma 

Pinna, fibrosarcoma 

Fibrosarcoma 

Fibroma 

carcinoma 

++++++++++ 

++++++++++ 

1 

1 

1 

1 

60 

60 

1 

~ 

Sarcoma 

Skeletal muscle 

Hemangiosarcoma 

Bone 

Musculoskeletal System 

~~ 

1 

1 

1 

59 

Spinal cord 

Brain 

Nervous System 

++++++++++ 

1 

60 
Lesions in Female Mice 277 

(continued) 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 1.2 mg/m3 

TABLE D2 

Number of Days on Study 

4905973000818023253922333 

7344906888802335780233333 

0234455555566666667771777 

Carcass ID Number 

6224190049629372659413285 

2257457347010326511137034 

4444444444444444444444444 

Lung 

Larynx 

Respiratory System 

......................... 

++++++++++++++++++++++I ++ 

Histiocytic sarcoma 

Hepatocellular carcinoma, metastatic, liver 

Alveolar/bronchiolar carcinoma 

Alveolar/bronchiolar adenoma 

X 

X 

Nose ......................... Hemangioma 

Trachea +M+++++++++M+++++++++++++ 

Harderian gland 

Ear 

Special Senses System 

Adenoma 

t 

X 

+ 

Urinary bladder 

Urethra 

Kidney 

Urinary System 

......................... Histiocytic sarcoma X 

+ 

++++++++++++++A++++++++++ 

Multiple organs 

Systemic Lesions 

Lymphoma malignant 

Histiocytic sarcoma 

......................... X X 

X X 
278 Nickel Subsulfide, NTP TR 453 

(continued) 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 1.2 mg/m3 

TABLE D2 

Number of Days on Study 

3333334444444555555555555 

3333333333333333333333333 

1117711711111171111771111 

6716188094914173822383480 

4456710223356001134445556 

4444444444444444444444444 

Carcass ID Number 

Lung 

Larynx 

Respiratory System 

X 

+.+++++++++++++++++++++++t 

+I +++I +++++++I ++++++++MI + 

Histiocytic sarcoma 

Hepatocellular carcinoma, metastatic, liver 

Alveolar/bronchiolar carcinoma 

Alveolar/bronchiolar adenoma 

Hemangioma 

xx 

Trachea 

Nose 

......................... X 

......................... 

Harderian gland 

Ear 

Special Senses System 

Adenoma 

+ 

~~ 

Urinary bladder 

Urethra 

Kidney 

Urinary System 

Histiocytic sarcoma 

......................... 

......................... 

Multiple organs 

Systemic Lesions 

Lymphoma malignant 

Histiocytic sarcoma 

++++++++++++++++++++++++t 

X 
Lesions in Female Mice 279 

(continued) 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide: 1.2 mg/m3 

TABLE D2 

Number of Days on Study 

5566666666 

3333333333 

7777777777 

Carcass ID Number 

8531560535 

6702235566 

4444444444 

Tumors 

Tissues/ 

Total 

Lung 

Larynx 

Respiratory System 

++++++++++ 

I +++++++++ 

1 

60 

53 

Histiocytic sarcoma 

Hepatocellular carcinoma, metastatic, liver 

Alveolar/bronchiolar carcinoma 

Alveolar/bronchiolar adenoma 

Hemangioma 

Trachea 

Nose ++++++++++ 

++++++++++ 

1 

1 

2 

58 

1 

60 

Harderian gland 

Ear 

Special Senses System 

Adenoma 1 

1 

2 

Urinary bladder 

Urethra 

Kidney 

Urinary System 

Histiocytic sarcoma 

++++++++++ 

++++++++++ 59 

1 

1 

60 

Multiple organs 

Systemic Lesions 

~~ ~~ ~ 

Lymphoma malignant 

Histiocytic sarcoma 

~~~ ~ 

++++++++++ 

X xx 6 

2 

60 
280 Nickel Subsulfide, NTP TR 453 

Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide 

TABLE D3 

O mg/m3 0.6 mglm3 1.2 mg/m3 

Fisher exact test 

Cochran-Armita e testd 

Logistic regression testd 

Life table testd 

First incidence (days) 

Terminal rate' 

Adjusted rateb 

Overall ratea 

Liver: Hepatocellular Adenoma 

5 P=0.248N 

P=0.252N 

P=0.242N 

508 

9/36 (25 %) 

31.7% 

13/58 (22 %) 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Liver: Hepatocellular Carcinoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Liver: Hepatocellular Adenoma or Carcinoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Lung: Alveolarlbronchiolar Adenoma 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Lung: Alveolarlbronchiolar Carcinoma 

P=0.034N 

P=0.032N 

P=0.043N 

603 

4/36 (11%) 

25.0% 

11/58 (19%) 

P=0.075N 

P=0.077N 

P=0.087N 

508 

12/36 (33 %) 

44.8% 

20158 (34%) 

P=0.194N 

P=0.194N 

P=0.193N 

683 

2/36 (6%) 

7.8% 

3/58 (5%) 

P=\033N 

P=0.033N 

PC0.033N 

683 

6/36 (17%) 

18.7% 

7/58 (12%) 

P=0.315N 

P=0.365N 

520 

7/34 (21 %) 

25.7% 

10160 (17%) 

P=0.289N 

P=0.345N 

P=0.434N 

552 

3/34 (9%) 

21.2% 

9/60 (15%) 

P=0.371N 

P=0.409N 

P=0.477N 

520 

10/34 (29%) 

41.6% 

18/60 (30%) 

c 

'P=0.373N 

P=0.308N 

P=0.324N 

733 (T) 

1/34 (3%) 

2.9% 

1/59 (2%) 

P=0.303N 

P=0.033N 

P=0.040N 

552 

0/34 (0%) 

1.9% 

1/59 (2%) 

P=0.029N 

P=0.293N 

P=0,277N 

563 

7/38 (18%) 

23.8% 

10160 (17%) 

P=0.289N 

P=0,042N 

P=0.051N 

563 

1/38 (3%) 

8.4% 

4/60 (7%) 

P=0.041N 

P=0.091N 

P=0.099N 

563 

8/38 (21 %) 

29.3% 

13/60 (22 %) 

P=0.089N 

P=0.294N 

P=0.290N 

733 (T) 

1/38 (3%) 

2.6% 

1/60 (2%) 

P=0.297N 

P=0.071N 

P=0.070N 

733 (T) 

2/38 (5%) 

5.3% 

2/60 (3 X) 

P=0.074N 
Lesions in Female Mice 281 

Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide (continued) 

TABLE D3 

0 mg/m3 0.6 mg/m3 1.2 mg/m3 

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 

P=0.027N 

P=0.027N 

P=0.026N 

683 

8/36 (22 %) 

24.1% 

9/58 (16%) 

P=0.028N 

P=0.035N 

552 

1 134 (3 %) 

4.8% 

2/59 (3 %) 

P=0.050N 

P=0.051N 

733 (T) 

3/38 (8%) 

7.9% 

3/60 (5 %) 

P=0.025N P=0.055N 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Mammary Gland: Carcinoma 

P=0.367N 

P=0.365N 

P=0.361N 

733 (T) 

1/36 (3%) 

2.8% 

1/58 (2%) 

P=O.297 

P=O.294 

71 1 

2/34 (6%) 

8.3% 

3/60 (5 %) 

0138 (0%) 

0.0% 

0160 (0%) 

P=0.489N 

P=0.489N 

- e 

P=O.322 P=0.492N 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Pituitary Gland (Pars Distalis): Adenoma 

P=0.067N 

P=0.068N 

P=0.066N 

536 

7/36 (19%) 

21.0% 

8/58 (14%) 

P=0.290N 

P=0.309N 

712 

3/34 (9%) 

13.6% 

5/57 (9%) 

P=0.094N 

P=0.089N 

607 

1/38 (3%) 

7.0% 

3/60 (5 %) 

P=0.290N P=0.092N 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Thyroid Gland (Follicular Cell): Adenoma 

P=O.433 

P=O.436 

P=O.445 

69 1 

1/36 (3%) 

5.1% 

2/58 (3%) 

P=O.516 

P=O.522 

733 (T) 

3/38 (8%) 

7.9% 

3/60 (5 %) 

P=O.331 

P=O.324 

711 

3/34 (9%) 

11.2% 

4/60 (7%) 

P=O.356 P=O.516 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Thyroid Gland (Follicular Cell): Adenoma or Carcinoma 

P=0.562N 

P=0.558N 

P=0.550N 

69 1 

2/36 (6%) 

7.9% 

3/58 (5%) 

P=O.492 

P=O.480 

711 

3/34 (9%) 

11.2% 

4/60 (7 %) 

P = 0.646N 

P=0.639N 

733 (T) 

3/38 (8%) 

7.9% 

3/60 (5 %) 

P=O.519 P=0.644N 
282 Nickel Subsulfide, NTP TR 453 

TABLE D3 

Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide (continued) 

O mg/m3 0.6 mg/m3 1.2 mg/m3 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Hemangiosarcoma 

P=0.562N 

P=0.564N 

P=0.554N 

680 

2/36 (6%) 

7.8% 

3/58 (5%) 

P=O.502 

P=O.482 

623 

2/34 (6%) 

10.1% 

4/60 (7%) 

P=0.647N 

P=0.641N 

733 (T) 

3/38 (8%) 

7.9% 

3/60 (5 %) 

P=O.519 P=0.644N 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

AU Organs: Hemangioma or Hemangiosarcoma 

P=0.553N 

P=0.556N 

P=0.544N 

680 

3/36 (8%) 

10.5% 

4/58 (7%) 

P=0.642N 

P=O.621 

623 

2/34 (6%) 

10.1% 

4/60 (7%) 

P=0.623N 

P=0.617N 

733 (T) 

4/38 (11%) 

10.5% 

4/60 (7 %) 

P=0.622N P=0.622N 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Malignant Lymphoma 

P=O.464 

P=O.460 

P=O.474 

679 

4/36 (1 1 %) 

13.1% 

5/58 (9%) 

P=O.400 

P=O.365 

425 

2/34 (6%) 

15.8% 

7/60 (12 %) 

P=O.521 

P=O.532 

653 

4/38 (1 1 %) 

14.6% 

6/60 (10%) 

P=O.405 P=O.524 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Benign Neoplasms 

P=0.070N 

P=0.073N 

563 

15/38 (39 %) 

47.1% 

21/60 (35%) 

P=0.060N 

P=0.061N 

P=0.066N 

508 

21/36 (58%) 

65.3% 

29/58 (50%) 

P=O. 117N 

P=0.196N 

116 

13/34 (38 %) 

49.6% 

22/60 (37 %) 

P=O.lOlN P=0.072N 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Malignant Neoplasms 

P=0.296N 

P=0.314N 

445 

11/38 (29%) 

44.8% 

23/60 (38%) 

P=0.266N 

P=0.286N 

P=0.293N 

419 

15/36 (42%) 

54.8% 

26/58 (45 X) 

P=O.558 

P=O.455 

206 

9/34 (26%) 

51.3% 

27/60 (45 %) 

P=O.566 P=0.299N 
Lesions in Female Mice 283 

TABLE D3 

Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Inhalation Study of Nickel Subsulfide (continued) 

0 mg/m3 0.6 mg/m3 1.2 mg/m3 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

All Organs: Benign or Malignant Neoplasms 

P=0.169N 

P=0.176N 

P=0.223N 

419 

29/36 (81 %) 

85.8% 

43/58 (74%) 

P=0.205N 

P=0.408N 

116 

18/34 (53 %) 

70.5% 

39/60 (65 %) 

P=0.200N 

P=0.228N 

445 

24/38 (63 %) 

73.4 % 

39/60 (65 X) 

P=0.190N P=0.190N 

a Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for liver, lung, pituitary 

(T)Terminal sacrifice 

compare directly the overall incidence rates. For all tests, a negative trend or a lower incidence in an exposure group is indicated by N. 

(directly or indirectly) the cause.of death. The logistic regression test regards these lesions as nonfatal. The Cochran-Armitage and Fisher exact tests 

painvise comparisons between the controls and that exposed group. The life table test regards neoplasms in animals dying prior to terminal kill as being 

Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding to 

Observed incidence at terminal kill 

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

gland, and thyroid gland; for other tissues, denominator is number of animals necropsied. 

e Not applicable; no neoplasms in animal group 
284 Nickel Subsulfide, NTP TR 453 

Historical Incidence of Alveolar/bronchiolar Neoplasms in Untreated Female B6C3Fl Micea 

TABLE D4 

Incidence in Controls 

Study Adenoma Carcinoma Adenoma or Carcinoma 

Historical Incidence at Lovelace Inhalation Toxicology Research Institute 

Talc 

Nickel Sulfate Hexahydrate 

Nickel Subsulfide 

Nickel Oxide 

3/46 

3/61 

3/58 

2/64 

Overall Historical Incidence in Inhalation Studies 

Range 

Standard deviation 

Total 

0%-14% 

3/1 % 

6/944 (6.5%) 

Overall Historical Incidence in Feed Studies 

Range 

Standard deviation 

Total 

0%-24% 

5.0% 

78/1,319 (5.9%) 

2/46 

4/61 

7/58 

4/64 

0%-12% 

3.2% 

38/944 (4.0%) 

0%-8% 

2.3% 

26/1,319 (2.0%) 

5/46 

7/61 

9/58 

6/64 

0%-16% 

3.7% 

971944 (10.3%) 

2%-26% 

5.3% 

102/1,319 (7.7%) 

a Data as of 17 June 1994 

\ 
Lesions in Female Mice 285 

of Nickel Subsulfidea 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Inhalation Study 

TABLE D5 

0 mg/mJ 0.6 mglm3 1.2 mglm’ 

Early deaths 

1s-Month interim evaluationb 

7-Month interim evaluationb 

Animals initially in study 

Disposition Summary 

Terminal sacrifice 

Natural deaths 

Moribund 

Animals examined microscopically 

Missexed 

Survivors 

10 

10 

80 

8 

14 

2 

36 

68 

10 

10 

80 

11 

15 

34 

70 

10 

10 

80 

9 

13 

38 

70 

Lymph node 

Hematopoietic System 

?-Month Interim Evaluation 

Lymph node, bronchial 

Pigmentation 

Lymph node, mandibular 

Hyperplasia, macrophage 

Hyperplasia, lymphoid 

Hyperplasia, lymphoid 

Hyperplasia 

(5) 

(3) 

2 (67%) 1 (33%) 

1 (33%) 

1 (20%) 

(3) 

Lung 

Respiratory System 

Interstitium, infiltration cellular 

Alveolus, pmteinosis 

Alveolus, hyperplasia, macrophage 

Inflammation, chronic active 

Olfactory epithelium, degeneration 

Olfactory epithelium, atrophy 

Nose 

1 (20%) 

1 (20%) 

(5) 

4 (80%) 

(5) 

5 (100%) 

4 (80%) 

5 (100%) 

2 (40%) 

(5) 

Urinary System 

Special Senses System 

Nervous System 

Musculoskeletal System 

Integumentary System 

Genital System 

General Body System 

Endocrine System 

Cardiovascular System 

Alimentary System 

Systems Examined With No Lesions Observed 

a Number of animals examined microscopically at the site and the number of animals with lesion 

Five animals in each exposure group were histopathologically examined at the interim evaluations. 
286 Nickel Subsulfide, NTP TR 453 

of Nickel Subsulfide (continued) 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Inhalation Study 

TABLE D5 

0 mg/m3 0.6 mg/m3 1.2 mg/m3 

Intestine large, cecum 

Alimentary System 

IS-Month Interim Evaluation 

Intestine small, ileum (5) 

(5) 

Basophilic focus 

Hyperplasia, lymphoid 

Hyperplasia 

Hyperplasia, lymphoid 

1 (20%) 

1 (20%) 

1 (20%) 

1 (20%) 

Liver (5) 

Uterus 

Clitoral gland 

Genital System 

Endometrium, hyperplasia 

Ectasia 

Hyperplasia, lymphoid 

Lymph node, bronchial 

Hematopoietic System 

Hyperplasia, macrophage 

Hyperplasia, lymphoid 

Lymph node, mesenteric 

Hyperplasia, lymphoid 

Lymph node, mandibular 

Hematopoietic cell proliferation 

Spleen (5) 

2 (40%) 

Lung 

Larynx 

Respiratory System 

Inflammation, chronic 

Interstitium, infiltration cellular 

Bronchus, hyperplasia, lymphoid 

Alveolus, proteinosis 

Alveolus, hyperplasia, macrophage 

Bronchialization 

Inflammation, chronic active 

Olfactory epithelium, degeneration 

Nose 

1 (20%) 

1 (20%) 

4 (80%) 

(5) 

(5) 

(4) 

1 (25%) 

5 (100%) 

4 (80%) 

5 (100%) 

5 (100%) 

2 (40%) 

4 (80%) 

(5) 

(5) 

5 (100%) 

4 (80%) 

5 (100%) 

5 (100%) 

5 (100%) 

5 (100%) 

(5) 

Ear 

Special Senses System 

External ear, ulcer 

(1) 

1 (100%) 
Lesions in Female Mice 287 

of Nickel Subsulfide (continued) 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Inhalation Study 

TABLE D5 

0 mg/m3 0.6 mg/mJ 1.2 mg/m3 

Urinary System 

Nervous System 

Musculoskeletal System 

Integumentary System 

General Body System 

Endocrine System 

Cardiovascular System 

Systems Examined With No Lesions Observed 

15-Month Interim Evaluation (continued) 

2-Year Study 

Alimentary System 

Intestine small, duodenum 

Intestine large, cecum 

Gallbladder 

Atrophy 

Lymphoid tissue, hyperplasia, lymphoid 

Epithelium, hyperplasia 

Atrophy 

Atrophy 

Inflammation 

Peyer’s patch, hyperplasia, lymphoid 

Pigmentation, hemosiderin 

Necrosis 

Mixed cell focus 

Inflammation 

Infiltration cellular, lymphocyte 

Infarct 

Fibrosis 

Eosinophilic focus 

Degeneration 

Cyst 

Basophilic focus 

Atrophy 

Angiectasis 

Artery, inflammation 

Necrosis 

Inflammation 

Mesentery 

Acinus, atrophy 

Inflammation 

Infiltration cellular, lymphocyte 

Cyst 

Atrophy, focal 

Intestine small, ileum 

Intestine small, jejunum 

Liver 

Pancreas 

(53) 

(54) 

(53) 

1 (2%) 

1 (2%) 

1 (2%) 

(54) 

(54) 

1 (2%) 

1 (2%) 

(58) 

3 (i%) 

1 (2%) 

1 (2%) 

4 (7%) 

2 (3%) 

1 (2%) 

1 (2%) 

2 (3%) 

(3) 

1 (33%) 

2 (3%) 

1 (2%) 

2 (3%) 

1 (33%) 

(58) 
288 Nickel Subsulfide, NTP TR 453 

of Nickel Subsulfide (continued) 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Inhalation Study 

TABLE D5 

0 mg/m3 0.6 mg/m3 1.2 mg/m3 

Stomach, forestomach 

Alimentary System (continued) 

%Year sf" (continued) 

Inflammation 

Hyperplasia, squamous 

Hyperplasia, focal, squamous 

Inflammation 

Developmental malformation 

Inflammation 

Tooth 

Stomach, glandular 

2 (4%) 

(58) 

1 (2%) 

Heart 

Cardiovascular System 

Atrium, thrombosis 

Artery, inflammation 

Inflammation 

Cardiomyopathy 

2 (3%) 

1 (2%) 

1 (2%) 

(60) 

1 (2%) 

(59) 

1 (2%) 

Endocrine System 

Hyperplasia, focal 

Hyperplasia 

Hematopoietic cell proliferation 

Degeneration 

Atrophy 

Angiectasis 

Adrenal cortex 

Pituitary gland 

Hyperplasia 

Angiectasis 

Adrenal medulla 

Inflammation 

Pars distalis, hyperplasia 

Angiectasis 

Follicular cell, hyperplasia 

Follicle, atrophy 

Artery, inflammation 

cyst 

Thyroid gland 

(58) 

1 (2%) 

(58) 

1 (2%) 

20 (31%) 

1 (2%) 

1 (2%) 

28 (47%) 

(60) 

1 (2%) 

1 (2%) 

3 (5%) 

1 (2%) 

2 (3%) 

37 (62%) 

Thoracic, inflammation 

Tissue NOS 

General Body System 
Lesions in Female Mice 289 

of Nickel Subsulfide (continued) 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Inhalation Study 

TABLE D5 

0 mg/m3 0.6 mglm’ 1.2 mg/m’ 

Clitoral gland 

Genital System 

&Year Study (continued) 

Inflammation 

Ectasia 

Atrophy 

Periovarian tissue, cyst 

Hemorrhage 

cyst 

Abscess 

Ovary 

Endometrium, hyperplasia 

Inflammation 

Cyst, multiple 

cyst 

Angiectasis 

Abscess 

Uterus 

(55) 

2 (4%) 

14 (24%) 

(59) 

2 (3%) 

1 (2%) 

1 (2%) 

8 (14%) 

1 (2%) 

(58) 

(60) 

1 (2%) 

19 (32%) 

1 (2%) 

2 (3%) 

3 (5%) 

19 (32%) 

3 (5%) 

3 (5%) 

Bone marrow 

Hematopoietic System 

Myeloid cell, hyperplasia 

Myeloid cell, atrophy 

Erythroid cell, hyperplasia 

Inflammation 

Renal, inflammation, granulomatous 

Renal, inflammation 

Renal, hyperplasia, lymphoid 

Pancreatic, inflammation 

Pancreatic, hyperplasia 

Inguinal, inflammation 

Inguinal, hyperplasia, lymphoid 

Iliac, hyperplasia, lymphoid 

Inflammation, granulomatous 

Inflammation 

Hyperplasia, macrophage 

Hyperplasia, lymphoid 

Lymph node, bronchial 

Inflammation 

Hyperplasia, plasma cell 

Hyperplasia, lymphoid 

Lymph node, mandibular 

Inflammation 

Hyperplasia, lymphoid 

Ectasia 

Lymph node, mesenteric 

Inflammation 

Hyperplasia, lymphoid 

Lymph node, mediastinal 

Lymph node 

(57) 

1 (2%) 

7 (12%) 

(9) 

1 (11%) 

1 (11%) 

1 (11%) 

(50) 

(57) 

1 (2%) 

10 (20%) 

1 (2%) 

2 (4%) 

18 (32%) 

3 (5%) 

4 (7%) 

1 (2%) 

1 (7%) 

(55) 

(15) 

1 (8%) 

1 (8%) 

2 (17%) 

1 (8%) 

1 (2%) 

1 (2%) 

44 (77%) 

46 (81%) 

1 (2%) 

7 (12%) 

(57) 

(59) 

1 (2%) 

3 (27%) 

1 (9%) 

1 (9%) 

(59) 

47 (80%) 

52 (88%) 

(60) 

4 (7%) 

(57) 

1 (3%) 

5 (17%) 

2 (4%) 

6 (11%) 

(30) 
290 Nickel Subsulfide, NTP TR 453 

of Nickel Subsulfide (continued) 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Inhalation Study 

TABLE D5 

0 mg/m3 0.6 mg/m3 1.2 mg/m3 

Spleen 

Hematopoietic System (continued) 

2bYeut Study (continued) 

Red pulp, atrophy 

Lymphoid follicle, atrophy 

Inflammation 

Hyperplasia, lymphoid 

Hematopoietic cell proliferation 

Atrophy 

Hyperplasia, lymphoid 

Hyperplasia 

Degeneration 

Thymus 

(58) 

1 (2%) 

2 (3%) 

1 (2%) 

17 (29%) 

14, (24%) 

2 (3%) 

8 (14%) 

(58) 

(60) 

27 (45%) 

15 (25%) 

1 (2%) 

5 (9%) 

1 (2%) 

(57) 

2 (4%) 

(60) 

19 (32%) 

9 (15%) 

1 (2%) 

(54) 

8 (15%) 

Integumentary System 

Inflammation 

Hyperplasia, cystic 

Hyperplasia 

Mammary gland 

Pinna, inflammation 

Pinna, hyperkeratosis 

Pinna, cyst epithelial inclusion 

Inflammation 

Hemorrhage 

Fibrosis 

Skin 

(57) 

1 (2%) 

8 (14%) 

(58) 

1 (2%) 

3 (5%) 

2 (3%) 

(59) 

11 (19%) 

1 (2%) 

(60) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

Bone 

Musculoskeletal System 

Tibia, fracture 

Maxilla, hyperostosis 

Femur, hyperostosis 

Inflammation 

Hyperostosis 

(57) 

10 (18%) 

1 (2%) 

1 (2%) 

1 (2%) 

9 (15%) 

(59) 

1 (2%) 

10 (17%) 

1 (2%) 

1 (2%) 

Thalamus, degeneration, focal 

Brain 

Nervous System 

Degeneration 

Abscess 

Spinal cord 

Larynx 

Respiratory System 

Epithelium, hyperplasia 

Inflammation 

(54) (55) 

1 (2%) 

1 (2%) 

(53) 
Lesions in Female Mice 291 

of Nickel Subsulfide (continued) 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Inhalation Study 

TABLE D5 

Lung 

Respiratory System (continued) 

&Year Study (continued) 

Interstitium, inflammation 

Interstitium, infiltration cellular 

Bronchus, hyperplasia, lymphoid 

Alveolus, proteinosis 

Alveolus, hyperplasia, macrophage 

Bronchializtion 

Inflammation, chronic active 

Inflammation, acute 

Fibrosis 

Bronchiectasis 

Respiratory epithelium, degeneration 

Olfactory epithelium, degeneration 

Olfactory epithelium, atrophy 

Inflammation, acute 

Nose 

5 (9%) 

3 (5%) 

1 (2%) 

1 (2%) 

19 (33%) 

34 (59%) 

(58) 

1 (2%) 

26 (45%) 

1 (2%) 

(59) 

53 (90%) 

26 (44%) 

54 (92%) 

57 (97%) 

53 (90%) 

46 (78%) 

1 (2%) 

7 (12%) 

1 (2%) 

(59) 

3 (5%) 

11 (19%) 

11 (19%) 

17 (28%) 

58 (97%) 

34 (57%) 

59 (98%) 

60 (100%) 

58 (97%) 

58 (97%) 

(60) 

12 (20%) 

41 (68%) 

14 (23%) 

Ear 

Special Senses System 

Cataract 

External ear, inflammation 

Inflammation 

EY e 

(2) 

1 (50%) 

1 (50%) 

1 (50%) 

(2) 

Urinary System 

Renal tubule, pigmentation, hemosiderin 

Pelvis, inflammation 

Glomerulus, inflammation 

Nephropathy 

Metaplasia, osseous 

Inflammation 

Infiltration cellular, lymphocyte 

Infarct 

Hydronephrosis 

Kidney 

Infiltration cellular, lymphocyte 

Urinary bladder 

1 (2%) 

9 (16%) 

1 (2%) 

5 (9%) 

(56) 

1 (2%) 

(60) 

1 (2%) 

12 (20%) 

2 (3%) 

(55) 

3 (5%) 

1 (2%) 

1 (2%) 

1 (2%) 

2 (3%) 

1 (2%) 

(59) 
292 Nickel Subsulfide, NTP TR 453 
293 

GENETIC TOXICOLOGY 

APPENDIX E 

SALMONELL4 TYPHlMURlUM MUTAGENICITY TEST PROTOCOL .......................... 294 

TABLE E2 Frequency of Micronuclei in Mouse Peripheral Blood Erythrocytes 

TABLE El Mutagenicity of Nickel Subsulfide in Salmonella typhimurium 

MOUSE PERIPHERAL BLOOD MICRONUCLEUS TEST PROTOCOL ......................... 

300 

296 

294 

RESULTS ........................................................... 295 

................. 

Following Treatment with Nickel Subsulfide by Inhalation for 13 Weeks .......... 
294 Nickel Subsulfide, NTP TR 453 

GENETIC TOXICOLOGY 

incubation at 37" C. All tests were repeated using either the same or different S9 concentrations. 

plates. Histidine-independent mutant colonies arising on these plates were counted following 2 days 

added, and the contents of the tubes were mixed and poured onto the surfaces of minimal glucose agar 

Syrian hamster liver) for 20 minutes at 37" C. Top agar supplemented with Z-histidine and d-biotin was 

(metabolic activation enzymes and cofactors from Aroclor 1254-induced male Sprague-Dawley rat or 

typhimunum tester strains TA97, TA98, TA100, TA102, and TA1535, either in buffer or S9 mix 

as a coded aliquot from Radian Corporation (Austin, TX). It was incubated with the Salmonella 

Testing was performed as reported by Zeiger et ai. (1992). Nickel subsulfide was sent to the laboratory 

SALMONELLA TYPHZMURZUM MUTAGENICITY TEST PROTOCOL 

five doses of nickel subsulfide. In the absence of toxicity, 10,000 pglplate was selected as the high dose. 

Each trial consisted of triplicate plates of concurrent positive and negative controls and of at least 

percentage or fold increase required for a chemical to be judged positive or weakly positive. 

increase in revertant colonies was observed following chemical treatment. There was no minimum 

magnitude to support a determination of mutagenicity. A negative response was obtained when no 

defined as an increase in revertants that is not dose related, not reproducible, or is of insufficient 

independent (revertant) colonies in any one strain/activation combination. An equivocal response is 

In this assay, a positive response is defined as a reproducible, dose-related increase in histidinedose group. The criteria of Schmid (1976) were used to define micronuclei. 

coded. From 6,400 to 7,400 normochromatic erythrocytes (NCEs) were scored in up to 10 animals per 

a chromatin-specific fluorescent dye mixture of Hoechst 33258lpyronin Y (MacGregor et aZ., 1983) and 

were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with 

toxicity study, peripheral blood samples were obtained from male and female B6C3F, mice and smears 

A detailed discussion of this assay is presented in MacGregor et ai. (1990). At the end of the 13-week 

MOUSE PERIPHERAL BLOOD MICRONUCLEUS TEST PROTOCOL 

reproducibility of any effects observed, and the magnitudes of those effects. 

the final call is determined by the scientific staff after considering the results of statistical analyses, 

micronucleus induction is preferably based on reproducibly positive trials (as noted above). Ultimately, 

less than or equal to 0.025/N where N equals the number of exposure groups. A final call of positive for 

1) the trend test P value is less than or equal to 0.025 or 2) the P value for any single exposure group is 

proportion to the excess variation. In the micronucleus test, an individual trial is considered positive if 

by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in 

and the control group (Margolin et al., 1990). In the presence of excess binomial variation, as detected 

one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each exposure group 

analyzed by a statistical software package that tested for increasing trend over exposure groups using a 

plus or minus the standard error of the mean. The frequency of micronucleated cells among NCEs was 

The results were tabulated as the mean of the pooled results from all animals within a treatment group, 
Genetic Toxicology 295 

micronucleated NCEs in peripheral blood samples (Table E2). 

by inhalation for 13 weeks. Nickel subsulfide did not induce an increase in the frequency of 

Nickel subsulfide was also tested for induction of micronuclei in NCEs of male and female mice exposed 

mutagenic responses were observed in strains TA97, TA98, TA102, or TA1535, with or without S9. 

with the most consistent demonstration of mutagenicity noted in the presence of 10% rat liver S9. No 

strain TA100. Varied indications of mutagenic activity were observed with and without S9 in TA100, 

(Table El). Results of these tests were considered to be equivocal overall due to the response seen in 

Nickel subsulfide was tested for induction of gene mutations in five strains of SuZmoneZZu typhimurium 

RESULTS 
Nickel Subsulfide, NTF' TR 453 

Mutagenicity of Nickel Subsulfide in Salmonella typhimuriuma 

TABLE El 

~~~ ~~ 

+hamster S9 

Revertants/plateb 

Strain Dose -s9 +rat S9 

(pglplate) Trial 1 Trial 2 10% 30% 10% 30% 

TA102 0 227 f 4.2 161 f 13.6 211 f 8.7 249 f 2.5 207 f 12.7 312 f 30.0 

100 230 f 14.3 148 f 3.2 224 f 11.2 234 f 11.2 212 f 5.9 351 f 15.4 

333 236 f 19.8 160 f 18.4 220 f 12.5 276 * 14.6 197 f 13.8 338 f 5.5 

1,000 210 f 10.7 154 f 3.1 209 f 13.8 281 f 16.7 196 f 8.2 352 f 12.2 

3,333 151 f 4.7' 163 f 2.6' 210 f 2.1' 279 f 15.8 198 f 16.7' 321 f 15.5' 

10,000 154 f 3.8' 151 f 2.6' 233 f 5.0' 300 f 5.8 250 f 8.1' 350 f 17.9' 

Trial summary Negative Negative Negative Negative Negative Negative 

Positive controld 813 f 13.5 737 f 7.8 534 f 59.0 926 f 74.0 1,110 f 17.2 1,236 f 18.7 

Revertantdplate 

Trial 1 Trial 2 Trial 3 

-s9 +5% hamster S9 

Trial 1 Trial 2 Trial 3 

TAlOO 

333 

100 

0 

10,000 

6,666 

3,333 

1.666 

1,000 

Positive control 

Trial summary 

131 f 3.2 111 f 2.8 143 f 17.3 

110 f 7.5 111 f 12.5 

132 f 11.6 131 f 8.6 132 f 9.5 

127 f 6.1 145 f 8.8 162 f 3.5 

126 f 6.9' 183 f 9.1' 178 f 5.8 

199 * 4.4 

98 f 5.5' 156 f 9.0' 184 f 11.7 

Negative Weak positive Equivocal 

429 f 7.8 390 f 20.3 295 f 7.5 

147 f 5.2' 133 f 2.0' 

129 f 5.5' 

155 f 7.9' 136 f 4.4' 131 f 10.0' 

152 f 6.0 

122 f 14.0 138 f 7.3 128 f 6.1 

110 f 2.7 120 f 13.4 120 f 10.7 

105 f 17.0 111 * 11.2 

100 f 5.7 109 rt 15.4 145 f 13.9 

Equivocal Equivocal Negative 

922 f 44.3 897 f 57.6 1.270 rf: 30.7 

Revertantshlate 

Trial 1 Trial 2 Trial 3 

10% +hamster S9 

Trial 1 Trial 2 Trial 3 

30% 

10.000 

6,666 

3,333 

1.666 

1,000 

333 

(continued) 100 

TAlOO 0 

Positive control 

Trial summary 

102 f 7.2 147 f 6.4 137 f 13.1 

128 f 8.6 129 f 8.2 134 f 9.3 

121 f 2.7 130 f 3.5 147 * 3.1 

129 f 6.9 136 f 7.3 160 f 4.6 

140 f 3.5' 126 f 13.7' 171 f 5.8' 

144 f 1.9' 146 f 1.5' 154 f 7.5' 

Equivocal Negative Negative 

577 f 10.9 760 f 22.6 764 f 17.2 

124 f 4.7 127 + 12.8 126 * 15.3 

125 f 5.3 130 f 3.9 146 f 5.1 

137 f 5.8 132 2.5 141 * 3.2 

164 f 7.2 146 rt 7.9 129 + 21.5 

160 * 10.6' 131 f 6.7' 151 + 4.4' 

137 f 7.2' 

142 f 2.3' 147 f 10.2' 141 f 11.9' 

157 * 2.7 

Equivocal Negative Negative 

462 f 31.2 457 f 22.2 703 f 13.1 
Genetic Toxicology 297 

Mutagenicity of Nickel Subsulfide in Salmonella typhimurium (continued) 

TABLE El 

Revertantslplate 

(Irglplate) 

Strain Dose 

Trial 1 Trial 2 Trial 3 

+5% rat S9 

10.000 

3,333 

1.000 

333 

(continued) 100 

TAlOO 0 

Positive control 

Trial summary 

10,000 

6,666 

3,333 

1,000 

333 

(continued) 100 

TAlOO 0 

Positive control 

Trial summary 

10.000 

3,333 

1,000 

333 

(continued) 100 

TAlOO 0 

Positive control 

Trial summary 

125 * 5.8' 145 f 8.4' 

141 f 8.9' 151 f 13.0' 152 f 5.2' 

133 f 2.1 153 f 8.2 156 f 2.8 

132 f 4.7 131 f 9.3 144 f 5.7 

125 f 16.5 138 f 2.4 

104 f 3.8 119f 11.1 154 f 5.8 

Negative Equivocal Negative 

909 f 16.2 1,011 f 25.0 940 f 11.2 

Revertantdplate 

+lo% rat S9 

Trial 1 Trial 2 Trial 3 Trial 4 

145 f 2.1' 154f 5.3' 162 f 7.2' 185 f 3.4 

134 f 9.8 152 f 6.1 163 f 8.2 151 f 6.6 

129 f 4.3 139 f 4.8 149 f 1.5 143 f 4.7 

110 f 6.8 136 f 2.6 130 f 12.8 

124 f 11.0 140 f 15.4 122 f 14.4 132 f 9.3 

147 f 8.5' 146 f 12.1' 187 f 11.4' 188 f 7.3 

. 187 f 10.0 

Negative Negative Weak positive Weak positive 

556 f 32.4 589 f 41.9 441 f 9.8 585 f 32.7 

Revertantdplate 

Trial 1 Trial 2 Trial 3 

+30% rat S9 

140 f 9.9 141 f 9.1 133 f 13.9 

131 f 19.6 131 f 11.1 112 f 6.7 

161 f 4.6 141 f 12.3 140 f 16.0 

202 f 19.0 152 f 4.9 122 * 3.8 

196 f 9.0' 159 f 3.7' 146 f 8.4' 

180 f 6.1' 164 f 20.4' 168 f 9.2' 

Equivocal Negative Negative 

244 f 6.7 521 f 412 626 f 17.3 
298 Nickel Subsulfide, NTP TR 453 

Mutagenicity of Nickel Subsulfide in Salmonella typhimurium (continued) 

TABLE El 

Revertantdplate 

Strain Dose 

Trial 1 

-s9 +hamster S9 

(&plate) 5% 10% 30% 

333 

100 

TA1535 0 

10,000 

3,333 

1,000 

Positive control 

Trial summary 

10,000 

3,333 

1,000 

333 

(continued) 100 

TA1535 0 

Positive control 

Trial summary 

TA97 

333 

100 

0 

10,000 

3,333 

1.000 

Positive control 

Trial summary 

7 f 1.9 

14 f 2.5 

17 f 0.9. 

16 f 2.0 

16 f 0.6 

21 f 1.2 

9 f 1.9' 

8 f 0.9' 8 f 0.3' 

6 f 0.3 11 f 1.5 

7 f 0.7 10 * 0.9 

8 f 1.2 10 f 0.6 

8 f 1.9 9 f 1.8 

11 f 1.8' 

719 f 54.9 

Negative Negative Negative 

131 f 4.8 119 f 4.8 

Revertantdplate 

Trial 1 Trial 2 Trial 3 

+30% rat S9 

13 f 1.9 14 f 2.5 14 f 0.3 

11 f 1.3 9 f 0.6 21 f 3.7 

7 f 0.3 9 f 2.2 11 f 1.9 

10 f 1.5 11 f 3.2 15 f 0.7 

12 f 2.5' 10 f 0.6' 14 f 1.8' 

9 f 2.6' 7 f 1.2' 14 f 1.5' 

Negative Negative Negative 

181 f 18.8 163 f 7.8 144 f 3.5 

9 f 2.0 

6 f 1.2 

7 f 1.5 

11 f 1.5 

10 f 0.9' 

14 f 2.0' 

296 f 10.5 

Negative 

Revertantdplate 

-s9 +lo% hamster S9 +lo% rat S9 

Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2 

162 f 5.0 

176 f 3.3 

173 f 5.5 

169 f 11.1 

150 5.8 

153 & 7.1 

497 f 34.3 

Negative 

158 f 6.6 

163 f 8.7 

161 f 5.5 

171 f 2.3 

182 f 9.1' 

174 f 6.0 

374 f 4.7 

Negative 

157 f 10.7 

165 f 13.8' 

158 f 2.0 

153 f 7.8 

153 f 9.5 

147 f 6.0 

541 f23.7 

Negative 

149 f 15.6 

164 * 8.7' 

154 f 9.0 

164 f. 8.4 

145 f 10.2 

152 f 4.5 

554 f 16.8 

Negative 

176 f 5.3 

174 f 0.9' 

153 f 7.5 

171 f 7.8 

169 f 4.4 

164 f 6.4 

324 f 10.1 

Negative 

172 f 9.2 

165 -+ 19.1' 

188 * 12.1 

159 f 17.9 

182 f 15.1 

188 f 6.1 

343 f 13.5 

Negative 
Genetic Toxicology 299 

Mutagenicity of Nickel Subsulfide in Salmonella typhimurium (continued) 

TABLE El 

Revertantslplate 

Strain Dose -s9 +hamster S9 +rat S9 

(&plate) Trial 1 Trial 2 10% 30% 10% 30% 

TA98 

333 

100 

0 

10,000 

3,333 

1.000 

Positive control 

Trial summary 

24 f 3.5 

22 f 3.9 

22 f 1.5 

20 f 1.7 

15 f 1.8' 

372 f 12.5 

Negative 

8 f 3.5 15 f 0.7' 

20 f 2.3' 

29 f 4.4 

28 f 1.8 

15 f 0.3 

16 f 1.5 

521 f 13.1 

Negative 

24 f 2.6 

22 f 1.0 

20 f 0.9 

23 f 2.6 

29 f 1.9' 

27 f 3.5' 

614 f 29.2 

Negative 

40 f 1.5 

28 f 2.7 

32 f 3.3 

42 f- 7.9 

32 f 3.1' 

27 f 4.5' 

495 f 21.7 

Negative 

24 f 2.7' 

23 f 1.8' 

30 f 1.2 

25 f 3.5 

26 f 1.2 

31 f 1.3 

286 f 10.7 

Negative 

32 f 0.6 

30 f 1.8 

31 f 2.9 

21 f 2.1 

27 f 0.7' 

23 f 4.7' 

104 f 5.9 

Negative 

2-aminoanthracene. 

4-nitro-0-phenylenediamine (TA98). mitomycin C (TA102). The positive control for metabolic activation with all strains was 

The positive controls in the absence of metabolic activation were sodium azide (TA100 and TA1535). 9-aminoacridine (TA97), 

Precipitate on plate 

Revertants are presented as mean f standard error from three plates. 

solvent control. 

Study performed at SRI, International. The detailed protocol and these data are presented in Zeiger et al. (1992); 0 pglplate dose is the 
300 Nickel Subsulfide, NTP TR 453 

with Nickel Subsulfide by Inhalation for 13 Weeksa 

Frequency of Micronuclei in Mouse Peripheral Blood Erythrocytes Following Treatment 

TABLE E2 

Erythrocytes/1,000 Cellsb 

Normochromatic 

Micronucleated 

of Mice 

Number 

Male 

Trend test 

Female 

Trend test 

Trend test 

Urethaned 

1.2 

0.6 

0 

1.2 

0.6 

0 

0.2 

1.07 * 0.13 

1.11 f 0.18 

1.14 * 0.15 

P=O.634' 

0.66 * 0.14 

0.81 * 0.14 

0.66 0.14 

P=O.522 

P=0.000* 

16.76 f 0.73 

10 

10 

10 

10 

10 

8 

3 

Urethane was used as the positive control and is presented as parts per million. 

Significance of percent micronucleated NCE determined by a one-tailed trend test. 

Data presented as mean rt standard error. NCE = normochromatic erythrocyte. 

scored per animal. 

Slides scored at SRI, International. The detailed protocol is presented in MacGregor et n1..(1990); a minimum of 6.400 NCEs were 

P<O.OOl 
301 

AND ORGAN-WEIGHT-TO-BODY-WEIGHT RATIOS 

ORGAN WEIGHTS 

APPENDIX F 

TABLE F3 

TABLE F2 

TABLE F1 

TABLE F4 

TABLE F7 

TABLE F6 

TABLE F5 

TABLE F'S 

of Nickel Subsulfide 

at the 15-Month Interim Evaluation in the 2-Year Inhalation Study 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice 

of Nickel Subsulfide 

at the 7-Month Interim Evaluation in the 2-Year Inhalation Study 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice 

in the 13-Week Inhalation Study of Nickel Subsulfide 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice 

in the 16-Day Inhalation Study of Nickel Subsulfide 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice 

of Nickel Subsulfide 

at the 15-Month Interim Evaluation in the 2-Year Inhalation Study 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats 

of Nickel Subsulfide 

at the 7-Month Interim Evaluation in the 2-Year Inhalation Study 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats 

in the 13-Week Inhalation Study of Nickel Subsulfide 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats 

in the 16-Day Inhalation Study of Nickel Subsulfide 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats 

....................... 

303 

302 

...................... 

........................................... 304 

........................................... 

307 

306 

305 

....................... 

...................... 

........................................... 309 

........................................... 310 
302 Nickel Subsulfide, NTP TR 453 

of Nickel Subsulfidea 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in the 16-Day Inhalation Study 

TABLE F1 

0 mg/m3 0.6 mg/m3 1.2 mg/m3 2.5 mg/m3 5 mg/m3 10 mg/m3 

Brain 

Necropsy body wt 

Male 

n 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Heart 

R. Kidney 

Liver 

Lung 

Testes 

Thymus 

Brain 

Necropsy body wt 

Female 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Heart 

R. Kidney 

Liver 

Lung 

Thymus 

5 

216 f 13 

8.33 f 0.59 

1.767 f 0.027 

1.43 f 0.16 

0.315 f 0.044 

6.05 f 0.58b 

1.253 f 0.048b 

5.32 f 0.42 

1.130 f 0.052 

53.45 f 4.10 

11.711 f 1.401 

3.90 f 0.51 

0.831 f 0.107 

3.52 f 0.11 

0.753 f 0.028 

151 f 4 

3.63 0.12 

0.549 f 0.030 

10.49 f 0.29 

1.580 f 0,042 

48.96 f 2.82 

7.399 f 0.508 

3.80 f 0.10 

0.573 f 0.019 

1.80 f 0.15b 

0.272 f 0.015b 

5.43 f 0.31 

0.821 f 0.060 

5 

236 f 4 

5.32 f 0.12 

1.253 f 0.017 

5.97 f 0.31 

1.406 f 0.067 

52.29 f 3.48 

12.304 f 0.722 

3.94 f 0.15 

0.929 f 0.033 

3.41 f 0.20 

0.802 f 0.040 

7.45 f 0.13 

1.755 f 0.021 

1.52 f 0.11 

0.358 f 0.023 

149 f 4 

46.76 f 1.69 

7.005 f 0.427 

4.09 f 0.06 

0.610 f 0.015 

3.75 f 0.12 

0.558 f 0.009 

11.14 f 0.24 

1.659 f 0.017 

7.58 f 0.54** 

1.124 f 0.056** 

2.20 f 0.11 

0.330 f 0.024 

5 

226 f 6 

56.28 f 1.44b 

12.872 f 0.414b 

4.09 f 0.14 

0.923 f 0.040 

3.73 f 0.18 

0.842 f 0.044 

8.01 f 0.22 

1.802 f 0.013 

5.88 f 0.14 

1.323 f 0.019 

7.11 f 1.05 

1.602 f 0.244* 

1.69 f 0.17 

0.383 f 0.045 

147 f 5 

45.01 f 1.27 

6.636 f 0.408 

4.18 f 0.12* 

0.614 f 0.029 

3.77 f 0.21 

0.555 f 0.040 

11.39 f 0.26 

1.669 f 0.040 

7.62 f 0.41** 

1.115 f 0.044** 

1.97 f 0.09 

0.290 f 0.019 

5 

198 f 6 

4.30 f 0.10 

0.849 f 0.030 

3.80 f 0.08 

0.750 f 0.022 

8.78 f 0.23 

1.731 f 0.024 

54.80 f 2.23 

10.796 f 0.312 

8.06 f 0.11** 

1.593 f 0.054* 

1.61 f 0.12 

0.318 f 0.023 

6.11 f 0.05 

1.208 f 0.032 

138 f 1* 

47.92 f 1.54 

6.587 f 0.192 

4.19 f 0.09* 

0.576 f 0.014 

3.85 f 0.06 

0.530 f 0.012 

11.82 f 0.11* 

1.625 f 0.008 

9.86 * 0.33** 

1.356 f 0.047** 

2.33 f 0.26 

0.320 f 0.036 

5 5 

155 f 8** 113 f 5** 

1.679 f 0.054*b 1.657 f 0.016** 

11.14 f 0.85**b 14.76 f 0.51** 

0.770 f 0.035 0.644 f 0.036 

5.03 f 0.40** 5.75 f 0.43** 

0.708 f 0.052 0.611 f 0.026* 

4.56 f 0.18 5.46 f 0.38** 

7.699 f 0.552** 4.916 f 0.472** 

49.49 f 1.17 43.32 f 3.11* 

1.820 f 0.120** 1.536 f 0.033** 

11.84 f 0.86** 13.68 * 0.49** 

1.166 f 0.048 0.813 f 0.029** 

7.54 f 0.12** 7.21 f 0.13** 

0.184 f 0.045* 0.056 f 0.007** 

1.15 f 0.25 0.49 f 0.04** 

117 f 6** 86 f 1** 

0.569 f 0.023 0.473 It 0.014 

4.87 f 0.07** 5.53 f 0.17** 

0.511 f 0.030 0.458 f 0.019** 

4.37 f 0.17** 5.35 f 0.17** 

5.661 f 0.585** 3.682 f 0.140** 

47.87 f 2.51 42.99 f 1.31 

1.422 f 0.049** 1.253 f 0.050** 

12.26 f 0.74** 14.64 f 0.54** 

0.243 f 0.047 0.100 f 0.013** 

2.02 f 0.30 1.17 f 0.15 

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weightlg body weight 

** P10.01 

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

n=4 

(mean f standard error). 
Organ Weight Analyses 303 

of Nickel Subsulfidea 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in the 13-Week Inhalation Study 

TABLE F2 

0 mg/m3 0.15 mg/m3 0.3 mg/m3 0.6 mg/m3 1.2 mg/m3 2.5 mg/m3 

Brain 

Necropsy body wt 

n 

Male 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Heart 

R. Kidney 

Liver 

Lung 

R. Testis 

Thymus 

Brain 

Necropsy body wt 

n 

Female 

Relative 

Absolute 

Heart 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

R. Kidney 

Liver 

Lung 

Thymus 

10 

352 f 4 

0.71 f 0.03 

0.251 f 0.013 

4.09 f 0.11 

1.438 f 0.035 

3.78 f 0.06 

1,330 f 0.015 

35.95 f 0.41 

12.670 f 0.240 

3.28 f 0.05 

1.156 f 0.024 

2.95 f 0.11 

1.040 f 0.040 

5.49 f 0.06 

1.933 f 0.008 

10 

202 f 3 

8.85 f 0.09 

1.786 f 0.020 

0.673 f 0.029 

3.36 f 0.07 

0.678 f 0.019 

3.33 f 0.14 

5.01 f 0.16 

1.010 f 0.028 

33.95 f 0.67 

6.860 f 0.189 

1.08 f 0.06 

0.218 f 0.015 

10 10 

354 f 5 339 f 4 

1.909 f 0.014 1.917 f 0.016 

5.40 f 0.07 5.66 f 0.08 

1.010 f 0.051 1.030 f 0.033 

2.84 f 0.11 3.04 f 0.11 

1.153 f 0.023 1.110 f 0.024 

3.26 f 0.06 3.28 f 0.06 

12.830 f 0.213 12.040 f 0.225 

36.27 f 0.45 

4.92 f 0.12** 

1.740 f 0.052** 

4.18 f 0.05 

1.478 f 0.032 

0.64 f 0.03 

0.225 f 0.011 

10 

204 f 4 

8.76 f 0.14 

1.785 f 0.019 

0.681 f 0.023 

3.34 f 0.11 

3.33 f 0.06 

0.680 f 0.015 

34.41 f 0.63 

7.020 f 0.170 

1.290 f 0.023** 

6.33 f 0.12** 

0.99 f 0.06 

0,201 f 0.010 

5.40 f 0.07** 

1.830 f 0.037** 

35.52 f 0.43 

0.72 f 0.04 

0.244 f 0.013 

4.34 f 0.07 

1.470 f 0.023 

205 f 4 

10 

3.44 0.05 

0.704 f 0.012 

3.24 f 0.10 

0.662 f 0.023 

8.80 f 0.20 

1.795 f 0.018 

33.14 f 0.58 

6.790 f 0.188 

6.80 f 0.19** 

1.390 f 0.035** 

0.97 f 0.03 

0.199 f 0.007 

9 10 

328 f 6 344 f 4 

1.906 f 0.025 1.934 f 0.016 

5.82 f 0.12 5.63 f 0.03 

0.998 f 0.034 0.949 f 0.028 

3.07 f 0.13 2.76 f 0.07 

1.112 f 0.019 1.122 f 0.019 

3.37 f 0.07 3.27 f 0.04 

11.430 f 0.277** 11.890 f 0.159** 

35.01 f 0.52 

7.01 f 0.29** 

2.300 f 0.067** 

4.22 f 0.08 

1.385 f 0.020 

0.71 f 0.03 

0.232 f 0.010 

10 

199 f 2 

9.02 f 0.09 

1.797 f 0.015 

3.28 f 0.09 

0.655 f 0.021 

3.48 f 0.09 

0.693 0.019 

33.12 f 0.66 

6.610 f 0.177 

9.13 f 0.22** 

1.820 f 0.044** 

1.00 f 0.04 

0.200 f 0.008 

7.66 f 0.10** 

2.630 f 0.030** 

34.62 f 0.41 

0.71 f 0.04 

0.246 f 0.015 

4.39 f 0.04* 

1.507 f 0.018 

196 f 4 

10 

9.25 f 0.14 

1.810 f 0.014 

33.40 f 0.73 

6.550 f 0.177 

3.48 f 0.04 

0.683 f 0.013 

3.32 f 0.08 

0.650 f 0.019 

9.44 f 0.14** 

1.850 f 0.037** 

0.98 f 0.04 

0.192 f 0.007 

10 

335 f 11 

5.90 * 0.20* 

1.958 f 0.034 

3.15 f 0.11 

1,051 f 0.044 

3.39 f 0.06 

1.132 f 0.036 

11.580 f 0.387** 

34.69 f 0.82 

7.25 f 0.11** 

2.420 f 0.068** 

1.477 f 0.026 

4.45 f 0.13** 

0.64 f 0.02 

0.215 f 0.007 

10 

194 * 2 

9.41 f 0.17* 

1.817 f 0.014 

3.40 f 0.08 

0.657 f 0.016 

3.56 f 0.06 

0.688 f 0.012 

32.57 f 0.82 

6.300 f 0.167* 

9.36 f 0.20** 

1.810 f 0.038** 

1.01 f 0.05 

0.195 f 0.009 

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weighdg body weight 

** P50.01 

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

(mean f standard error). 
304 Nickel Subsulfide, NTP TR 453 

in the 2-Year Inhalation Study of Nickel Subsulfidea 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats at the 7-Month Interim Evaluation 

TABLE F3 

O mg/m3 0.15 mg/m3 1 mglm’ 

n 5 5 5 

Male 

Necropsy body wt 391 f 9 397 f 12 365 f 6 

Brain 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

R. Kidney 

Liver 

Lung 

Spleen 

Thymus 

5.29 f 0.09 

2.066 f 0.026 

5.02 f 0.11 

1.988 & 0.032 

5.47 f 0.07 

1.994 f 0.034 

3.54 f 0.11 

1.382 f 0.032 

3.27 f 0.02* 

1.298 f 0.041 

3.49 f 0.05 

1.274 f 0.029 

33.00 f 0.63 

12.920 f 0.454 

34.24 f 0.32 

13.584 f 0.426 

36.60 f 1.33* 

13.372 f 0.692 

4.79 f 0.14 

1.874 f 0.078 

5.98 f 0.18** 

2.376 f 0.108** 

9.55 f 0.22** 

3.478 f 0.050** 

1.99 f 0.03 

0.776 f 0.010 

1.96 f 0.06 

0.774 f 0.018 

2.13 * 0.05 

0.776 f 0.025 

0.58 f 0.02 

0.227 f 0.006 

0.65 f 0.04 

0.257 f 0.017 

0.71 f 0.03* 

0.257 f 0.008 

Female 

Necropsy body wt 221 f 3 219 f 11 218 f 4 

Brain 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

R. Kidney 

Liver 

Lung 

Spleen 

Thymus 

8.28 f 0.10 

1.830 f 0.01 1 

8.51 f 0.33 

1.854 f 0.062 

8.27 f 0.19 

1.804 f 0.043 

3.56 f 0.10 

0.788 f 0.026 

3.81 f 0.13 

0.830 f 0.032 

3.81 f 0.14 

0.830 f 0.021 

36.34 f 3.11 

8.004 f 0.568 

35.47 f 0.60 

7.758 f 0.338 

34.61 f 2.00 

7.544 f 0.415 

5.94 f 0.16 

1.314 f 0.039 

8.01 f 0.38** 

1.746 f 0.078** 

11.87 rt 0.59** 

2.586 f. 0.114** 

2.48 f 0.20 

0.546 f 0.036 

2.26 f 0.07 

0.494 f 0.019 

2.32 f 0.15 

0.506 f 0.033 

0.90 f 0.04 

0.199 f 0.012 

0.91 5 0.09 

0.199 f 0.024 

0.84 * 0.05 

0.184 f 0.014 

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weighvg body weight 

** P10.01 

* Significantly different (P10.05) from the control group by Williams’ or Dunnett’s test 

(mean f standard error). 
Organ Weight Analyses 305 

in the 2-Year Inhalation Study of Nickel Subsulfidea 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats at the 15-Month Interim Evaluation 

TABLE F4 

O mg/m3 0.15 mg/m3 1 mg/m3 

n 5 5 5 

Male 

Necropsy body wt 469 f 15 462 f 8 443 f 9 

Brain 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

R. Kidney 

Liver 

Lung 

Spleen 

Thymus 

3.49 f 0.14 

1.634 f 0.075 

4.45 f 0.12 

2.076 f 0.030 

4.53 f 0.07 

2.092 f 0.018 

3.59 f 0.18 

1.590 f 0.081 

4.69 f 0.11 

2.074 f 0.020 

3.53 f 0.12 

1.634 f 0.071 

38.58 f 3.12 

18.116 +_ 1.674 

36.75 f 2.33 

17.036 +_ 1.289 

36.83 f 1.15 

16.300 f 0.501 

4.84 f 0.13 

2.268 f 0.090 3.308 f 0.219** 

7.14 f 0.39 15.47 f 1.65** 

6.838 f 0.697** 

2.12 f 0.09 

0.992 f 0.059 

1.93 f 0.05 

0.892 f 0.024 

2.92 f 0.95 

1.284 f 0.409 

0.57 * 0.01 

0.266 f 0.027 

0.56 f 0.09 

0.259 f 0.037 

0.56 f 0.06 

0.246 f 0.024 

Female 

Necropsy body .wt 300 f 4 299 f 12 268 f 11 

Brain 

Relative 

Absolute 

Relative 

Absolute 

R. Kidney 

Liver 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Lung 

Spleen 

Thymus 

3.15 f 0.11 

0.946 f 0.034 

6.30 f 0.04 

1.890 f 0.017 

6.20 f 0.20 

1.842 * 0.033 

6.99 f 0.32 

1.860 f 0.040 

3.41 f 0.15 

1.018 f 0.069 

3.84 f 0.19* 

1.022 f 0.043 

31.60 f 0.59 

9.496 f 0.288 

34.04 f 1.43 

10.212 f 0.813 

34.27 f 0.96 

9.172 f 0.435 

5.06 f 0.28 

1.524 f 0.106 

8.43 * 0.80** 

2.516 f 0.252** 

15.54 f 0.30** 

4.140 f 0.105** 

1.98 f 0.06 

0.596 It 0.023 

1.99 f 0.13 

0.592 f 0.037 

2.16 f 0.18 

0.572 f 0.034 

0.81 f 0.09 

0.243 f 0.026 

0.84 f 0.15 

0.250 f 0.047 

0.82 f 0.08 

0.218 * 0.018 

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weightlg body weight 

** P 10.01 

* Significantly different (PlO.05) from the control group by Williams’ or Dunnett’s test 

(mean f standard error). 
306 Nickel Subsulfide, NTP TR 453 

of Nickel Subsulfidea 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice in the 16-Day Inhalation Study 

TABLE F5 

0 mg/m3 0.6 mg/m3 1.2 mg/m3 2.5 mg/m3 5 mg/m3 

Brain 

Necropsy body wt 

n 

Male 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Heart 

R. Kidney 

Liver 

Lung 

Testes 

Thymus 

Brain 

Necropsy body wt 

n 

Female 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Heart 

R. Kidney 

Liver 

Lung 

Thymus 

23.6 f 1.7 

5 

5.56 f 0.41 

0.129 f 0.006 

18.60 f 0.55 

0.435 f 0.023 

0.224 f 0.016 

9.56 f 0.33 

61.42 f 4.48 

1.478 f 0.186 

1.71 f 0.29 

0.042 f 0.008 

4.04 f 0.09 

0.096 f 0.008 

9.40 f 1.11 

0.215 f 0.015 

20.3 f 2.0 

5 

22.05 f 2.07 

0.433 f 0,014 

2.13 f 0.43 

0.045 f 0.011 

10.18 f 0.62 

0.203 f 0.014 

62.81 f 5.88 

1.322 f 0.212 

8.00 f 0.33 

0.160 f 0.011 

5.81 f 0.50 

0.117 f 0.015 

5 5 5 5 

25.0 f 0.4 22.8 f 1.8 23.4 f 0.5 21.8 f 0.5 16.7 f 0.4**b 

5 

0.442 f 0.004 0.454 f 0.009 0.431 f 0.010 0.424 f 0.007 0.385 f 0.023* 

17.72 f 0.36 20.35 f 1.55 18.47 f 0.34 19.47 f 0.66 23.17 f 1.86* 

0.150 f 0.013 0.147 f 0.022 0.159 * 0.011 0.135 f 0.011 0,162 f 0.013 

6.00 f 0.46 6.54 f 0.92 6.83 f 0.54 6.15 If: 0.44 9.70 f 0.92** 

0.233 f 0.007 0.207 f 0.020 0.227 f 0.007 0.193 f 0.004 0,169 f 0.014** 

9.33 f 0.24 9.00 f 0.23 9.72 f 0.25 8.85 f 0.06 10.12 f 0.80 

1.624 f 0.017 1.346 f 0.201 1.485 f 0.041 1.246 f 0.036 0.886 f 0.100** 

65.02 f 0.64 57.33 f 5.73 63.54 f 0.80 57.11 f 0.82 53.03 f 6.03 

0.202 f 0.016 0.22 f 0.151 0.278 f 0.013 0.305 f 0.022** 0.380 f 0.036** 

8.09 f 0.56 15.90 f 6.12 11.96 f 0.70 13.94 f 0.80 22.78 f 2.29** 

4.33 f 0.22 4.42 f 0.11 4.61 f 0.17 4.63 f 1.37 

0,095 f 0.006 0.099 f 0.009 0.103 f 0.002 0.100 f 0.003 0.079 f 0.025 

3.80 f 0.21 

0.048 f 0.004 0.045 f 0.009 0.046 f 0.005 0.033 f 0.002 0.013 f 0.005** 

1.91 f 0.18 1.89 f 0.32 1.97 f 0.23 1.52 f 0.08 0.76 f 0.29* 

5 5 5 5 

21.6 f 0.2 21.1 f 0.4 20.6 f 0.3 20.1 f 0.6 13.5 f 0.8**b 

4 

0.438 * 0.004. 0.450 f 0.006 0.437 f 0.007 0.434 f 0,001 0.408 f 0,014 

20.31 f 0.18 21.36 f 0.43 21.22 f 0.60 21.62 f 0.58 30.55 f 1.44** 

0.140 * 0.014 0.122 f 0.007 0.118 f 0.005 0.117 f 0.005 0.124 f 0.014 

6.47 * 0.67 5.80 f 0.34 5.71 f 0.23 5.84 f 0.28 9.32 f 1.26** 

0.165 f 0.004 0.159 0.005 0.156 f 0.006 0.154 f 0.008 0.114 f 0.003** 

7.66 f 0.19 7.55 f 0.14 7.56 f 0.21 7.65 f 0.35 8.57 f 0.48 

1.473 f 0.027 1.429 f 0.073 1.334 f 0.040 1.201 f 0,048 0.732 f 0.075** 

68.26 f 1.41 67.54 f 2.12 64.67 f 1.05 59.72 f 1.79 54.03 f 2.67 

0.206 f 0.009 0.215 f 0.008 0.271 f 0.010 0.356 f 0.048* 0.252 f 0.045 

9.52 f 0.42 10.17 f 0.30 13.14 f 0.50 17.60 f 2.02** 18.77 f 3.32** 

0.062 f 0.005 0.065 f 0.003 0.063 f 0.003 0.058 f 0.006 0.012 f 0.003** 

2.86 f 0.22 3.10 f 0.16 3.07 f 0.13 2.87 f 0.29 0.86 f 0.15* 

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weightlg body weight 

** P10.01 

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

All animals in this exposure group died early. 

(mean f standard error). 
Organ Weight Analyses 307 

of Nickel SubsuIfidea 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice in the 13-Week Inhalation Study 

TABLE F6 

0 mg/m3 0.15 mg/m3 0.3 mg/m3 0.6 mg/m3 1.2 mg/m3 2.5 mg/m3 

Brain 

Necropsy body wt 

n 

Male 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Heart 

R. Kidney 

Liver 

Lung 

R. Testis 

Thymus 

28.8 f 0.8 

8 

5.48 f 0.28 

0.158 f 0.009 

15.80 f 0.59 

0.451 f 0.007 

1.08 f 0.05 

0.031 f 0.002 

3.83 f 0.08 

0.110 f 0.003 

6.53 f 0.44 

0.188 f 0.013 

52.85 * 1.72 

1.525 f 0.080 

9.97 f 0.21 

0.286 f 0.008 

10 

29.6 f 0.5 

5.11 f 0.17 

0.151 f 0.006 

15.26 f 0.22 

0.450 f 0.004 

6.78 f 0.11 

0.200 f 0.000 

53.11 f 1.65 

1.570 f 0.058 

9.28 f 0.25 

0.275 f 0.011 

1.17 f 0.14 

0.034 f 0.004 

3.62 f 0.18 

0.107 f 0.005 

10 

31.0 f 0.7 

1.21 f 0.06 

0.037 f 0.001 

3.93 f 0.14 

0.121 f 0.002 

7.10 * 0.61 

0.220 f 0.020 

57.19 f 1.49 

1.780 f 0.077* 

9.75 f 0.17 

0.303 f 0.011 

5.42 f 0.17 

0.168 f 0.006 

14.72 f 0.28 

0.455 f 0.006 

28.9 f 0.4 

8 

16.13 f 0.25 

0.465 f 0.005 

1.45 f 0.13 

0.042 f 0.004 

3.90 f 0.20 

0.113 f 0.006 

7.37 f 0.45 

0.213 f 0.013 

55.40 f 0.68 

1.600 f 0.038 

9.44 f 0.27 

0.273 f 0.009 

5.36 f 0.22 

0.155 f 0.007 

9 10 

28.8 f 0.8 27.1 f 0.4 

0.462 f 0.004 0.455 f 0.007 

16.15 f 0.49 16.85 f 0.49 

0.159 f 0.007 0.146 f 0.006 

5.52 f 0.18 5.40 f 0.23 

0.270 f 0.014 0.256 f 0.007 

9.33 f 0.27 9.45 f 0.25 

1.567 f 0.055 1.300 f 0.033* 

54.33 f 0.82 48.06 * 1.37 

0.233 f 0.017* 0.280 f 0.013** 

8.20 f 0.72* 10.39 f 0.58** 

0.120 f 0.003 0.121 f 0.008 

4.19 f 0.15 4.50 f 0.34* 

0.043 f 0.005 0.035 f 0.003 

1.52 f 0.22 1.8 f 0.11 
308 Nickel Subsulfide, NTP TR 453 

of Nickel Subsulfide (continued) 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice in the 13-Week Inhalation Study 

TABLE F6 

0 mg/m3 0.15 mg/m3 0.3 mg/m3 0.6 mg/m3 1.2 mg/mg 2.5 mg/m3 

Brain 

Necropsy body wt 

n 

Female 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Heart 

R. Kidney 

Liver 

Lung 

Thymus 

24.1 f 0.8 

9 

5.38 f 0.15 

0.128 f 0.003 

19.05 * 0.89 

0.455 f 0.007 

56.02 f 1.94 

1.360 f 0.070 

7.66 f 0.20 

0.187 f 0.008 

7.98 f 0.63 

0.190 f 0.010 

1.59 f 0.15 

0.039 f 0.004 

8 

24.5 f 0.4 

19.01 f 0.25 

0.465 f 0.005 

5.52 f 0.27 

0.135 f 0.007 

7.14 f 0.66 

0.175 f 0.016 

55.22 f 1.11 

1.350 f 0.019 

7.90 f 0.19 

0.194 f 0.007 

1.70 f 0.14 

0.042 k 0.004 

25.0 f 0.7 

10 

18.73 f 0.54 

0.465 f 0.005 

5.50 f 0.27 

0.137 f 0.007 

7.45 f 0.31 

0.185 f 0.005 

1.77 f 0.11 

0.044 f 0.003 

8.06 f 0.23 

0.200 f 0.000 

63.67 k 4.21 

1.580 f 0.096 

9 

23.9 f 0.4 

19.39 f 0.45 

0.463 f 0.008 

7.52 f 0.15 

0.180 f 0.005 

5.70 f 0.44 

0.136 f 0.009 

8.84 f 0.48 

0.211 f 0.011 

53.82 f 1.54 

1.289 f 0.045 

1.59 f 0.10 

0.038 * 0.002 

10 8 

24.1 f 0.3 22.7 f 0.5 

0.456 f 0.005b 0.455 f 0.005 

19.00 f 0.17b 20.08 f 0.54 

0.134 f 0.010 0.134 f 0.007 

5.53 f 0.33 5.90 f 0.32 

0.176 f 0.005 0.176 f 0.005 

7.30 f 0.18 7.75 f 0.17 

1.280 f 0.039 1.213 f 0.055 

53.10 f 1.29 53.18 f 1.63 

0.260 k 0.022** 0.288 f 0.013** 

10.74 f 0.87** 12.62 f 0.43** 

0.038 f 0.003 0.038 f 0.003 

1.58 f 0.11 1.66 f 0.14 

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weighug body weight 

** P<O.Ol 

* Significantly different (PCO.05) from the control group by Williams’ or Dunnett’s test 

n=9 

(mean f standard error). 

. 
Organ Weight Analyses 309 

in the 2-Year Inhalation Study of Nickel Subsulfidea 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice at the 7-Month Interim Evaluation 

TABLE F7 

0 mg/m3 0.6 mg/m3 1.2 mg/m3 

n 5 5 5 

Male 

Necropsy body wt 36.6 f 0.6 35.6 f 1.0 34.4 f 0.5 

Brain 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

R. Kidney 

Liver 

Lung 

Spleen 

Thymus 

13.08 f 0.26 

0.478 f 0.004 

13.31 f 0.35 

0.472 f 0.002 

13.72 f 0.20 

0.472 f 0.002 

10.56 f 0.31 

0.386 f 0.009 

10.30 f 0.40 

0.366 f 0.016 

11.28 f 0.44 

0.388 f 0.012 

48.78 f 2.88 

1.786 f 0.111 

50.60 f 0.66 

1.798 f 0.040 

47.98 f 1.77 

1.650 f 0.045 

6.57 f 0.62 

0.240 f 0.022 

7.71 f 0.33 

0.274 f 0.013 

9.92 f 0.57** 

0.342 f 0.022** 

2.35 f 0.06 

0.086 f 0.002 

2.30 f 0.07 

0.082 f 0.004 

2.27 f 0.12 

0.078 f 0.004 

0.92 f 0.09 

0.034 k 0.003 

0.97 f 0.12 

0.034 f 0.003 

1.08 -I 0.09 

0.037 f 0.003 

Female 

Necropsy body wt 29.8 f 1.2 30.8 f 1.5 29.9 f 0.6 

Brain 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

R. Kidney 

Liver 

Lung 

Spleen 

Thymus 

16.43 f 0.63 

0.486 f 0.008 

15.75 f 0.81 

0.480 f 0.007 

15.96 f 0.35 

0.476 f 0.002 

7.55 f 0.27 

0.224 f 0.009 

8.16 f 0.70 

0,248 f 0.013 

7.90 f 0.34 

0.236 f 0.011 

48.95 f 1.28 

1.456 f 0.067 

48.42 f 2.17 

1.478 f 0.009 

50.16 f 1.15 

1.500 f 0.053 

6.25 f 0.23 

0.186 f 0.011 

8.37 * 0.44* 

0.256 f 0.011* 

9.62 f 0.74** 

0.288 f 0.025** 

3.33 f 0.23 

0.100 f 0.008 

3.43 f 0.18 

0.102 f 0.007 

3.27 f 0.14 

0.100 f 0.003 

1.31 f 0.14 

0.040 f 0.005 

1.16 f 0.17 

0.036 f 0.006 

1.20 f 0.10 

0.036 f 0.004 

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weightlg body weight 

** P10.01 

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

(mean f standard error). 
310 Nickel Subsulfide, NTP TR 453 

Necropsy body wt 

Male 

39.5 f 0.7 39.2 f 0.7 39.1 f 1.9 

Brain 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

R. Kidney 

Liver 

Lung 

Spleen 

Thymus 

12.02 f 0.31 

0.474 f 0.010 

11.95 f 0.25 

0.468 f 0.006 

11.84 f 0.69 

0.458 f 0.005 

10.10 f 0.57 

0.398 f 0.021 

10.48 f 0.38 

0.410 f 0.009 

10.58 f 0.56 

0.410 f 0.009 

48.10 f 2.90 

1.892 f 0.087 

49.82 f 2.02 

1.948 f 0.051 

56.07 f 5.65 

2.198 f 0.273 

5.90 f 0.31 

0.232 f 0.009 

10.27 f OS** 

0.402 f 0.020** 

10.39 f 1.13** 

0.408 f 0.048** 

1.88 * 0.10 

0.074 f 0.004 

2.15 f 0.17 

0.084 f 0.005 

2.14 f 0.12 

0.084 f 0.007 

0.93 f 0.11 

0.037 f 0.004 

1.00 f 0.12 

0.040 f 0.005 

1.03 f 0.09 

0.041 f 0.005 

Female 

Necropsy body wt 37.4 f 2.1 34.7 f 1.8 33.5 f 1.5 

Brain 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

Relative 

Absolute 

R. Kidney 

Liver 

Lung 

Spleen 

Thymus 

13.25 f 0.77 

0.490 f 0.004 

14.50 f 0.52 

0.500 f 0.009 

14.86 f 0.55 

0.494 f 0.005 

6.81 f 0.42 

0.252 f 0.007 

52.73 f 2.21 

1.836 f 0.140 

7.53 f 0.23 

0.260 f 0.008 

7.41 f 0.21 

0.248 f 0.013 

51.42 f 2.92 

1.906 f 0.070 

49.02 f 1.71 

1.644 f 0.106 

6.94 f 0.69 

0.256 f 0.021 

11.49 f 1.33** 

0.390 f 0.025** 

14.85 f 0.55** 

0.496 f 0.023** 

3.66 f 0.35 

0.136 f 0.012 

1.35 f 0.05 

0.047 f 0.002 

3.28 f 0.06 

0.114 f 0.006 

3.54 f 0.16 

0.118 f 0.006 

1.41 f 0.14 

0.053 f 0.006 

1.33 f 0.10 

0.045 f 0.004 

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weightlg body weight 

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

(mean f standard error). 
311 

HEMATOLOGY RESULTS 

APPENDIX G 

in the 2-Year Inhalation Study of Nickel Subsulfide 

TABLE G4 Hematology Data for Mice at the 15-Month Interim Evaluation 

of Nickel Subsulfide 

TABLE 63 Hematology Data for Mice in the 13-Week Inhalation Study 

in the 2-Year Inhalation Study of Nickel Subsulfide 

TABLE 62 Hematology Data for Rats at the 15-Month Interim Evaluation 

of Nickel Subsulfide 

TABLE G1 Hematology Data for Rats in the 13-Week Inhalation Study 

........................................ 

317 

315 

314 

312 

.................... 

........................................ 

.................... 
312 Nickel Subsulfide, NTP TR 453 

Hematology Data for Rats in the 13-Week Inhalation Study of Nickel Subsulfidea 

TABLE G1 

0 mg/m3 0.15 mg/m3 0.3 mg/m3 0.6 mg/m3 1.2 mg/mJ 2.5 mg/m3 

Male 

n 10 10 

Nucleated erythrocytes (103/pL) 

Eosinophils (103/pL) 

Monocytes (103/pL) 

Lymphocytes (103/pL) 

Segmented neutrophils ( 103/pL) 

Leukocytes (103/pL) 

Reticulocytes (106/pL) 

Mean cell hemoglobin concentration (gldL) 

Mean cell volume (fL) 

Erythrocytes (106/pL) 

Hemoglobin (gldL) 

Hematocrit (%) 

42.9 f 0.3 43.4 f 0.6 

15.5 f 0.2 15.7 f 0.2 

8.20 f 0.07 8.34 f 0.11 

51.7 f 0.3 51.4 f 0.3 

36.1 f 0.3 36.2 f 0.2 

0.7 f 0.1 0.7 f 0.1 

2.19 f 0.18 2.85 f 0.26 

0.63 _+ 0.06 1.16 f 0.10** 

1.50 f 0.15 1.62 f 0.19 

0.03 f 0.01 0.05 f 0.02 

0.03 f 0.01 0.02 f 0.01 

0.06 f 0.02 0.10 f 0.03 

9 

36.2 f 0.3 

51.9 f 0.4 

8.24 f 0.09 

15.6 f 0.2 

43.2 f 0.6 

0.05 f 0.01 

1.63 f 0.15 

1.06 f 0.09** 

2.19 f 0.23 

0.6 f 0.1 

0.08 f 0.02 

0.03 f 0.01 

10 

8.34 f 0.22 

15.9 f 0.4 

43.7 f 0.1 

1.19 f 0.14** 

3.04 f 0.37 

0.8 f 0.2 

36.4 & 0.4 

52.1 * 1.0 

0.09 f 0.03 

0.01 f 0.01 

0.02 f 0.01 

1.82 * 0.26 

10 

37.0 f 0.2* 

50.6 f 0.2** 

8.55 f 0.05** 

16.2 f 0.1** 

43.7 f 0.3 

0.11 f 0.03 

0.02 f 0.01 

0.04 f 0.02 

1.80 f 0.26 

1.08 f 0.14** 

2.93 _+ 0.41 

0.8 f 0.1 

8 

45.1 f 0.4** 

16.7 f 0.2** 

3.58 2 0.47* 

0.7 f 0.1 

37.1 f 0.2** 

51.1 f 0.9* 

8.73 f 0.14** 

0.15 rfI 0.06 

0.02 * 0.01 

0.05 f 0.02 

1.69 f 0.17 

1.82 f 0.33** 
Hematology Results 313 

Hematology Data for Rats in the 13-Week Inhalation Study of Nickel Subsulfide (continued) 

TABLE G1 

0 mg/m3 0.15 mg/m3 0.3 mg/m3 0.6 mg/m3 1.2 mg/m3 2.5 mg/m3 

Female 

n 10 10 

Nucleated erythrocytes ( 103/pL) 

Eosinophils ( 103/pL) 

Monocytes (103/pL) 

Lymphocytes ( 103/pL) 

Segmented neutrophils (103/pL) 

Leukocytes (103/pL) 

Reticulocytes (106/pL) 

Mean cell hemoglobin concentration (g/dL) 

Mean cell volume (fL) 

Erythrocytes (106/pL) 

Hemoglobin (gldL) 

Hematocrit (%) 

40.8 f 0.5 41.0 f 0.3 

14.8 f 0.2 14.9 f 0.1 

7.27 f 0.08 7.38 f 0.06 

55.6 f 0.2 55.0 f 0.2 

36.3 f 0.2 36.3 f 0.2 

0.6 f 0.1 0.5 f 0.1 

2.06 f 0.22 2.72 f 0.33 

0.44 f 0.11 0.82 f 0.12** 

1.55 f 0.17 1.85 f 0.23 

0.03 f 0.01 0.02 f 0.01 

0.04 f 0.01 0.03 f 0.01 

0.05 f 0.01 0.09 f 0.02 

10 

7.51 f 0.10** 

15.2 f 0.2 

41.4 f 0.5 

0.62 f 0.09* 

2.30 f 0.21 

0.7 f 0.1 

36.6 f 0.2 

54.6 f 0.2** 

0.13 f 0.04 

0.02 * 0.01 

0.03 f 0.01 

1.63 f 0.14 

10 

36.5 f 0.3 

54.7 f 0.4* 

7.49 & 0.12** 

15.1 f 0.2 

41.3 f 0.5 

0.07 f 0.03 

0.03 * 0.01 

0.03 f 0.01 

1.48 f 0.06 

0.67 f 0.06* 

2.21 f 0.10 

0.7 f 0.1 

10 

41.7 0.4 

15.4 f 0.2* 

0.19 f 0.03** 

0.03 f 0.01 

0.02 f 0.01 

1.35 f 0.11 

0.84 f 0.08** 

2.24 f 0.18 

0.5 f 0.0 

37.0 f 0.3* 

54.2 f 0.3** 

7.63 f 0.06** 

10 

37.2 f 0.3* 

53.8 f 0.3** 

7.79 f 0.06** 

15.8 f 0.1** 

42.3 f 0.4* 

0.14 f 0.03** 

0.03 0.01 

0.04 f 0.01 

1.59 f 0.23 

1.01 f 0.11** 

2.66 f 0.32 

0.6 f 0.1 

* Significantly different (P (0.05) from the control group by Dunn’s or Shirley’s test ** PSO.01 a Mean f standard error; statistical tests were performed on unrounded data. 
314 Nickel Subsulfide, NTP TR 453 

of Nickel Subsulfidea 

Hematology Data for Rats at the 15-Month Interim Evaluation in the 2-Year Inhalation Study 

TABLE 62 

O mg/m3 0.15 mg/m3 1 mg/m3 

n 5 5 5 

Male 

Mean cell hemoglobin 

Mean cell hemoglobin (pg) 

Mean cell volume (fL) 

Erythrocytes (106/pL) 

Hemoglobin (g/dL) 

Hematocrit (%) . 

Eosinophils (103/pL) 

Monocytes (103/pL) 

Lymphocytes ( 103/pL) 

Segmented neutrophils (103/pL) 

Leukocytes (103/pL) 

Reticulocytes (106/pL) 

concentration (gldL) 

Female 

Mean cell hemoglobin 

Mean cell hemoglobin (pg) 

Mean cell volume (a) 

Erythrocytes (106/pL) 

Hemoglobin (g/dL) 

Hematocrit (%) 

Eosinophils (103/pL) 

Monocytes (103/pL) 

Lymphocytes (103/pL) 

Segmented neutrophils (103/pL) 

Leukocytes (103/pL) 

Reticulocytes (106/pL) 

concentration (gldL) 

15.9 f 0.2 

53.6 f 0.5 

10.06 f 0.18 

15.9 f 0.1 

51.3 f 0.7 

0.06 & 0.02 

0.24 f 0.05 

4.32 f 0.26 

1.10 f 0.13 

5.68 f 0.15 

0.3 f 0.0 

31.1 f 0.3 

17.6 $- 0.2 

57.8 f 0.4 

8.86 f 0.10 

15.6 0.1 

48.9 f 0.5 

0.04 f 0.02 

0.16 & 0.05 

4.18 f 0.53 

1.04 f 0.19 

5.94 f 0.73 

0.2 f 0.0 

31.9 f 0.2 

15.8 f 0.1 

53.2 & 0.4 

10.42 f 0.08 

16.5 * 0.2 

52.8 f 0.6 

0.06 0.02 

0.18 f 0.05 

4.32 f 0.38 

1.86 f 0.29 

6.40 f 0.46 

0.2 f 0.0 

31.2 f 0.2 

17.5 * 0.1 

57.4 f 0.4 

8.84 f 0.14 

15.4 f 0.2 

48.6 f 0.8 

0.06 f 0.04 

0.22 f 0.05 

3.16 f 0.16 

1.80 f 0.64 

5.24 f 0.58 

0.2 f 0.0 

31.8 f 0.1 

16.2 f 0.1 

54.6 f 0.5 

10.80 f 0.19** 

17.5 f 0.4** 

56.1 f 0.8** 

0.08 f 0.04 

0.64 f 0.49 

5.30 f 0.85 

1.58 f 0.27 

7.60 f 1.53 

0.3 f 0.1 

31.1 & 0.4 

17.9 f 0.2 

59.0 f 0.6 

9.20 f 0.10 

16.4 f 0.2* 

51.8 f 0.7* 

31.8 0.1 

0.04 f 0.02 

0.12 f 0.02 

3.32 f 0.37 

1.14 f 0.19 

4.64 * 0.53 

0.1 f 0.0 

* Significantly different (PlO.05) from the control group by DUM’S or Shirley’s test ** P10.01 a Mean f standard error; statistical tests were performed on unrounded data. 
Hematology Results 315 

Hematology Data for Mice in the 13-Week Inhalation Study of Nickel Subsulfidea 

TABLE 63 

0 mg/m3 0.15 mg/m3 0.3 mg/m3 0.6 mg/m3 1.2 mg/m3 2.5 mg/m3 

Male 

n 4 4 

39.5 f 1.7 35.5 f 7.4 

13.7 f 0.7 12.3 f 2.5 

7.79 f 0.35 6.92 f 1.41 

50.0 f 0.4 50.0 f 0.8 

34.6 f 0.5 34.7 f 0.4 

0.5 f 0.1 0.4 f 0.1 

Nucleated erythrocytes (103/pL) 

Eosinophils (103/pL) 

Monocytes (103/pL) 

Lymphocytes (103/pL) 

Segmented neutrophils (lO’IpL) 

Leukocytes (103/pL) 

Reticulocytes (106/pL) 

Mean cell hemoglobin concentration (g/dL) 

Mean cell volume (a) 

Erythrocytes (106/pL) 

Hemoglobin (g/dL) 

Hematocrit (%) 

0.50 f 0.11 0.43 f 0.23 

0.15 f 0.02 0.19 f 0.13 

0.34 f 0.12 0.23 f 0.11 

0.01 f 0.01 0.00 f 0.00 

0.00 f 0.00 0.00 f 0.00 

0.01 f 0.01 0.02 f 0.01 

40.6 f 1.4 

5 

49.6 f 0.7 

8.09 f 0.34 

14.3 * 0.4 

0.03 f O.OOb 

0.00 f 0.00 

0.03 f 0.02 

0.57 f 0.08 

0.59 f 0.14 

1.18 f 0.18 

0.7 f 0.2 

35.2 f 0.4 34.8 f 0.3 

49.7 f 0.3 

8.17 f 0.08 

14.3 f 0.1 

41.2 f 0.4 

3 

0.01 f 0.01 

0.01 f 0.01 

0.01 f 0.01 

0.27 f 0.01 

0.48 f 0.18 

0.17 f 0.17 

0.6 f 0.0 

8.18 f 0.17 

14.5 f 0.4 

41.4 f 1.0 

5 

0.6 f 0.1 

35.1 f 0.1 

50.0 f 0.3 

0.10 f 0.02 

0.01 f 0.01 

0.01 f 0.01 

1.14 f 0.20* 

0.55 f 0.09 

1.66 f 0.22 

8.50 f 0.38 

14.3 f 0.5 

42.2 f 1.9 

5 

49.0 f 0.0 

34.1 f 1.2 

0.04 f 0.02 

0.00 f 0.00 

0.00 f 0.00 

0.73 f 0.19* 

0.29 f 0.05 

1.02 f 0.22 

0.7 f 0.1 
316 Nickel Subsulfide, NTP TR 453 

Hematology Data for Mice in the 13-Week Inhalation Study of Nickel Subsulfide (continued) 

TABLE 63 

0 mg/m3 0.15 mg/m3 0.3 mg/m3 0.6 mg/m3 1.2 mg/m3 2.5 mg/m3 

Female 

n 

0.6 f 0.1 0.4 f 0.1 

35.6 f 0.5 35.5 f 0.9 

49.4 f 0.5 49.7 f 0.3 

7.65 f 0.16 8.11 f 0.14 

13.6 f 0.3 14.5 f 0.2 

38.3 f 1.1 40.9 f 0.8 

5 3 

Eosinophils (103/pL) 

Monocytes (103/rL) 

Lymphocytes (103/pL) 

Segmented neutrophils (103/pL) 

Leukocytes (103/pL) 

Reticulocytes (106/pL) 

Mean cell hemoglobin concentration (g/dL) 

Mean cell volume (E) 

Erythrocytes ( 106/pL) 

Hemoglobin (gldL) 

Hematocrit (%) 

1.00 f 0.24 0.77 f 0.03 

0.23 f 0.09 0.14 f 0.01 

0.75 f 0.19 0.58 f 0.04 

0.03 f 0.01 0.02 f 0.01 

0.01 f 0.01 0.02 f 0.01 

5 

41.1 f 0.3 

14.7 f 0.1** 

8.27 f 0.09** 

49.2 f 0.2 

35.8 f 0.2 

0.6 f 0.2 

0.82 f 0.20 

0.27 f 0.06 

0.02 f 0.01 

0.00 f 0.00 

0.53 f 0.15 

1.66 f 0.51 

0.8 f 0.4 

35.8 f 0.2 

49.3 f 0.2 

8.18 f 0.12* 

14.6 f 0.2* 

40.8 * 0.5 

5 

0.00 * 0.00 

0.02 f 0.01 

1.12 f 0.38 

0.47 & 0.22 

14.8 f 0.1** 

41.6 f 0.7 

5 

8.40 f 0.13** 

1.66 f 0.44 

2.1 f 1.2 

35.5 f 0.4 

48.8 f 0.4 

0.71 f 0.20 

0.00 f 0.00 

0.00 f 0.00 

0.95 f 0.28 

8.66 f 0.20** 

15.0 f 0.4** 

42.1 & 1.1 

5 

1.3 f 0.5 

35.7 f 0.1 

48.2 f 0.4 

0.00 f 0.00 

0.03 0.02 

0.82 f 0.21 

0.34 & 0.05 

1.30 f 0.34 

* Significantly different (P10.05) from the control group by Dum’s or Shirley’s test 

** P10.01 a Mean f standard error; statistical tests were performed on unrounded data. 

n=3 
Hematology Results 317 

of Nickel Subsulfidea 

Hematology Data for Mice at the 15-Month Interim Evaluation in the 2-Year Inhalation Study 

TABLE G4 

O mg/m3 0.6 mg/m3 1.2 mg/m3 

Male 

n 

Mean cell hemoglobin 

Mean cell hemoglobin (pg) 

Mean cell volume (fL) 

Erythrocytes (106/pL) 

Hemoglobin (g/dL) 

Hematocrit (%) 

Eosinophils ( 103/pL) 

Monocytes (103/pL) 

Lymphocytes ( 103/pL) 

Segmented neutrophils (103/pL) 

Leukocytes (103/pL) 

Reticulocytes (106/pL) 

concentration (gldL) 

Female 

n 

Mean cell hemoglobin 

Mean cell hemoglobin (pg) 

Mean cell volume (E) 

Erythrocytes (106/pL) 

Hemoglobin (g/dL) 

Hematocrit (X) 

Eosinophils (103/pL) 

Monocytes (103/pL) 

Lymphocytes ( 103/pL) 

Segmented neutrophils ( 103/pL) 

Leukocytes (103/pL) 

Reticulocytes (106/pL) 

concentration (g/dL) 

5 

14.7 f 0.1 

49.8 f 0.4 

10.56 k 0.15 

15.5 f 0.1 

50.2 f 0.5 

30.9 f 0.3 

0.2 f O.ob 

0.08 f 0.02 

0.10 f 0.00 

2.42 f 0.44 

1.28 f 0.20 

3.88 f 0.58 

5 

14.5 f 0.2 

48.6 f 0.2 

10.69 f 0.43 

15.5 f 0.5 

49.4 f 1.8 

0.04 f 0.02 

0.04 f 0.02 

1.92 f 0.32 

1.04 013

3.04 f 0.40 

0.2 f 0.0 

31.4 f 0.2 

5 

15.0 f 0.2 

51.0 f 0.7 

10.69 f 0.24 

16.0 f 0.3 

52.1 f 1.5 

0.04 f 0.02 

0.02 f 0.02 

2.28 f 0.20 

1.12 * 0.22 

3.48 f 0.36 

0.2 f 0.0 

30.8 f 0.4 

4 

14.6 f 0.3 

48.8 f 0.6 

10.91 4 0.18 

15.9 f 0.1 

50.8 f 0.5 

0.13 * 0.03 

0.15 f 0.05 

2.90 f 0.35 

1.28 f 0.15 

4.45 f 0.51 

0.1 * 0.0 

31.3 f 0.3 

5 

14.5 f 0.2 

49.2 f 0.7 

10.93 f’0.43 

15.9 f 0.6 

51.5 f 1.5 

0.06 f 0.04 

0.12 f 0.07 

2.56 f 0.32 

1.16 f 0.21 

3.92 f 0.59 

0.2 f 0.0 

30.8 f 0.2 

5 

14.6 f 0.1 

49.2 f 0.6 

11.17 f 0.07 

16.3 f 0.1 

52.6 f OS* 

0.14 f 0.05 

0.18 f 0.06* 

3.02 f 0.16* 

1.56 0.07** 

4.88 f 0.16* 

0.2 f 0.1 

31.0 f 0.3 

* Significantly different (PlO.05) from the control group by Dum’s or Shirley’s test ** PSO.01 a Mean f standard error; statistical tests were performed on unrounded data. 

n=4 
318 Nickel Subsulfide, NTP TR 453 
319 

TISSUE BURDEN IN RATS 

APPENDIX H 

TABLE H6 Kidney Burden in Rats in the 2-Year Inhalation Study 

TABLE H5 Lung Weight and Lung Burden in Rats in the 2-Year Inhalation Study 

TABLE H4 Kidney and Testes Burden in Male Rats in the 13-Week Inhalation Study 

TABLE H3 Lung Weight and Lung Burden in Rats in the 13-Week Inhalation Study 

TABLE H2 Kidney Weight and Kidney Burden in Rats in the 16-Day Inhalation Study 

TABLE H1 Lung Weight and Lung Burden in Rats in the 16-Day Inhalation Study 

of Nickel Subsulfide 

of Nickel Subsulfide 

of Nickel Subsulfide 

of Nickel Subsulfide 

of Nickel Subsulfide 

of Nickel Subsulfide ........................................... 

324 

323 

322 

321 

320 

320 

........................................... 

........................................... 

........................................... 

........................................... 

........................................... 
320 Nickel Subsulfide, NTP TR 453 

Lung Weight and Lung Burden in Rats in the 16-Day Inhalation Study of Nickel SubsuIfidea 

TABLE H1 

0 mg/m3 0.6 mg/m3 2.5 mg/m3 IO mg/m3 

n 

Male 

pg Ni/g control lung 

pg Ni/g lung 

pg Nillung 

Absolute lung wt (9) 

Female 

pg Nilg control lung 

pg Ni/g lung 

pg Ni/lung 

Absolute lung wt (g) 

3 

0.827 f 0.009 - 

b 

- 

- 

0.810 f 0.169 - 

- 

- 

3 

1.000 f 0.000 

9 f 1.0* 

7 f 0.9* 

7 f. 0.9* 

0.883 f 0.041 

12 f 2.0* 

9 f 1.3* 

8 f 1.3* 

3 

30 f 0.6** 

18 f 0.3** 

25 f 0.6** 

1.370 f 0.012** 

1.263 f 0.078* 

36 f 3.0** 

19 f 1.6** 

24 f 1.9** 

3 

127 f 3.3** 

67 f 8.7** 

103 f 2.9** 

1.577 f 0.150** 

133 -f 13.3** 

77 f 12.1** 

87 f 9.3** 

1.167 0.087 

a Mean f standard error; statistical tests were performed on unrounded data. 

** P10.01 

* Significantly different (PsO.05) from the control group by Dunnett’s or Williams’ test (lung weight) or Shirley’s test (lung burden 

parameters) 

Results were below 0.093 pg Ni (the limit of detection). 

Kidney Weight and Kidney Burden in Rats in the 16-Day Inhalation Study of Nickel Subsulfidea 

TABLE H2 

O mg/m3 2.5 mg/m3 10 mg/m3 

Male 

pg Ni/g kidney 

pg Nilkidney 

Absolute kidney wt (9) 1.96 f 0.12 

0.209 0.022* 

0.362 f 0.028* 

1.75 f 0.07 

1.993 f 0.964** 

2.658 f 1.473** 

1.27 f 0.12** 

- b 

- 

Female 

pg Ni/g kidney 

pg Ni/kidney 

Absolute kidney wt (9) 

0.115 f 0.058 

1.33 f 0.11 

0.187 f 0.017* 

0.263 f 0.012* 

1.42 f 0.10 1.01 f 0.05 

1.269 k 0.327** 

1.242 f 0.273** 

- 

a Mean f standard error; statistical tests were performed on unrounded data. 

** P 10.01 

* Significantly different (P10.05) from the control group by Dunnett’s or Williams’ test (kidney weight) or Shirley’s test 

(kidney burden parameters) 

Results were below 0.093 pg Ni (the limit of detection). 
Tissue Burden in Rats 321 

Lung Weight and Lung Burden in Rats in the 13-Week Inhalation Study of Nickel Subsulfidea 

TABLE H3 

Male 

n 6 6 6 6 

4 weeks 

pg Nilg control lung 

pg Ni/g lung 

3 f 0.2** 

3 f 0.2** 6 f 0.7** 15 f 1.4** 

8 f 0.9** 23 f 2.2** 

9 weeks 

pg Ni/g control lung 

pg Ni/g lung 5 f 0.2** 

6 f 0.2** 

7 f 0.6** 16 f 1.0** 

12 f 1.2** 31 f 1.7** 

13 weeks 

pg Ni/g control lung 

pg Nilg lung 

pg Ni/lung 

Absolute lung wt (g) 1.15 f 0.03 

- 

- 

- 

1.56 f 0.01** 

7 f 0.3** 

5 * 0.2** 

8 f 0.3** 

2.23 f 0.08** 2.38 f 0.03** 

17 f 1.0** 42 f 1.3** 

7 f 0.4** 18 f 0.5** 

14 f 0.9** 36 f 1.2** 

Female 

n 6 6 6 5 

13 weeks 

pg Nilg control lung 

pg Ni/g lung 

pg Nillung 

Absolute lung wt (g) 0.849 f 0.026 - 

- 

- 

1.234 f 0.053** 

7 f 0.3** 

5 f 0.1** 

6 f 0.2** 

1.570 f 0.034** 1.607 f 0.045** 

11 f 0.3** 

13 f 0.4** 33 f 1.7** 

7 f 0.1** 17 f 0.7** 

28 f 1.4** 

a Mean f standard error; statistical tests were performed on unrounded data. 

** Significantly different (P10.01) from the control group by Williams’ test (lung weight) or Shirley’s test (lung burden parameters) 

Results were below 0.218 pg Ni (the limit of detection). 
322 Nickel Subsulfide, NTP TR 453 

Kidney and Testes Burden in Male Rats in the 13-Week Inhalation Study of Nickel Subsulfidea 

TABLE H4 

O mg/m3 0.6 mg/m3 2.5 mg/m3 

n 

13 weeks 

Kidney 

pg Ni/g kidney 

13 weeks 

Testes 

pg Nilg testes 

6 

- b 

0.480 f 0.041 

6 6 

- 0.140 f 0.063* 

0.048 f 0.048** 0.465 f 0.007 

a Mean f standard error; statistical tests were performed on unrounded data. 

** PSO.01 

* Significantly different (PIO.05) from the control group by Dunn’s test 

Results were below 0.218 pg Ni (the limit of detection). 
Tissue Burden in Rats 323 

Lung Weight and Lung Burden in Rats in the 2-Year Inhalation Study of Nickel Subsulfidea 

TABLE H5 

0 mg/m3 0.15 mg/m3 1 mg/m3 

n 

Male 

pg Ni/g control lung 

pg Nilg lung 

pg Nillung 

Absolute lung wt (g) 

?'-Month interim evalu&tion 

n 

pg Ni/g control lung 

pg Ni/g lung 

pg Nillung 

Absolute lung wt (9) 

15-Month interim evaluation 

n 

Female 

pg Nilg control lung 

pg Nilg lung 

pg Nillung 

Absolute lung wt (9) 

744011th interim evaluation 

n 

pg Nilg control lung 

pg Ni/g lung 

pg Nillung 

Absolute lung wt (8) 

15,Month interim evaluation 

1.53 f 0.13 

7 

- 

b 

- 

2.27 f 0.09 

5 

- 

- 

- 

- 

1.10 f 0.03 

7 

- 

- 

1.52 f 0.11 

5 

- 

- 

- 

- 

7 

8 f 0.3** 

6 f 0.2** 

12 f OS** 

1.97 f 0.09** 

5 

6 f 0.3** 

4 f 0.2** 

14 f 0.6** 

3.31 f 0.22** 

7 

1.48 f 0.04** 

8 f 0.4** 

6 f 0.2** 

9 f 0.4** 

5 

2.52 f 0.25** 

6 f 0.5** 

4 f 0.5** 

9 f 0.8** 

7 

18 f 1.2** 

9 f OS** 

28 f 1.8** 

3.21 f 0.08** 

5 

6.84 f 0.70** 

9 f 0.7** 

3 f 0.1 

21 f 1.5** 

7 

21 f 0.6** 

9 f 0.4** 

23 f 0.7** 

2.44 f 0.07** 

5 

19 f 0.6** 

7 f 0.2** 

29 f 1.0** 

4.15 f 0.10** 

a Mean f standard error; statistical tests were performed on unrounded data. 

** Significantly different (PSO.01) from the control group by Williams' test (lung weight) or Dunn's or Shirley's test (lung burden 

interim evaluation. 

Results were below 0.035 pg Ni (the limit of detection) for the ?'-month interim evaluation and below 0.119 pg Ni for the 15-month 

parameters) 
324 Nickel Subsulfide, NTP TR 453 

Kidney Burden in Rats in the 2-Year Inhalation Study of Nickel Subsulfidea 

TABLE H6 

O mg/m3 0.15 mg/m3 1 mg/m3 

Male 

7-Month interim evaluation 

n 

pg Nilg kidney 

15-Month interim evaluation 

n 

pg Nilg kidney 

Female 

7-Month interim evaluation 

n 

pg Ni/g kidney 

15-Month interim evaluation 

n 

pg Ni/g kidney 

7 

- 

b 

5 

7 

0.307 f 0.101 

5 

0.044 f 0.005** 

7 7 

- 0.036 f 0.003** 

5 

0.522 f 0.123* 

5 

- 

7 

0.312 k 0.091 

5 

0.224 * 0.023** 

7 

0.217 f 0.011** 

5 

0.757 k 0.094** 

a Mean f standard error; statistical tests were performed on unrounded data. 

** P10.01 

* Significantly different (PSO.05) from the control group by DUM’S or Shirley’s test 

interim evaluation. 

Results were below 0.033 pg,Ni (the limit of detection) for the 7-month interim evaluation and below 0.113 pg Ni for the 15-month 
325 

TISSUE BURDEN IN MICE 

APPENDIX I 

TABLE I1 Lung Weight and Lung Burden in Mice in the 16-Day Inhalation Study 

TABLE I2 Kidney Weight and Kidney Burden in Mice in the 16-Day Inhalation Study 

TABLE I3 Lung Weight and Lung Burden in Mice in the 13-Week Inhalation Study 

TABLE 14 Lung Weight and Lung Burden in Mice in the 2-Year Inhalation Study 

TABLE 15 Kidney Burden in Mice in the 2-Year Inhalation Study 

of Nickel Subsulfide 

of Nickel Subsulfide 

of Nickel Subsulfide 

of Nickel Subsulfide 

of Nickel Subsulfide ........................................... 

330 

329 

328 

327 

326 

........................................... 

........................................... 

........................................... 

........................................... 
326 Nickel Subsulfide, NTP TR 453 

Lung Weight and Lung Burden in Mice in the 16-Day Inhalation Study of Nickel Subsulfidea 

TABLE I1 

0 mg/m3 0.6 mg/m3 2.5 mg/m3 10 mg/m3 

Male 

n 

pg Nilg control lung 

pg Nilg lung 

pg Nillung 

Absolute lung wt (g) 

Female 

n 

pg Ni/g control lung 

pg Ni/g lung 

pg Nillung 

Absolute lung wt (g) 

3 

0.138 f 0.004 - b 

3 

0.125 f 0.002 - 

- 

- 

3 

0.145 f 0.006 

10 f 1.0* 

10 f 0.7* 

1 f 0.2* 

3 

0.153 f 0.011 

10 f 1.0* 

8 f 0.4 

1 f 0.1* 

3 

30 f 6.8** 

20 f 3.2** 

4 f 0.9** 

0.200 f 0.014** 

3 

0.197 f 0.006* 

31 f 4.1** 

20 f 2.6** 

4 f 0.5** 

2 

31 f 6.0* 

13 f 2.5* 

4 f 0.9* 

0.335 f 0.005** 

3 

22 f 3.9* 

8 f 1.9 

3 f 0.5* 

0.380 f. 0.040** 

a Mean f standard error; statistical tests were performed on unrounded data. 

** P40.01 

* Significantly different (PlO.05) from the control group by Williams’ test (lung weight) or Dunn’s or Shirley’s test (lung burden 

parameters) 

Results were below 0.155 pg Ni (the limit of detection). 
Tissue Burden in Mice 327 

Kidney Weight and Kidney Burden in Mice in the 16-Day Inhalation Study of Nickel Subsulfidea 

TABLE I2 

0 mg/m3 

Male 

n 

pg Nilg kidney 

pg Nilkidney 

Absolute kidney wt (g) 

Female 

n 

pg Ni/g kidney 

pg Nilkidney 

Absolute kidney wt (g) 

3 3 

0.472 f 0.019 

0.122 f 0.122 

0.054 f 0.054 

0.442 f 0,010 - b 

- 

3 3 

0.328 f 0.017 

0.457 f 0.269 

0.146 f 0.080 

0.332 f 0.021 - 

- 

n 

1 

4.440 f 2.680* 

1.142 f 0.648* 

0.265 f 0.015** 

3 

1.283 f 0.041* 

0.276 0.040* 

0.213 f 0.024* 

a Mean f standard error; statistical tests were performed on unrounded data. 

** P10.01 

* Significantly different (PSO.05) from the control group by Dunnett’s test (kidney weight) or Dum’s or Shirley’s test (kidney burden 

parameters) 

Results were below 0.155 pg Ni (the limit of detection). 
328 Nickel Subsulfide, NTP TR 453 

Lung Weight and Lung Burden in Mice in the 13-Week Inhalation Study of Nickel Subsulfidea 

TABLE I3 

0 mg/m3 0.15 mg/m3 0.6 mgh3 2.5 mgh3 

Male 

n 

pg Ni/g control lung 

pg Ni/g lung 

pg Ni/lung 

Absolute lung wt (9) 

Female 

n 

pg Ni/g control lung 

pg Nilg lung 

pg Ni/lung 

Absolute lung wt (9) 

6 5 6 6 

0.158 f 0.005 0.178 f 0.010 0.195 f 0.010* 0.258 f 0.013** - b 1 f 0.2* 2 f 0.1** 4 f 0.5** 

3 f 0.9* 11 f 0.8** 17 f 1.6** 

4 f 1.0* 14 f 0.8** 28 f 3.2** 

- 

- 

6 5 5 6 

5 f 0.4** 14 f 0.8** 41 f 2.9** 

6 f 0.4** 13 f 1.0** 23 f 1.4** 

1 f 0.1** 2 f 0.1** 7 f 0.5** 

0.163 f 0.004 0.139 f 0.007 0.170 f 0.011 0.298 f 0.012** - 

- 

- 

a Mean f standard error; statistical tests were performed on unrounded data. 

** P10.01 

* Significantly different (P10.05) from the control group by Williams’ test (lung weight) or Shirley’s test (lung burden parameters) 

Results were below 0.255 pg Ni (the limit of detection). 
Tissue Burden in Mice 329 

Lung Weight and Lung Burden in Mice in the 2-Year Inhalation Study of Nickel Subsulfidea 

TABLE I4 

0 mg/m’ 0.6 mg/m3 1.2 mg/m3 

n 

Male 

pg Nilg control lung 

pg Ni/g lung 

pg Nillung 

Absolute lung wt (g) 

7-Month interim evaluation 

pg Ni/g control lung 

pg Nilg lung 

pg Ni/lung 

Absolute lung wt (g) 

15-Month interim evaluation 

Female 

pg Ni/g control lung 

pg Ni/g lung 

pg Nillung 

Absolute lung wt (g) 

7-Month interim evaluation 

pg Nilg control lung 

pg Nilg lung 

pg Nillung 

Absolute lung wt (g) 

15-Month interim evaluation 

5 

0.219 2 0.015b 

- C 

0.184 f 0.009 

- 

- 

- 

0.247 f 0.026 

- 

- 

- 

0.178 f 0.011 

- 

- 

- 

5 

15 jI OS** 

10 f 0.2** 

3 2 0.1** 

0.311 f 0.016** 

24 f 1.9** 

12 +_ 0.7** 

4 f 0.3** 

0.354 f 0.017** 

0.325 f 0.020* 

13 f 0.4** 

10 f 0.6** 

3 f 0.1** 

0.346 f 0.019** 

30 f 4.9** 

15 f 0.7** 

5 f 0.9** 

5 

0,406 f 0.018** 

20 f 1.6** 

11 f 1.2* 

4 f 0.3** 

44 f 4.0** 

20 & 0.9** 

8 f 0.7** 

0.410 f 0.017** 

0.358 f 0.012** 

19 f 1.0** 

14 * 1.0** 

5 * 0.2** 

61 f 7.4** 

26 f 2.2** 

11 f 1.3** 

0.414 f 0.020** 

a Mean f standard error; statistical tests were performed on unrounded data. 

** PSO.01 

* Significantly different (P 50.05) from the control group by Williams’ test (lung weight) or Dunn’s or Shirley’s test (lung burden 

parameters) 

interim evaluation. 

Results were below 0.0037 pg Ni (the limit of detection) for the 7-month interim evaluation and below 0.0034 Kg Ni for the 15-month 

n=4 
330 Nickel Subsulfide, NTP TR 453 

Kidney Burden in Mice in the 2-Year Inhalation Study of Nickel Subsulfidea 

TABLE I5 

0 mg/mJ 0.6 mg/m3 1.2 mg/m3 

Male 

7-Month interim evaluation 

n 

pg Nilg kidney 

15-Month interim evaluation 

n 

pg Ni/g kidney 

Female 

’7-Month interim evaluation 

n 

pg Ni/g kidney 

15-Month interim evaluation 

n 

pg Ni/g kidney 

0.015 f 0.012 

5 

0.025 f 0.018 

4 

0.097 f 0.028 

5 

0.874 f 0.632 

5 

0.061 f 0.014* 

5 

0.065 f 0.014 

5 

0.076 f 0.006** 

5 

0.082 f 0.014 

5 

0.097 f 0.024 

5 

0.130 f 0.061 

5 

0.258 f 0.054* 

5 

0.364 f 0.107 

5 

a Mean f standard error; statistical tests were performed on unrounded data. 

** P10.01 

* Significantly different (PlO.05) from the control group by Dum’s or Shirley’s test 
331 

AND ESTROUS CYCLE CHARACTERIZATION 

REPRODUCTIVE TISSUE EVALUATIONS 

APPENDIX J 

TABLE J1 Summary of Reproductive Tissue Evaluations and Estrous Cycle 

of Nickel Subsulfide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 

Characterization for Mice in the 13-Week Inhalation Study 

of Nickel Subsulfide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332 

Characterization for Rats in the 13-Week Inhalation Study 

TABLE 52 Summary of Reproductive Tissue Evaluations and Estrous Cycle 
332 Nickel Subsulfide, NTP TR 453 

in the 13-Week Inhalation Study of Nickel Subsulfidea 

Summary of Reproductive Tissue Evaluations and Estrous Cycle Characterization for Rats 

TABLE J1 

O mg/m3 0.15 mg/m3 0.6 mg/m3 2.5 mg/m3 

Male 

n 10 10 10 10 

Weights (9) 

R. testis 

R. epididymis 

R. cauda 

1.438 f 0.035 

0.513 f 0.015 

0.126 f 0.005 

1.478 f 0.032 

0.519 f 0.011 

0.122 * 0.006 

1.385 f 0.020b 

0.490 f 0.021 

0.137 * 0.008 

1.477 f 0.026 

0.533 f O.OIOb 

0.126 f 0.007 

Epididymal spermatozoal parameters 

Concentration (106/g cauda epididymal tissue) 

Abnormal (%) 

Motility (%) 

713 f 39 

0.640 f 0.154 

94.81 f 0.73 

0.500 f 0.091 

93.83 f 0.98 

0.500 f 0.061 

94.16 & 1.03 

0.500 f 0.080 

93.72 f 0.53 

696 * 53 736 f 50 742 f 33 

Female 

Estrous stages (% of cycle) 

Estrous cycle length (days)‘ 

n 9 

5.00 f 0.29d 

Metestrus 

Estrus 

Proestrus 

Diestrus 

12.9 

30.0 

21.4 

35.7 

9 10 10 

4.89 f 0.26d 5.20 f 0.20 5.00 f 0.21 

21.4 

24.3 

18.6 

35.7 

18.6 

30.0 

18.6 

32.9 

17.1 

38.6 

12.9 

31.4 

a Data are presented as mean f standard error. Differences from the control group for all study parameters are not significant by Dunn’s 

n=9 

or Dunned’s test. 

‘ There is no evidence of any differences between the exposed and control groups in cycle length or in relative length of time spent in 

Estrous cycle was longer than 7 days or was unclear in 1 of 10 animals. 

estrous stages. 
Sperm Morphology and Vaginal Cytology Evaluations 333 

in the 13-Week Inhalation Study of Nickel Subsulfidea 

Summary of Reproductive Tissue Evaluations and Estrous Cycle Characterization for Mice 

TABLE 52 

0 mg/m3 0.15 mg/m3 0.6 mg/m3 2.5 mg/m3 

Male 

n 8 10 8 10 

Weights (g) 

R. testis 

R. epididymis 

R. cauda 

0.110 .003 0.107 * 0.005 0.113 + 0.006 0.121 f 0.008 

0.048 f 0.002 0.046 f 0.003 0.046 f 0.002c 0.044 f 0.001 

0.013 f 0.001 0.011 0.012 f 0.001b 0.012 f 0.000c f 0.000 

Epididymal spermatozoal parameters 

Concentration (1OVg cauda epididymal tissue) 

Abnormal (%) 

Motility (%) 90.70 f 0.38 91.93 f 0.73 91.75 f 0.94 91.55 f 0.84 

1.53 f 0.16 1.02 f 0.10 1.30 f 0.23 1.26 f 0.14 

1,228 f 173 1,244 k 140 1,396 f 111 1,406 f 48 

Female 

Estrous stages (W of cycle) 

Estrous cycle length (days)d 

n 10 6 9 6 

4.50 f 0.31 4.00 * 0.26e 4.22 f 0.15 4.50 * 0.22f 

Metestrus 

Estrus 

Proestrus 

Diestrus 

24.3 

37.1 

12.9 

25.7 

21.4 

26.8 

14.3 

37.5 

23.8 

42.9 

12.7 

20.6 

18.4 

51.0 , 

14.3 

16.3 

Estrous cycle was longer than 7 days or was unclear in 1 of 7 animals. 

Estrous cycle was longer than 7 days or was unclear in 2 of 8 animals. 

estrous stages. 

There is no evidence of any differences between the exposed and control groups in cycle length or in relative length of time spent in 

n=7 

n=9 

or Dunnett’s test. 

Data are presented as mean f standard error. Differences from the control group for all study parameters are not significant by Dunn’s 
334 Nickel Subsulfide, NTP TR 453 
335 

GENERATION OF CHAMBER CONCENTRATIONS 

CHEMICAL CHARACTERIZATION AND 

APPENDIX K 

PROCUREMENT AND CHARACTERIZATION OF NICKEL SUBSUL~E 

FIGURE K7 Daily Mean Filter Concentrations and Standard Deviations 

FIGURE K6 2-Year Nickel Subsulfide Inhalation Suite 

FIGURE W 13-Week Nickel Subsulfide Inhalation Suite 

FIGURE K4 Schematic of the HlOOO and H2000 Exposure Chambers 

FIGURE K3 Schematic of the Nickel Subsulfide Aerosol Delivery System 

FIGURE K2 Schematic of the Generation System 

FIGURE K1 Infrared Absorption Spectrum of Nickel Subsulfide 

CHAMBER ATMOSPHERE CHARACTEREATION 

AEROSOL CONCENTRATION MONITORING 

TABLE K4 Summary of Aerosol Size Measurements for the 0.6 and 1.2 mg/m3 Mouse Chambers 

TABLE K3 Summary of Aerosol Size Measurements for the 0.15 and 1 mg/m3 Rat Chambers 

TABLE K2 Summary of Aerosol Size . Measurements for the Rat and Mouse Chambers 

TABLE K1 Summary of Aerosol Size Measurements for the Rat and Mouse Chambers 

FIGURE K10 Weekly Mean Filter Concentrations and Standard Deviations 

FIGURE K9 Weekly Mean Filter Concentrations and Standard Deviations 

FIGURE K8 Daily Mean Filter Concentrations and Standard Deviations 

....................... 336 

345 

344 

343 

342 

341 

340 

339 

338 

352 

351 

350 

350 

349 

348 

347 

346 

AEROSOL GENERATION AND EXPOSURE SYSTEM .................................. 337 

....................................... .................................... ....................... ................................. ................. .................... ............................ 

............................. 

in the 2-Year Inhalation Study of Nickel Subsulfide 

in the 2-Year Inhalation Study of Nickel Subsulfide 

in the 13-Week Inhalation Studies of Nickel Subsulfide 

in the 16-Day Inhalation Studies of Nickel Subsulfide 

in the 2-Year Inhalation Study in Mice 

in the 2-Year Inhalation Study in Rats 

in the 13-Week Inhalation Study in Mice 

in the 13-Week Inhalation Study in Rats .............................. 

.............................. 

............................... 

............................... 

...................... 

..................... 

....................... 

....................... 
336 Nickel Subsulfide, NTP TR 453 

CHEMICAL CHARACTERIZATION AND 

GENERATION OF CHAMBER CONCENTRATIONS 

Institute of Environmental Health Sciences. 

MO). Reports on analyses performed in support of the nickel subsulfide studies are on file at the National 

analyses were conducted by the analytical chemistry laboratory, Midwest Research Institute (Kansas City, 

blend of the three lots (identified as lot M082288) was used during the 2-year studies. Identity and purity 

(HF-1517, I080786 and 1072588). Lot HF-1517 was used during the 16-day and 13-week studies, and a 

Nickel subsulfide was obtained from International Nickel Company, Ltd. (Toronto, Ontario), in three lots 

PROCUREMENT AND CHARACTERIZATION OF NICKEL SUBSULFIDE 

purity of the bulk chemical. 

800 ppm before and after milling. These analyses indicated that milling did not significantly alter the 

difference was not considered significant. Spark source mass spectrometry indicated total impurities of 

before and after milling indicated that there was a higher content of sulfur oxides after milling, but this 

elemental analyses before and after milling were in agreement. Results of analysis by ion chromatography 

by Accu-Labs Research, Inc. (Wheat Ridge, CO), before and after milling of the chemical. Results of 

chromatography by Galbraith Laboratories, Inc. (Knoxville, TN), and by spark source mass spectrometry 

Mills, Inc., Boston, MA). Analyses of lot HF-15 17 were performed by elemental analyses and ion 

All lots were subjected to particle size reduction with a 4-inch micronizer prior to use (Sturtevant 

literature. 

visible spectrum was consistent with that expected for the structure. No spectra were found in the 

spectroscopy. No absorbance was observed with infrared spectroscopy (Figure Kl), and the ultraviolet/ 

acid. The chemical was also identified as nickel subsulfide by infrared and ultraviolet/visible 

The chemical, a gray or black powder with a melting point greater than 300" C, was soluble in nitric 

and a calomel reference electrode filled with saturated potassium chloride. 

aqueous 0.1 N iron (111) chloride. The titration was monitored with a platinum disc indicating electrode 

hydroxide. Ethylenediaminetetraacetate (EDTA) was added, and the samples were back-titrated with 

70% nitric acid for 1 hour, then adjusted to pH 5 with sodium acetate trihydrate and ammonium 

chromatography, and chelometric titration. For chelometric titration, samples were dissolved in 

The purity of lot HF-1517 was determined by elemental analyses, Karl Fischer water analysis, ion 

97.0% _+ 1.1 % nickel subsulfide. The overall purity of lot HF-15 17 was determined to be at least 97 % 

thiosulfate were detected by ion chromatography. Chelometric titration indicated a purity of 

indicated 0.09% f 0.01 % water. Only trace amounts (less than 70 ppm) of sulfate, sulfite, and 

sulfur were in agreement with the theoretical values for nickel subsulfide. Karl Fischer water analysis 

Elemental analysis values for nickel in lot HF-15 17 were slightly low. Elemental analysis results for 

98.0% & 1.7% nickel subsulfide. The overall purity of lot M082288 was determined to be at least 98%. 

phosphorus (335 ppm), and chromium (300 ppm). Chelometric titration indicated a purity of 

or equal to 2,620 ppm; the major inorganic impurities were silicon (1,200 ppm), iron (470 ppm), 

indicated 0.06% +_ 0.01 % water. Spark source mass spectrometry indicated total impurities of less than 

values were slightly low for nickel and high for sulfur in lot M082288. Karl Fischer water analysis 

same as described for lot HF-1517 except that the samples were adjusted to pH 6. Elemental analysis 

analysis, spark source mass spectrometry, and chelometric titration. The chelometric titration was the 

The purity of the combined lot M082288 was determined by elemental analyses, Karl Fischer water 
Chemical Characterization and Generation of Chamber Concentrations 337 

This insoluble material was identified as sulfur by spark source mass spectrometry. 

An insoluble impurity ( - 1 %) was found in both lots during the ultraviolet/visible spectroscopic analyses. 

protected from light under an inert gas headspace at room temperature. 

ensure stability, the analytical chemistry laboratory recommended that the bulk chemical be stored 

properties as evidenced by the high melting point and extreme conditions necessary for dissolution. To 

A chemical stability study was not performed for nickel subsulfide because of its inert refractory 

there was no degradation of the bulk chemical during the studies. 

analytical chemistry laboratory. The result was considered to be an aberration, and it was concluded that 

occasion out of 10 analyses, values for nickel in the samples fell outside the range recommended by the 

every 3 to 4 months during the 2-year studies by elemental analyses for nickel and sulfur. On one 

chemical was performed by Huffman Laboratories, Inc. (Golden, CO), prior to and after all studies and 

The bulk chemical was stored in amber glass bottles at room temperature. Periodic monitoring of the bulk 

aerosol delivery system is shown in Figure K3. 

in hood. Air was circulated through HEPA filters to remove suspended particles in the enclosure. The 

depended on the aerosol concentration desired. The aerosol generation assembly was enclosed in a walkbed and maintain a stable aerosol concentration. The amount of nickel subsulfide added to each bed 

loaded into a V-tube mixer and was mixed with a sufficient amount of nickel subsulfide to replenish the 

depleted stainless steel bed material, collected in an enclosed container at the base of the generator, was 

was continually drained from the FBG through an overflow port located at the side of the generator. The 

generator from a hopper located above the generator; the nickel subsulfide-depleted stainless steel powder 

the exposure chambers. Fresh nickel subsulfide-containing bed material was constantly added to the 

subsulfide aerosol was mixed with diluting air to achieve the desired concentrations and was delivered to 

Kr-85 discharger was placed in the generator to reduce the electrical charge on the aerosol. The nickel 

when air was passed through the FBG released the much smaller nickel subsulfide particles into the air. A 

the flow of air. The nickel subsulfide was added to the stainless steel bed, and the motion of the bed 

FBG contained a bed of stainless steel powder of a diameter too heavy to be carried from the generator in 

Figure K2 shows the schematic of the FBG with the gravity feed and catch pan collection systems. The 

Nickel subsulfide aerosol was generated from 2-inch fluid bed generators (FBGs) during all studies. 

AEROSOL GENERATION AND EXPOSURE SYSTEM 

was placed in the aerosol delivery line to remove coarse aerosol particles. Animal cages were rotated 

aerosol entrance to further mix the aerosol as it entered the chamber. During the 2-year studies, a cyclone 

2107FL-1002, Newark Electronics, Chicago, IL) with a flow rate of 60 ft3/min was placed below the 

was diluted in a radial diluter prior to introduction into the chamber, and a small boxer fan (Model WS 

reduce the spatial variation of aerosol concentration and to increase the uniformity of mixing, the aerosol 

chambers, corresponding to 5.99 to 21.63 (rats) and 12.02 to 18.06 (mice) air changes per hour. To 

studies, the air flow rate was 14.6 f 2.0 ft3/min in the rat chambers and 8.7 f 1.2 ft3/min in the mouse 

7 f. 1 ft3/min in the HlOOO chambers, corresponding to 12 k 2 air changes per hour. In the 2-year 

per hour. In the 13-week studies, the air flow rate was 12 f 2 ft3/min in the H2000 chambers and 

were used to expose the mice. The air flow rate in the 16-day studies corresponded to 10 & 2 air changes 

groups. In the 2-year studies, the H2000 chambers were used to expose the rats, and the HlOOO chambers 

0, 0.15, 0.6, and 2.5 mg/m3 groups, and the HlOOO chambers were used for the 0.3 and 1.2 mg/m3 

used for the 1.2 and 5 mg/m3 groups. During the 13-week studies, the H2000 chambers were used for the 

the H2000 chambers were used for the 0, 0.6, 2.5, and 10 mg/m3 groups, and the HlOOO chambers were 

Aberdeen, MD) were used to expose the rats and mice in these studies (Figure K4). In the 16-day studies, 

Stainless steel, multi-tiered, whole-body exposure chambers (H1000 and H2000, Hazleton Systems, 
338 Nickel Subsulfide, NTP TR 453 

Figures K5 and K6, respectively. I 

the 13-week and 2-year studies. Diagrams of the 13-week and 2-year exposure suites are shown in 

weekly to reduce the variation of concentrations of aerosols that the animals were being exposed to during 

particle/m3 in the mouse control chamber. 

suspended particles were 0.02 f 0.01 mg particle/mg3 in the rat control chamber and 0.0 1 f 0.0 1 mg 

0.08 mg particles/m3 in the 13-week studies. In the 2-year studies, the mean concentrations of total 

suspended particles in the control chamber was 0.03 mg particles/m3 in the 16-day studies and was 

from the control chambers during the 16-day and 13-week studies. The mean concentration of total 

exposure day of the 2-year studies by collecting one 6-hour filter sample; samples were collected overnight 

background concentrations of total suspended particles in the control chambers were monitored each 

determined from two 3-hour samples (4.5 Umin flow rate), during each 6-hour exposure day. The 

2-hour samples (3 L/min flow rate) from each exposure chamber, except the 0.15 mg/m3 chambers were 

In the 13-week and 2-year studies, the aerosol concentrations were determined gravimetrically from three 

AEROSOL CONCENTRATION MONITORING 

exposure concentrations for the %-year studies are presented in Figures K9 and K10. 

exposure concentrations for the 13-week studies are presented in Figures K7 and K8. Weekly mean 

means and standard deviations of each chamber were calculated for each exposure day. Daily mean 

concentrations were calculated by dividing the mass increment (mg) by the volume sampled (m3); the 

Cahn Instruments, Cerrito, CA) before and after the collection of the samples. The aerosol mass 

on the filters was determined gravimetrically by weighing the filters with an electrobalance (Cahn 29, 

(Zefluor, Gelman, Ann Arbor, MI) during the 2-year studies. The quantity of nickel subsulfide collected 

during the 13-week studies and with 25 mm, Teflon@-coated, fiberglass filters with a pore size of 0.1 pm 

concentration, samples were collected with 25 mm fiberglass filters (Type AE, Gelman, Ann Arbor, MI) 

aerosol generation. The flow rate was monitored with calibrated rotameters. To determine aerosol 

a flow rate of 3 L/min; all samples in the 2-year studies were collected after the initial 8 minutes of 

All samples in the 13-week studies were collected after the initial 15 minutes (Tw) of aerosol generation at 

studies. The RAM-S unit has a self-contained sampling system which operates at 2 Wmin. 

during the 16-day studies and at least 5 minutes of every exposure hour during the 13-week and 2-year 

exposures. The RAM-S was used to monitor each chamber for at least 10 minutes of every exposure hour 

of the aerosol concentrations and to determine the need to adjust the aerosol generation system during 

A continuous aerosol monitor (Model RAM-S, GCA, Co., Bedford, MA) was used to monitor the stability 

sample was at the front of each chamber, and the probe for the RAM-S was at the back of each chamber. 

the middle level of the H2000 and HlOOO chambers above the animal cage. The probe for the filter 

used as an indication of aerosol stability for each exposure day. RAM-S and filter samples were taken at 

each exposure day for each chamber. The coefficient of variation from the RAM-S measurement was 

regression line fitted to the data. The mean and standard deviation of the'concentrations were calculated 

readings (volts) were converted to mass concentration (mg/m3) based on the slope and intercept of the 

gravimetrically. Linear regression analysis was performed monthly on these data, and the RAM-S voltage 

taken during a filter-sampling period were plotted versus the aerosol concentration determined 

against the mass concentration obtained gravimetrically. The average of three RAM-S voltage readings 

Aerosol concentration was also quantitated with the RAM-S. The RAM-S voltage output was calibrated 
Chemical Characterization and Generation of Chamber Concentrations 339 

rate. The results are presented in Tables K1 through K4. 

deviation were calculated from the mass data, effective cutoff diameter of each stage, and impactor flow 

after the sample was collected. The mass medium aerodynamic diameter and the geometric standard 

amount of nickel subsulfide on each stage was determined by the difference in stage weight before and 

concentration. Stainless steel shimstock coated with apiezon grease was used as impactor substrate. The 

rate of 15 L/min. The sampling period ranged from 1 to 6 hours depending on the chamber 

the 2-year studies for each exposure chamber with a Lovelace multijet cascade impactor operated at a flow 

Aerosol size distribution was determined once during the ldday and 13-week studies and monthly during 

CHAMBER ATMOSPHERE CHARACTERIZATION 

structure of the bulk chemical. 

Standards. The data supports the conclusion that the generation system does not change the crystal 

conformed to the standard pattern (# 8-126) published by the Joint Committee on Powder Diffraction 

diffraction analyses. The nickel contents of both samples were identical and their diffraction patterns 

The generated aerosol was sampled and compared to the bulk sample by atomic absorption and X-ray 

3.17 % , and the mean spatial variations were between 2.06 % and 2.60 % . and 3.09 % . For mice in the 2-year studies, the mean temporal variations were between 2.91 % and 

during exposure were between 1.63 % and 2.3 1 % , and the mean spatial variations were between 1.84 % 

1.7% to 8.8%. For rats in the 2-year studies, the mean temporal variations of aerosol concentration 

variation of the three reference samples. In the 13-week studies, the spatial variations ranged from 

variation of samples collected at different locations, and the temporal variation is the coefficient of 

collected for the other locations. The total variation of aerosol concentrations is the coefficient of 

reference point in each chamber at the start, middle, and end of the procedure. One sample each was 

checked quarterly during the 2-year studies. Three samples were collected with the RAM-S at a specified 

studies without animals in the chambers and with animals during the first week of exposure, and was 

Uniformity of aerosol concentration in the exposure chambers was measured prior to the start of the 

the 13-week studies, and 8 minutes was used during the 2-year studies. 

6 hours plus T,. A T, of 10 minutes was used during the 16-day studies, 15 minutes was used during 

The aerosol rise and fall time (T,) wa; determined with a RAM-S, and an exposure day was set at 

non-nickel-containing material. 

indicated that the aerosol collected on the filter was not solely nickel subsulfide, but consisted chiefly of 

November 1989). Results of chemical analysis of these filter samples by atomic absorption spectroscopy 

study exceeded 200 pg four times but was only analyzed for nickel content twice (August 1989 and 

the 2-year rat study never exceeded 200 pg. The mass of the particles collected during the 2-year mouse 

spectroscopy. The mass of the particles collected on the filter during the 16-day and 13-week studies and 

was greater than 200 pg, the material collected was analyzed for nickel content by atomic absorption 

rat chamber and from the 1.2 mg/m3 mouse chamber. If the weight of the material collected on the filter 

chamber during the 13-week studies. During the 2-year studies, samples were collected from the 1 mg/m3 

overnight (about 15 hours) at a flow rate of 3 Wmin. Samples were collected from the 2.5 mg/m3 

2 weeks of exposure and quarterly thereafter during the 2-year studies. The filter samples were collected 

gravimetrically once during the 16-day studies, once during the 13-week studies, and once during the first 

Residual concentration of nickel subsulfide in the chambers during nonexposure hours was evaluated 
340 Nickel Subsulfide, NTP TR 453 

100 

110 I I l~llll I I I I I I 

- 

80 - - 

h 

$- 

C 

- 

E 

? 40- 

.g 60 - 

C 

v) 

v) .- 

- 

20 - - 

- 

4001 3600 3200 2800 2400 2000 1600 1200 800 651 

0 11, IIIIII I 1 I I I I 

Wavenumber 

SAMPLE 

252 N 

S. Tippin 

CONCENTRATION OPERATOR DATE 

Potassium 

Lot No.: M08228 

Nickel subsulfide -2% in KBr 1 I1 8/89 

REFERENCE NO. 

ORIGIN 

bromide pellet 

FIGURE K1 

Infrared Absorption Spectrum of Nickel Subsulfide 
Chemical Characterization and Generation of Chamber Concentrations 341 

FEED 

HOPPPER 

COMPRESSED 

METER 

ORIFICE 

DISTRIBUTION PLATE 

PRESSURE P CHARGER 

DISK r-85_ 

\\ 

AEROSOL 

Schematic of the Generation System 

FIGURE K2 
342 Nickel Subsulfide, NTP TR 453 

AEROSOL DYUT)<)N/OELIVERY SYSTEM 

I 

1 T 

r-1 - EXt4AUSl 

FIGURE K3 

Schematic of the Nickel Subsulfide Aerosol Delivery System 
Chemical Characterization and Generation of Chamber Concentrations 

11 

INPUT AIR 

1 DlSTRlBUTlON PLATE An 

343 

RODENT CAGE UNITEXCRETACATCHPAN 1 

EXHAUST '+r 

EXHAUST 

Schematic of the HlOOO and H2000 Exposure Chambers 

FIGURE K4 
344 Nickel Subsulfide, NTP TR 453 

ROOM 257 

r 

CH 1 CH 2 CH 3 CH 4 CH 5 

FOR CONTROL CHAMBER 

FLUID BED GENERATOR 

,BENCH TOP 

/BALANCE 

13-Week Nickel Subsulfide Inhalation Suite 

FIGURE KS 
Chemical Characterization and Generation of Chamber Concentrations 345 

ROOM 257 

n 

CH4 CH5 I 

DOOR 1 

WALK-IN HOOD BENCH TOP V r 

0 FBG3 OFBG2 \ 

AND BLOWERS 

HEPA FILTERS 0 i 

BALANCE/ 

-FBG 1 

2-Year Nickel Subsulfide Inhalation Suite 

FIGURE K6 
346 Nickel Subsulfide, NTP TR 453 

4 

0 

C I T 

EXPOSURE DAY 

in the 13-Week Inhalation Study in Rats 

Daily Mean Filter Concentrations and Standard Deviations 

FIGURE K7 
Chemical Characterization and Generation of Chamber Concentrations 347 

A 1 

EXPOSURE DAY 

in the 13-Week Inhalation Study in Mice 

Daily Mean Filter Concentrations and Standard Deviations 

FIGURE K8 
348 Nickel Subsulfide, NTP TR 453 

1.4 

1.2 

c 1.0. 

N 

0 

C 

N 

0 0.6. 

I 

T 

A 

R 0.8. 

T 

N 

E 

3 

M 

I 

C 0.4. 

M 

0.2 

0.0 

T 

20 40 60 80 1 bo 

WEEK ON STUDY 

in the 2-Year Inhalation Study in Rats 

Weekly Mean Filter Concentrations and Standard Deviations 

FIGURE K9 
Chemical Characterization and Generation of Chamber Concentrations 

T 1.0 

A 

A 

T 

N 

E 

C 

N 

0 

C 

3 

H 

I 

0.5 

40 

-- 60 80 1 bo 

WEEK ON STUDY 

349 

FIGURE K10 

in the 2-Year Inhalation Study in Mice 

Weekly Mean Filter Concentrations and Standard Deviations 
Nickel Subsulfide, NTP TR 453 

in the 16-Day Inhalation Studies of Nickel Subsulfide 

Summary of Aerosol Size Measurements for the Rat and Mouse Chambers 

TABLE K1 

(mg/m3) 

Concentration 

Target 

Diameter (pm) 

Aerodynamic 

Mass Median 

Deviation 

Standard 

Geometric 

10 

5 

2.5 

1.2 

0.6 

2.79 

2.65 

2.91 

2.93 

2.57 1.86 

1.87 

1.95 

2.12 

2.09 

in the 13-Week Inhalation Studies of Nickel Subsulfide 

Summary of Aerosol Size Measurements for the Rat and Mouse Chambers 

TABLE K2 

(mdm3) 

Concentration 

Target 

Diameter (pm) 

Aerodynamic 

Mass Median 

Deviation 

Standard 

Geometric 

0.15 

2.5 

1.2 

0.6 

0.3 

2.40 

2.71 

2.16 

2.37 

2.60 

2.70 

2.23 

2.17 

2.12 

1.99 
Chemical Characterization and Generation of Chamber Concentrations 

in the 2-Year Inhalation Study of Nickel Subsulfide 

Summary of Aerosol Size Measurements for the 0.15 and 1 mg/m3 Rat Chambers 

TABLE K3 

351 

Date 

0.15 mg/m’ 

Mass Median Geometric 

Aerodynamic Standard 

Diameter (pm) Deviation 

1 mdm’ 

Mass Median Geometric 

Aerodynamic Standard 

Diameter (pm) Deviation 

October 1990 

September 1990 

August 1990 

July 1990 

June 1990 

May 1990 

April 1990 

March 1990 

February 1990 

January 1990 

December 1989 

November 1989 

October 1989 

September 1989 

August 1989 

July 1989 

June 1989 

May 1989 

April 1989 

March 1989 

February 1989 

January 1989 

December 1988 

November 1988 

deviation 

Mean f standard 

1.98 

1.79 

1.98 

2.33 

1.76 

1 .I2 

2.55 

2.19 

1.87 

1.96 

2.58 

1.65 

2.17 

2.38 

1.91 

2.10 

2.11 

2.65 

2.19 

2.99 

1.99 

2.51 

2.21 

2.54 

2.50 

2.38 

2.24 

2.15 

2.76 

2.33 

2.08 

2.23 

2.78 

2.09 

2.51 

2.09 

2.51 

1.85 

3.47 

2.22 

1.82 

1.77 

2.93 

2.02 

2.69 

2.05 

2.18 

2.54 

2.17 f 0.34 2.34 f 0.39 

1.88 

1.97 

2.05 

1.83 

1.94 

1.95 

2.00 

2.15 

1.92 

2.26 

2.60 

2.06 

2.31 

2.17 

2.12 

1.89 

1.93 

1.91 

1.89 

1.80 

2.10 

2.06 

2.06 

2.01 

1.98 

1.95 

1.99 

1.98 

1.87 

1.90 

1.67 

2.02 

1.98 

2.06 

2.15 

1.86 

1.88 

1.97 

1.63 

1.91 

1.81 

2.22 

2.29 

2.03 

1.84 

2.26 

1.69 

1.89 

2.03 f 0.18 1.95 f 0.17 
Nickel Subsulfide, NTP TR 453 

in the 2-Year Inhalation Study of Nickel Subsulfide 

Summary of Aerosol Size Measurements for the 0.6 and 1.2 mg/m3 Mouse Chambers 

TABLE K4 

Date 

0.6 mg/m3 

Mass Median Geometric 

Aerodynamic Standard 

Diameter (pm) Deviation 

1.2 malm3 

Mass Median Geometric 

Aerodynamic Standard 

Diameter (pm) Deviation 

September 1990 

August 1990 

July 1990 

June 1990 

May 1990 

April 1990 

March 1990 

February 1990 

January 1990 

December 1989 

November 1989 

October 1989 

September 1989 

August 1989 

July 1989 

June 1989 

May 1989 

April 1989 

March 1989 

February 1989 

January 1989 

December 1988 

November 1988 

October 1988 

deviation 

Mean f standard 

2.24 

2.09 

2.19 

1.90 

2.14 

2.33 

1.99 

2.01 

1.84 

2.31 

1 .77 

1.96 

2.09 

2.28 

1.76 

2.27 

2.95 

2.31 

2.77 

2.74 

2.42 

2.39 

2.63 

2.35 

1.79 

1.94 

1.98 

1.93 

1.85 

1.82 

2.08 

2.16 

2.25 

2.32 

1.95 

1.90 

2.26 

1.98 

1.91 

2.03 

1.79 

2.06 

1.79 

1.99 

2.01 

2 .oo 

1.73 

1.91 

2.24 f 0.31 1.98 f 0.16 

2.34 

2.22 

1.86 

2.14 

2.05 

2.24 

1.98 

1.88 

2.08 

2.72 

2.06 

2.28 

2.05 

1.97 

1.93 

2.54 

2.09 

1.94 

2.38 

2.26 

2.19 

2.45 

2.37 

2.32 

1.76 

1.85 

2 .oo 

1.82 

1.85 

1.76 

1.99 

1.96 

2.04 

2.01 

1.88 

1.92 

2.05 

1.98 

2.00 

1.84 

1.66 

2.03 

1.99 

2.16 

1.74 

1.85 

1.80 

1.95 

2.18 * 0.22 1.91 f 0.12 
353 

AND CONTAMINANT LEVELS 

INGREDIENTS, NUTRIENT COMPOSITION, 

APPENDIX L 

IN NIH-07 RAT AND MOUSE RATION 

TABLE L1 Ingredients of NIH-07 Rat and Mouse Ration 

TABLE L2 Vitamins and Minerals in NIH-07 Rat and Mouse Ration 

TABLE L3 Nutrient Composition of NIH-07 Rat and Mouse Ration 

TABLE L4 Contaminant Levels in NIH-07 Rat and Mouse Ration 356 

355 

354 

........................... 354 

................... 

.................... 

..................... 
354 Nickel Subsulfide, NTP TR 453 

Ingredients of NIH-07 Rat and Mouse Rationa 

TABLE L1 

Ingredientsb Percent by Weight 

Premixes (vitamin and mineral) 

Salt 

Ground limestone 

Dicalcium phosphate 

Dry molasses 

Dried brewer’s yeast 

Soy oil 

Corn gluten meal (60% protein) 

Alfalfa meal (dehydrated, 17% protein) 

Dried skim milk 

Wheat middlings 

Fish meal (60% protein) 

Soybean meal (49% protein) 

Ground hard winter wheat 

Ground #2 yellow shelled corn 

10.00 

10.00 

12.00 

23.00 

24.50 

0.25 

0.50 

0.50 

1.25 

1 SO 

2 .oo 

2 SO 

3 .OO 

4.00 

5 .OO 

a NCI, 1976; NIH, 1978 

Ingredients were ground to pass through a U.S. Standard Screen No. 16 before being mixed. 

Vitamins and Minerals in NM-07 Rat and Mouse Rationa 

TABLE L2 

Amount Source 

Vitamins 

A 

K3 

D3 

Thiamine 

Riboflavin 

&Pantothenic acid 

Niacin 

Folic acid 

Choline 

d-a-Tocopheryl acetate 

Biotin 

Pyridoxine 

Bl2 

Cobalt 

Iodine 

Copper 

Zinc 

Manganese 

Iron 

Minerals 

140.0 mg 

1.7 g 

4,000 pg 

10.0 g 

3.4 g 

18.0 g 

30.0 g 

2.2 g 

560.0 g 

20,000 IU 

2.8 g 

4,600,000 IU 

5,500,000 IU 

16.0 g 

60.0 g 

120.0 g 

0.4 g 

1.4 g 

4.0 g 

Menadione 

D-activated animal sterol 

Stabilized vitamin A palmitate or acetate 

Choline chloride 

Thiamine mononitrate 

&Calcium pantothenate 

&Biotin 

Pyridoxine hydrochloride 

Cobalt carbonate 

Calcium iodate 

Copper sulfate 

Zinc oxide 

Manganous oxide 

Iron sulfate 

-~ ~ 

a Per ton (2,000 lb) of finished product 
Feed Analyses 

Nutrient Composition of NIH-07 Rat and Mouse Ration 

TABLE L3 

355 

Nutrient Deviation 

Mean f Standard 

Range Number of Samples 

Ash (% by weight) 

Crude fiber (% by weight) 

Crude fat (% by weight) 

Protein (% by weight) 

Amino Acids (% of total diet) 

Valine 

Tyrosine 

Tryptophan 

Threonine 

Phenylalanine 

Methionine 

Lysine 

Leucine 

Isoleucine 

Histidine 

Glycine 

Cystine 

Arginine 

Essential Fatty Acids (% of total diet) 

Linolenic 

Linoleic 

Vitamins 

Choline (ppm) 

Vitamin B,, (ppb) 

Biotin (ppm) 

Folic acid (ppm) 

Pyridoxine (ppm) 

Pantothenic acid (ppm) 

Niacin (pprn) 

Riboflavin (ppm) 

Thiamine (ppm) 

a-Tocopherol (ppm) 

Vitamin D (IU/kg) 

Vitamin A (IU/kg) 

Minerals 

Sulfur (%) 

Magnesium (%) 

Sodium (%) 

Chloride (%) 

Potassium (%) 

Phosphorus (%) 

Calcium (%) 

Cobalt (ppm) 

Chromium (ppm) 

Iodine (ppm) 

Copper (ppm) 

Zinc (ppm) 

Manganese (ppm) 

Iron (ppm) 

6.50 f 0.22 

3.60 f 0.43 

5.30 f 0.23 

23.20 f 0.70 

1.089 f 0.057 

0.677 f 0.105 

0.223 f 0.160 

0.896 f 0.055 

0.994 f 0.125 

0.431 f 0.115 

1.273 f 0.051 

1.972 f 0.052 

0.917 f 0.034 

0.580 f 0.024 

1.160 f 0.050 

0.306 f 0.075 

1.287 f 0.084 

0.277 f 0.036 

2.389 f 0.233 

19.20 f 2.40 

e36.92 -I 9.32 

4,450 f 1,382 

6,537 -I 2,005 

3.068 & 314 

40.14 If 20.04 

0.267 f 0.049 

2.51 f 0.64 

9.25 f 2.62 

30.30 f 3.60 

100.95 f 25.92 

7.92 f 0.93 

11.50 f 2.40 

58.14 f 9.91 

92.24 f 5.35 

356.2 f 90.0 

0.274 f 0.063 

0.168 f 0.008 

0.315 f 0.034 

0.526 f 0.092 

0.887 +_ 0.067 

0.95 f 0.03 

1.21 f 0.10 

0.797 f 0.23 

1.71 f 0.45 

3.70 f 1.14 

6.10 - 6.90 

2.80 - 4.30 

4.60 - 5.60 

21 .80 - 24.20 

0.962 - 1,170 

0.564 - 0.794 

0.107 - 0.671 

0,824 - 0.985 

0.665 - 1.110 

0.306 - 0.699 

1.200 - 1.370 

1.850 - 2.040 

0.867 - 0.965 

0.531 - 0.608 

1,060 - 1.220 

0.181 - 0.400 

1,100 - 1.390 

0.210 - 0.320 

1.830 - 2.570 

2,400 - 3,430 

10.6 - 65.0 

0.19 - 0.35 

1.80 - 3.70 

5.60 - 14.0 

23.0 - 34.6 

65.0 - 150.0 

6.10 - 9.00 

16.0 - 28.0 

22.5 - 48.9 

3,000 - 6,300 

4,180 - 12,140 

46.10 - 81.60 

81.70 - 99.40 

255.0 - 523.0 

0.208 - 0.420 

0.151 -0,180 

0.258 - 0.370 

0.380 - 0.635 

0.772 - 0.971 

0.89 - 1 .OO 

1.06 - 1.54 

0.49 - 1.15 

0.85 - 2.09 

1.52 - 5.83 

8.09 - 15.39 

22 

22 

22 

22 

10 

10 

10 

10 

10 

10 

10 

10 

10 

10 

10 

10 

10 

9 

9 

10 

10 

10 

10 

10 

9 

10 

22 

9 

4 

22 

9 

6 

9 

10 

10 

10 

10 

10 

10 

10 

10 

8 

8 

22 

22 
356 Nickel Subsulfide, NTP TR 453 

Contaminant Levels in NIH-07 Rat and Mouse Ratio* 

TABLE L4 

Deviationb 

Mean f Standard 

Range Number of Samples 

Nitrite nitro en (ppm)e 

Nitrate nitrogen (ppm)e 

Aflatoxins (ppb)c*d 

Selenium (ppm) 

Merculy (ppm)' 

Lead (ppm) 

Cadmium (ppm) 

Arsenic (ppm) 

Contaminants 

BHT (ppm)' 

BHA (ppm) F 

N-Nitrosopyrrolidine (ppb)g 

N-Nittosodimethylamine (ppb)g 

Total nitrosoamines (ppb)g 

Salmonella (MPN/g) 

Escherichia coli (MPN/g) 

Coliform (MPNlg) 

Aerobic plate count (CFU/g) 

Pesticides (ppm) 

&BHC 

7-BHC 

&BHC 

CX-BHC 

Endosulfan sulfate 

Endosulfan I1 

Endosulfan I 

Malathion 

Ethyl parathion 

Methyl parathion 

Diazinon 

Trithion 

Ethion 

Ronnel 

Estimated PCBs 

Toxaphene 

Chlordane 

Telodrin 

Endrin 

Dieldrin 

Methoxychlor 

Mirex 

HCB 

DDT 

DDD 

DDE 

Heptachlor epoxide 

Aldrin 

Heptachlor 

1.41 f 0.59 

1.41 f 0.59 

0.26 f 0.20 

17.00 f 4.20 

<'5.0 

0.43 f 0.25. 

0.04 * 0.02 

0.23 f 0.09 

0.08 f 0.02 

0.27 f 0.18 

4.7 f 5.6 

42,745 f 26.010 

Negative 

<3.00 

1.89 f 0.93 

6.00 f 2.74 

7.80 f 3.00 

<0.02 

<0.02 

<o. 1 

cO.05 

co.02 

<0.01 

<0.2 

<0.1 

<0.05 

<0.01 

<0.01 

<0.01 

<0.05 

<0.01 

<0.01 

<0.01 

<0.01 

<0.01 

<0.01 

<0.01 

<0.01 

<0.01 

<0.01 

<0.02 

<0.01 

<0.03 

<0.01 

<0.01 

0.23 f 0.23 

0.20 - 1.20 

0.02 - 0.1 1 

0.10 - 0.40 

0.05 - 0.10 

0.06 - 0.60 

6,700 - 120.000 

1 .OO - 3.00 

1 .OO - 3.00 

0.10 - 0.70 

8.60 - 24.0 

3 -23 

1.00 - 3.90 

2.60 - 13.00 

3.60 - 16.50 

< 0.05 - 1 .OO 

22 

22 

22 

22 

22 

22 

22 

21 

22 

22 

22 

22 

22 

22 

22 

22 

22 

22 

22 

22 

22 

22 

22 

22 

22 

22 

22 

22 

22 

22 

22 

22 

22 

22 

22 

22 

22 

22 

22 

22 

22 

22 

22 

22 

22 

g All values were corrected for percent recovery. 

e Sources of contamination: alfalfa, grains, and fish meal. 

' All values were less than the detection limit. 

a CFU = colony forming units, MPN = most probable number, BHC is hexachlorocyclohexane or benzene hexachloride 

Sources of contamination: soy oil and fish meal. 

No aflatoxin measurement was recorded for the lot milled October 2, 1989. 

For values less than the limit of detection, the detection limit is given as the mean. 
357 

SENTINEL ANIMAL PROGRAM 

APPENDIX M 

in the 13-Week and 2-Year Inhalation Studies of Nickel Subsulfide . . . . . . . . . . . . . 360 

TABLE M1 Murine Virus Antibody Determinations for Rats and Mice 

METHODS .......................................................... 358 
358 Nickel Subsulfide, NTP TR 453 

SENTINEL ANIMAL PROGRAM 

source and weanling groups as the animals used for the studies of chemical compounds. 

are subject to identical environmental conditions. The sentinel animals come from the same production 

serology on sera from extra (sentinel) animals in the study rooms. These animals and the study animals 

evaluation of chemical compounds. Under this program, the disease state of the rodents is monitored via 

Animal Program is part of the periodic monitoring of animal health that occurs during the toxicologic 

optimally clean facilities to eliminate potential pathogens that may affect study results. The Sentinel 

Rodents used in the Carcinogenesis Program of the National Toxicology Program are produced in 

METHODS 

performed are tabulated below; the times at which blood was collected during the studies are also listed. 

determination of antibody titers. The laboratory serology methods and viral agents for which testing was 

were processed appropriately and sent to Microbiological Associates, Inc. (Bethesda, MD), for 

studies. Blood from each animal was collected, allowed to clot and the serum separated. The samples 

Serum samples were collected from randomly selected rats and mice during the 13-week and 2-year 

Method and Test Time of Analysis 

ELISA 

13-Week Study 

RATS 

Mycoplasma arthritidis Study termination 

Mycoplasma pulmonis Study termination 

PVM (pneumonia virus of mice) Study termination 

RCV/SDA (rat coronavirus/sialodacryoadenitis virus) Study termination 

Sendai Study termination 

KRV (Kilham rat virus) 

H-1 (Toolan’s H-1 virus) 

Hemagglutination Inhibition 

ELISA 

2-Year Study 

Sendai 

RCV/SDA 

PVM 

M. pulmonis 

M. arthritidis 

Immunofluorescence Assay 

RCVISDA 

Hemagglutination Inhibition 

H- 1 

KRV 

Study termination 

Study termination 

7 months, 15 months, study termination 

7 months, 15 months, study termination 

7 months, 15 months, study termination 

Study termination 

Study termination 

7 months 

7 months, 15 months, study termination 

7 months, 15 months, study termination 
Sentinel Animal Program 359 

Method and Test Time of Analvsis 

Complement Fixation 

13-Week Study 

MICE 

LCM (lymphocytic choriomeningitis virus) 

ELISA 

Sendai 

Reovirus 3 

PVM 

M. pulmonis 

M. arthritidis 

MHV (mouse hepatitis virus) 

Mouse adenoma virus 

GDVII (mouse encephalomyelitis virus) 

Ectromelia virus 

Immunofluorescence Assay 

EDIM (epizootic diarrhea of infant mice) 

Hemagglutination Inhibition 

Polyoma virus 

MVM (minute virus of mice) 

K (papovavirus) 

ELISA 

2-Year Study 

Sendai 

Reovirus 3 

PVM 

M. pulmonis 

M. arthritidis 

MHV 

Mouse adenoma virus 

MVM 

LCM 

GDVII 

EDIM 

Ectromelia virus 

Immunofluorescence Assay 

Sendai 

Reovirus 3 

MHV 

Mouse adenoma virus 

MVM 

LCM 

EDIM 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

7 months, 15 months, study termination 

7 months, 15 months, study termination 

7 months, 15 months, study termination 

Study termination 

Study termination 

7 months, 15 months, study termination 

7 months, study termination 

7 months 

15 months, study termination 

7 months, 15 months, study termination 

Study termination 

7 months, 15 months, study termination 

Study termination 

7 months, study termination 

Study termination 

15 months 

15 months 

7 months, 15 months, study termination 

7 months, 15 months, study termination 
360 Nickel Subsulfide, NTP TR 453 

Method and Test 

Hemagglutination Inhibition 

2-Year Study (continued) 

MICE (continued) 

Polyoma virus 

MVM 

K 

Time of Analysis 

7 months, 15 months, study termination 

Study termination 

7 months, 15 months, study termination 

Results of the serology tests are presented in Table M1. 

of Nickel Subsulfide 

Murine Virus Antibody Determinations for Rats and Mice in the 13-Week and 2-Year Inhalation Studies 

TABLE M1 

Interval 

in Sentinel Animals 

Incidence of Antibody 

Reaction for 

Positive Serologic 

13-Week Studies 

Rats 

Study termination 

Mice 

Study termination 

2-Year Studies 

Rats 

Study termination 

15 Months 

7 Months 

Mice 

Study termination 

15 Months 

7 Months 

01 10 

0/8 

2112 

0/12 

0/12 

0112 

0112 

2/12 

None positive 

None positive 

M. arfhritidisa 

None positive 

None positive 

None positive 

None positive 

Reovirus 3 

a Further evaluation of samples positive for M. nrrhriridis by immunoblot and Western blot procedures indicated that the positive titers 

M. arrhriridis-positive titers were considered to be false positive. 

positive, and there were no clinical signs or histopathologic changes of M. nrrhriridis infection in rats with positive titers. Accordingly, 

may have been due to a cross reaction with antibodies of nonpathogenic Mycoplnsrnn or other agents. Only sporadic samples were 
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