National Cancer Institute

CARCINOGENESIS

Technical Report Series

No. 6

January 1977

BIOASSAYS OF

NITRILOTRIACETIC ACID (NTA) AND

NITRILOTRIACETIC ACID, TRISODIUM SALT,

MONOHYDRATE (Na3NTA•H2O)

FOR POSSIBLE CARCINOGENICITY

CAS No. 139-13-9 (NTA)

CAS No. 18662-53-8 (Na3NTA•H2O)

NCI-CG-TR-6

U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE

Public Health Service

National Institutes of Health

BIOASSAY OF
 

NITRILOTRIACETIC ACID AND
 

NITRILOTRIACETIC ACID, TRISODIUM SALT, MONOHYDRATE
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogen Bioassay and Program Resources Branch
 

Carcinogenesis Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

Bethesda, Maryland 20014
 

DHEW Publication No. (NIH) 77-806
 

BIOASSAY OF
 

NITRILOTRIACETIC ACID AND
 

NITRILOTRIACETIC ACID, TRISODIUM SALT, MONOHYDRATE
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Program, Division of Cancer Cause and Prevention
 

National Cancer Institute
 

CONTRIBUTORS: This report presents the results of carcinogen
 

bioassays of nitrilotriacetic acid and nitrilotriacetic acid,
 

trisodiura salt, monohydrate conducted by the Carcinogen Bioassay
 

and Program Resources Branch, Carcinogenesis Program, Division of
 

Cancer Cause and Prevention, National Cancer Institute (NCI),
 

Bethesda, Maryland. The bioassays were conducted at the Stanford
 

Research Institute, Menlo Park, California, and Litton Bionetics,
 

Inc., Kensington, Maryland, initially under direct contract to
 

the NCI and currently under a subcontract to Tracer Jitco, Inc.,
 

prime contractor for the NCI carcinogen bioassay program.
 

For the work performed at the Stanford Research Instiute (SRI)1,
 

the experimental design, including doses, was determined by
 

collaborative efforts of individuals at NCI (Dr. R. R. Bates1*8),
 

the University of California Medical School (Dr. R. M.
 

Elashoff2), and SRI (Dr. D. C. L. Jones1, Dr. D. P. Sasmore1,
 

Dr. G. W. Newell1, and Mr. W. E. Davis1). The principal
 

investigator for the contract was Dr. D. C. L. Jones; chemical
 

analyses were performed by Dr. R. Spanggord1; the technical
 

supervisor of animal treatment, observation, and data handling
 

was Mr. W. E. Davis1; necropsy and tissue fixation were
 

supervised by Dr. D. P. Sasmore1. Microscopic preparation and
 

histopathologic examination were performed by Dr. W. M. Busey^
 

and Dr. V. J. Rosen^, and the diagnoses included in this report
 

represent the interpretation of these pathologists. Dr. R. A.
 

Squire^ reviewed all diagnoses of tumors of the urinary tract and
 

concurred with the overall pathologic evaluation of the SRI
 

study.
 

At Litton Bionetics, Inc.6, the principal investigators for the
 

contract were Dr. B. M. Ulland^ and Dr. F. M. Garner**. The
 

iii
 
experimental design, including doses, was determined by Dr. B. M.
 

Ulland and the NCI Project Officer, Dr. E. K. Weisburger9.
 

Chemical analyses were performed by Mr. II. J. Paulin6; technical
 

supervisors of animal treatment and observation were Mr. J. D.
 

Farmer6, Ms. H. I. Ruckenbrod6, Mr. D. J. Howard6, and Mr. H. D.
 

Thornett6; data collection and transmittal was performed by
 

Ms. B. L. Herndon6 and Ms. M. J. McGowan6. The histopathologic
 

examinaton was supervised and conducted by Dr. F. M. Garner^ and
 

reviewed by Dr. C. N. Barron^O. Dr. R. A. Squire reviewed the
 

diagnoses of the tumors of the urinary system. Pathologists at
 

Tracor Jitco and NCI concurred with the overall pathologic
 

evaluation of the LBI study.
 

In studies at both laboratories, compilation of individual animal
 

survival and summary tables was performed by EG&G Mason Research
 

Institute^. Pathology tables were compiled by the different
 

research laboratories*»^,6# Statistical analyses were performed
 

by Dr. R. M. Elashoff2.
 

The results of this study were reviewed and this report was
 

prepared at Tracor Jitco. Those responsible for the report at
 

Tracor Jitco*0 were the toxicologist, Dr. J. F. Robens; the
 

technical editor, Dr. E. W. Gunberg; and the technical writers,
 

Ms. L. A. Waitz, Mr. W. D. Reichardt, and Dr. G. L. Miller. The 

final report was reviewed by Dr. Elashoff and members of the 

participating organizations*>6>9»*0. 

Stanford Research Institute, Menlo Park, California.
 

^Dr. Robert M. Elashoff, now with the Department of
 

Biomathematics, University of California, Los Angeles,
 

California.
 

^Experimental Pathology Laboratories, Inc., 17 Pine Street,
 

Herndon, Virginia.
 

^Dr. Victor J. Rosen, David M. Brotman Hospital, 3828 Hughes
 

Avenue, Culver City, California.
 

with Hazleton Laboratories, Inc., 9200 Leesburg Turnpike,
 

Vienna, Virginia.
 

iv
 
^Litton Bionetics, Inc., 5516 Nicholson Lane, Kensington,
 

Maryland.
 

7

EG&G Mason Research Institute, 1530 East Jefferson Street,
 

Rockville, Maryland.
 

&Now with United States Food and Drug Administration.
 

"Carcinogenesis Program, Division of Cancer Cause and Prevention,
 

National Cancer Institute, National Institutes of Health,
 

Bethesda, Maryland.
 

l0Tracor Jitco, Inc., 1776 East Jefferson Street, Rockville,
 

Maryland.
 

SUMMARY
 

Bioassays for the carcinogenicity of nitrilotriacetic acid,
 

trisodium salt, monohydrate (Na3NTA*H20) were conducted at
 

Stanford Research Institute (SRI), using Fischer 344 rats, and at
 

Litton Bionetics, Inc. (LBI), using both Fischer 344 rats, and
 

B6C3F1 mice. Similar bioassays, using rats and mice, were
 

conducted at LBI on the free acid, nitrilotriacetic acid (NTA).
 

Each chemical was mixed in respective diets and administered ad
 

libitum. The Na3NTA-H20 was tested in rats at SRI at 200, 2,000,
 

and 20,000 ppm for a 24-month period. It was also tested in rats
 

at LBI at 7,500 and 15,000 ppm and in mice at 2.500 and 5,000 ppm
 

using 18-month feeding periods for both species. The NTA was
 

tested in rats and mice at LBI at 7,500 and 15,000 ppm for the
 

18-month period. The numbers of animals used in tests at SRI
 

were 24 of each sex for each dose group and for the controls; at
 

LBI, 50 of each sex for each dose group and 20 of each sex for
 

the controls. Since equimolar quantities of Na3NTA*H20 and NTA
 

were not used, given concentrations of Na3NTA#H20 represented 30%
 

less NTA than did equal concentrations of the free acid.
 

Average weights attained by high-dose groups of rats and mice
 

were consistently lower than those of control groups. Less
 

difference was observed with the low-dose groups. Survival,
 

however, was not decreased by the compounds administered, except
 

in rats given 20,000 ppm Na3NTA H2O.
 

Lesions of the urinary tract were found in most treated groups of
 

both rats and mice. They were characterized, especially in the
 

high-dose groups, by primary tumors of epithelial origin. These
 

tumors were particularly significant since they were not found in
 

the urinary tract of the control mice and only rarely occur
 

spontaneously in the strains of animals on test. Lesions of the
 

urinary tract were also characterized by hydronephrosis and/or
 

nephritis in high-dose rats and by nephritis in both high- and
 

low-dose mice.
 

Statistical evidence of the carcinogenicity of Na3NTA*H20 and NTA
 

was provided by incidences of tumors at different sites in the
 

urinary tract. For example, among animals given 20,000 ppm
 

vii
 
at SRI, tumors of the kidney occurred in male
 

(treated, 9/24; untreated, 0/24; P = 0.001) and female (treated,
 

4/24; untreated, 0/24; P « 0.054) rats; tumors of the ureter, in
 

male (treated, 8/24; untreated, 0/24; P - 0.002) and female
 

(treated, 6/24; untreated, 0/24; P * 0.011) rats; and tumors of
 

the bladder, in female rats (treated, 5/24; untreated, 0/22; P =
 

0.031). Similarly, among animals given 15,000 ppm NTA at LBI,
 

tumors of the bladder occurred in female rats (treated, 12/48;
 

untreated, 0/18; P = 0.014) and tumors of the kidney occurred in
 

male mice (treated, 24/44; untreated, 0/20; P < 0.001).
 

Additional tests at LBI, using 15,000 and 7,500 ppm Na3NTA-U20
 

and 7,500 ppm NTA in male and female rats, 15,000 ppm NTA in
 

female mice, and 7,500 ppm NTA in male mice, also induced tumors
 

of the urinary tract, but in numbers too low to be statistically
 

significant. Metastatic tumors, appearing to have arisen from
 

primary tumors of the urinary tract, were found in 5/24 male and
 

5/24 female rats given 20,000 ppm Na3NTA*H20 at SRI and in one
 

male rat given 15, 000 ppm NTA at LBI; none were found in rats
 

given lower doses or in mice.
 

Thus, NTA and NA3NTA*H20 were shown to be carcinogenic to the
 

urinary tracts of both rats and mice at the higher doses tested.
 

Lower doses, as delineated in this report, did not induce
 

significant numbers of such lesions.
 

viii
 
TABLE OF CONTENTS
 

I.	 Introduction 1
 

II.	 Materials and Methods - Stanford Research Institute
 

(SRI) 3
 

A. Chemical	 3
 

B. Dosage Preparation and Analyses 3
 

C • Animals 4
 

D. Animal Maintenance	 5
 

E. Subchronic Study and Design of Chronic Study 5
 

F. Clinical and Pathologic Examinations	 7
 

G. Data Recording and Statistical Analyses	 8
 

III. Results - Stanford Research Institute ­

Na3NTA*H20 - Rats 11
 

A. Body Weights and Clinical Signs	 11
 

B. Survival	 11
 

C. Pathology	 14
 

D. Statistical Analyses of Results	 17
 

IV.	 Materials and Methods - Litton Bionetics, Inc.
 

(LBI) 27
 

A. Chemicals	 27
 

B. Dietary Preparation	 27
 

C. Animals	 28
 

D. Animal Maintenance	 29
 

E. Subchronic Study and Design of Chronic Study 30
 

F. Clinical and Pathologic Examinations	 31
 

G. Data Recording and Statistical Analyses	 33
 

V. Results - Litton Bionetics, Inc. - NTA	 35
 

A.	 Rats (NTA)
 

1. Body Weights and Clinical Signs	 35
 

2. Survival	 35
 

3. Pathology	 38
 

4. Statistical Analyses of Results	 42
 

ix
 
B. Mice (NTA)
 

1. Body Weights and Clinical Signs 48
 

2. Survival 48
 

3. Pathology 51
 

4. Statistical Analyses of Results 54
 

VI. Results - Litton Bionetics, Inc. ­

Na3NTA-H20 57
 

A. Rats (Na3NTA*H20)
 

1. Body Weights and Clinical Signs 57
 

2. Survival 57
 

3. Pathology 60
 

4. Statistical Analyses of Results (Rats) 64
 

B. Mice (Na3NTA-H20)
 

1. Body Weights and Clinical Signs 64
 

2. Survival 68
 

3. Pathology 68
 

4. Statistical Analyses of Results (Mice) 70
 

VII. Discussion 75
 

VIII. Bibliography 83
 

APPENDIXES
 

Appendix A Summary of the Incidence of Neoplasms
 

and Other Proliferative Lesions in
 

Rats Fed Na3NTA-H20 in the Diet
 

(Stanford Research Institute) 85
 

Table Al Male Rats 87
 

Table A2 Female Rats 90
 

Appendix B Summary of the Incidence of Neoplasms
 

and Other Proliferative Lesions in
 

Rats and Mice Fed NTA
 

(Litton Bionetics, Inc.) 93
 

Table Bl Male Rats 95
 
Page
 

Table B2 Female Rats 98 

Table B3 Male Mice 101 

Table B4 Female Mice 103 

Appendix C Summary of the Incidence of Neoplasms and 

Other Proliferative Lesions in Rats 

and Mice Fed Na3NTA-H20 

(Litton Bionetics, Inc.) 105 

Table C1 Male Rats 107 

Table C2 Female Rats 110 

Table C3 Male Mice 113 

Table C4 Female Mice 116 

Appendix D Summary of the Incidence of Nonneoplastic 

Lesions in Rats Fed Na3NTA-H20 in 

the Diet (Standford Research Institute) 119 

Table D1 Summary of the Incidence of Nonneoplastic 

Lesions in Male Rats Fed Na3NTA-H20 

in the Diet 121 

Table D2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Rats Fed Na3NTA-H20 

in the Diet 126 

Appendix E Summary of the Incidence of Nonneoplastic 

Lesions in Rats and Mice Fed NTA in 

in the Diet (Litton Bionetics, Inc.) 131 

Table E1 Summary of the Incidence of Nonneoplastic 

Lesions in Male Rats Fed NTA in 

the Diet 133 

Table E2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Rats Fed NTA 

in the Diet 142 

Table E3 Summary of the Incidence of Nonneoplastic 

Lesions in Male Mice Fed NTA 

in the Diet 152 

xi
 
Page
 

Table E4 Summary of the Incidence of Nonneoplastic 

Lesions in Female Mice Fed NTA 

in the Diet 159 

Appendix F Summary of the Incidence of Nonneoplastic 

Lesions in Rats and Mice Fed Na3NTA-H20 

in the Diet (Litton Bionetics, Inc.) 167 

Table F1 Summary of the Incidence of Nonneoplastic 

Lesions in Male Rats Fed Na3NTA-H20 

in the Diet 169 

Table F2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Rats Fed Na3NTA-H20 

in the Diet 174 

Table F3 Summary of the Incidence of Nonneoplastic 

Lesions in Male Mice Fed Na3NTA*H20 

in the Diet 179 

Table F4 Summary of the Incidence of Nonneoplastic 

Lesions in Female Mice Fed Na3NTA'H20 

in the Diet 182 

TABLES 

Table 1 Dosage Schedule for Chronic Study of 

Na3NTA-H20: SRI 6 

Table 2 Metastases of Urinary Tract Transitonal-cell 

Carcinomas in Rats Treated with 20,000 ppm 

Na3NTA*H20 16 

Table 3 Incidence of Tumors in Male Rats 

(SRI Na3NTA-H20) 18 

Table 4 Incidence of Tumors in Female Rats 

(SRI Na3NTA-H20) 22 

Table 5 Dosage Schedule for Chronic Studies of NTA 

and Na3NTA-H20: LBI 31 

Table 6 Incidence of Tumors in Male Rats (Litton NTA).... 43 

Table 7 Incidence of Tumors in Female Rats (Litton NTA). 44 

xii
 
Page 

Table 8 Incidence of Tumors in Male Mice (Litton NTA)... 55 

Table 9 Incidence of Tumors in Female Mice (Litton NTA). 56 

Table 10 Incidence of Tumors in Male Rats (Litton ­

Na3NTA-H20) 65 

Table 11 Incidence of Tumors in Female Rats (Litton ­

66 

Table 12 Incidence of Tumors in Male Mice (Litton ­

Na3NTA-H20) 71 

Table 13 Incidence of Tumors in Female Mice (Litton ­

Na3NTA-H20) 74 

Table 14 Summary of Primary Epithelial Neoplasms of the 

Urinary Tract - Rats 76 

Table 15 Summary of Primary Epithelial Neoplasms of the 

Urinary Tract - Mice 77 

FIGURES 

Figure 1

Figure 2

 Growth Curves for Rats, Na3NTA*H20 

Stanford Study

 Survival Curves for Rats, Na3NTA-H20 

 12 

Stanford Study 13 

Figure 3 Growth Curves for Rats, NTA - Litton Study 36 

Figure 4 Survival Curves for Rats, NTA - Litton Study 37 

Figure 5 Growth Curves for Mice, NTA - Litton Study 49 

Figure 6

Figure 7

 Survival Curves for Mice, NTA - Litton Study

 Growth Curves for Rats, 

Litton Study

 50 

 58 

Figure 8 Survival Curves for Rats, Na3NTA-H20

Litton Study

 ­

 59 

Figure 9 Growth Curves for Mice, Na3NTA-H20 -

Litton Study 67 

xiii 
Page 

Figure 10 Survival Curves for Mice, Na3NTA-H20 -

Litton Study 69 

xiv
 
I. INTRODUCTION
 

Nitrilotriacetic acid (NTA) is a synthetic amino-polycarboxylic
 

acid chelating agent used chiefly as a replacement for phosphates
 

in detergents, NTA sequesters magnesium and calcium ions present
 

in hard water, which would normally inhibit the activity of
 

detergent surfactants. In December, 1970, the detergent industry
 

voluntarily suspended such applications of NTA in the United
 

States, following an unpublished government report indicating
 

that the compound was teratogenic (Chernoff and Courtney, 1970).
 

During that year the annual production of NTA was 150 million
 

pounds, of which 86-92% was used in detergents. Major
 

nondetergent uses, for which,NTA is still being produced, include
 

water treatment, textile treatment, metal plating and cleaning,
 

and pulp and paper processing. To a lesser extent, NTA is used
 

in leather tanning, photographic development, synthetic rubber
 

production, the manufacture of pharmaceuticals, agriculture (in
 

herbicide formulations and micronutrient solutions), and in the
 

separation of rare-earth elements (Cureton, 1967). It has been
 

predicted that water-soluble chelates that survive biodegradation
 

in sewage treatment plants and natural water would be present in
 

domestic water supplies at an average concentration of less than
 

25 parts per billion (Thayer and Kensler, 1973). This estimate
 

was obtained under conditions of actual use of NTA-containing
 
detergents prior to 1972 and does not represent current exposure.
 

The trisodium salt of NTA (Na3NTA.H2O), the form used in
 

detergents, and NTA itself were both selected for testing in the
 

carcinogenesis bioassay program because toxicological evidence of
 

the incidence of tumors in rats administered NTA was inconclusive
 

(Saffiotti et al., unpublished ms.) and the exposure potential to
 

these compounds was significant. The bioassay of Na3NTA.H2O was
 

conducted in rats at Stanford Research Instiute (SRI); bioassays
 

of both Na3NTA.H2O and NTA were conducted at Litton Bionetics,
 

Inc. (LBI). The combined results of these bioassays are
 

summarized in this report.
 

2 
II. MATERIALS AND METHODS - STANFORD RESEARCH INSTITUTE (SRI)
 

A. Chemical
 

The test chemical in the SRI bioassay was nitrilotriacetic acid,
 

trisodium salt, monohydrate: N(CH2CO2Na)3

.H20. It was obtained
 

from Hampshire Chemical Division of W. R. Grace and Co., Nashua,
 

New Hampshire. The identity and purity of all batches used in
 

the bioassay were checked at SRI by elemental analysis (C,H,N)
 

and infrared and nuclear magnetic resonance spectrometry. No
 

impurities were detected. Elemental composition in all cases
 

matched the theoretical within normal error limits. Spectra were
 

as expected for this structure.
 

B. Dosage Preparation and Analyses
 

was administered by mixing the compound in the feed
 

(Lowfat Lab Chow% Ralston Purina Co.). A stock mixture containing 40,000 ppm Na3NTA.H2O was prepared every 2 weeks by premixing
 

the compound and the feed, adding corn oil as a dust suppressant,
 

and then mixing by machine. Every 2 weeks diets fed to the
 

animals were diluted from the stock mixture with control diet
 

containing corn oil and stored in covered plastic food containers
 

at room temperature until use. The concentration of corn oil in
 
the feed was 3%. The corn oil was purchased from the Staley
 

Manufacturing Company (Orange, California).
 

To check the concentration of test chemical in each stock mixture
 

immediately prior to preparation of the fed diets, SRI used the
 

method developed by Chau & Fox (1971) involving extraction,
 

purification, conversion to the tripropyl ester, and quantitation
 

by gas-liquid chromotography. All batches of stock mixture were
 

within the +4,000 ppm (+ 10%) tolerance limits of the theoretical
 

(40,000 ppm). Dosage mixtures that were stored for 2 weeks at
 

room temperature in a rat feeder showed no change in
 

concentration of the test chemical.
 

C. Animals:
 

Fischer 344 rats (Simonsen Laboratory; Gilroy, California) of
 

both sexes were used in these tests. Upon arrival at the
 

laboratory, all animals were observed, weighed, and quarantined
 

for 2 weeks. Following quarantine, all males gained less than
 

25 g, females gaining less than 15 g, and all sickly animals were
 

culled. The remaining animals were assigned to the test and
 

control groups in the following manner: they were assigned one
 

per cage until all cages were occupied; this procedure was
 

repeated until cages contained three animals. Cages were
 

numbered and assigned to test using computer-generated
 

randomization tables.
 

4
 
D. Animal Maintenance
 

All animals were housed in temperature- and humidity-controlled
 

rooms. Incoming air was filtered at a rate of 10 changes of room
 

air per hour. Fluorescent lighting was provided on a 12-hourper-day cycle. The rats were housed in polycarbonate cages
 

equipped with a disposable filter top of random-woven polyester
 

fiber. Clean cages with autoclaved bedding (Iso-Dri® hardwood
 

chips, manufactured by Becton, Dickinson, and Carworth,
 

Warrensburg, New York), were provided twice each week.
 

The chemical-feed mixture was supplied once per week in
 

hopper-type feeders that were kept filled during one week and
 

replaced the next. Water, which was softened, filtered, and
 

sterilized with ultraviolet light, was supplied automatically to
 

each cage. All diets and water were consumed ad libitum.
 

Neither cages nor racks were routinely rotated. The animals were
 

housed in a room in which N-nitroso-N-pentyl-1-pentamine,
 

N-methyl-N'-nitro-N-nitrosoguanidine, and 4-(butylnitrosoamino)­

1-butanol were also tested.
 

E. Subchronic Study and Design of Chronic Study
 

The study with Na3NTA.H2O at SRI was part of a larger
 

investigation of the effects of combinations of chemicals on
 

5 
their carcinogenicity. For this reason, the design of this study
 

varied from that of bioassays conducted at other laboratories.
 

To estimate the maximum tolerated dose for the chronic study, a
 

preliminary toxicity study was conducted in which Na3NTA.H2O was
 

administered in feed for 8 weeks at four doses to 15 animals of
 

each sex. At 20,000 ppm, the highest dose administered, there
 

was a weight decrement of approximately 10% in males and 0% in
 

females at 8 weeks. No mortality occurred and no other signs of
 

toxicity were apparent. Since Na3NTA.H2O was thus determined to
 

be relatively nontoxic, and since doses suitable for the chronic
 

study were also based on predicted effects of combinations of
 

compounds tested in the larger study, concentrations of 200,
 

2,000 and 20,000 ppm were selected for the Na3NTA.H2O. These
 

were administered throughout the chronic study (table 1). Rats
 

were started on the test at 53 + 2 days of age.
 

Table 1. Dosage Schedule for Chronic Study of Na3NTA.H2O: SRI
 

Concentration in Feed Treatment
 

Species No. /Sex (ppm) (mMa

/kg) Period (wks)
 

Fischer 344 24/M & 24/F 0 0	 104
 

Rat	 24 /M & 24/F 200 0.7 104
 

24/M & 24/F 2,000 7 104
 

24/M & 24/F 20,000 70 104
 

a

Milliraoles7
 

6 
The control animals were obtained from the same source as the
 

Na3NTA*H20-treated animals, given identical animal care, housed in the
 

same room, and administered the same quantity of corn oil in the feed,
 

F. Clinical
 

All animals were observed daily for deaths and for clinical signs.
 

They were weighed individually at the start and every 2 weeks for the
 

initial 12 weeks of the study and every 4 weeks thereafter. They were
 

also palpated for masses at each weighing. Those animals appearing
 

moribund at the time of clinical examination were killed and
 

necropsied.
 

The pathologic evaluation consisted of gross and microscopic
 

examination of all major tissues, organs, or gross lesions. The
 

following tissues and organs were taken from killed animals and, where
 

feasible, from animals found dead: all gross lesions, skin, mammary
 

gland, mandibular lymph node, salivary gland, sternal marrow,
 

costochondral rib, thymus, lung, heart, trachea, esophagus, thyroid,
 

parathyroid, stomach, cecum, colon, ileum, jejunum, duodenum,
 

pancreas, spleen, mesenteric lymph node, liver, bladder, prostate,
 

testes, ovaries, uterus, pituitary, kidneys, adrenals, and brain.
 

Tissues were preserved in 10% buffered formalin, embedded in paraffin,
 

sectioned, routinely stained with hematoxylin and eosin, and examined
 

histopathologically. An occasional section was subjected to special
 

staining techniques for more definitive diagnosis.
 

7
 
A few tissues were not examined for some animals, particularly for
 

those that showed early deaths. Also, some animals were missing,
 

cannibalized, or judged to be in such an advanced state of autolysis
 

as to preclude histopathologic interpretation. Thus, the number of
 

animals for which particular organs, tissues, or lesions were examined
 

microscopically, varies and does not necessarily represent the number
 

of animals that were placed on experiment in each group.
 

G. Data Recording and Statistical Analyses
 

Pertinent data for this experiment have been recorded in an automatic
 

data processing system, the Carcinogen Bioassay Data System (Linhart
 

et al., 1974). The data elements include descriptive information on
 

the chemicals, animals, experimental design, clinical observations,
 

survival, animal weight, and individual pathologic results, as
 

recommended by the International Union Against Cancer (Berenblum,
 

1969). Data tables were generated for verification of data transcription and for statistical review.
 

Survivorship analyses were made in the form of graphs for males and
 

females using the Kaplan-Meier techniques (1958). On each graph
 

survivorship curves were computed for each dose group and the
 

controls. The Thomas-Breslow-Gart program (1976) was used to obtain
 

these curves. Significance tests to compare survivorship curves for
 

the different dose groups were carried out using the Cox (1972) and
 

Tarone (1975) methods.
 

8
 
Analyses for the tumor pathology proceeded in the following order:
 

(1)	 For the incidence of tumor-bearing animals, (a)
 

each dose group was compared with the controls
 

using Fisher-Irwin one-tailed tests, (b) the
 

departure from the linear-trend statistic was
 

calculated (Cochran, 1954), (c) when the departure
 

statistic P value was greater than 0.10, the Cox
 

(1970) test for significance of linear trend was
 

carried out, and (d) exact two-sided 95%
 

confidence intervals for the odds ratio (Gart,
 

1970) were given when the result in (a) had a P
 

value less than 10% (values of the odds ratio
 

larger than 1 imply that the treated group has a
 

higher incidence of tumors than the controls).
 

These analyses were run on the incidence of tumors
 

using computer programs developed at the
 

University of California and at NCI (Thomas, 1975;
 

Thomas et al., 1976). In precise terms, the
 

incidence of total tumors for males is equal to
 

the proportion of animals with at least one tumor
 

(excluding benign testicular tumors) among the
 

number of animals autopsied and histologically
 

examined. The incidence of tumors for females is
 

9 
defined in the same way, except that benign
 

uterine tumors are excluded.
 

(2)	 For several sites, systems, and histology, the
 

same analyses were carried out as those reported
 

in (1) above. For example, analyses of the
 

incidence of urinary-tract tumors were done where
 

the incidence of urinary-tract tumors equaled the
 

number of animals with at least one primary
 

urinary-tract tumor among the animals autopsied
 

and histologically examined. Sites or systems, or
 

histologic types with no tumors or very few
 

tumors, are not reported but were statistically
 

analyzed.
 

10
 
III. RESULTS - STANFORD RESEARCH INSTITUTE - Na3NTA.H2O - RATS
 

A. Body Weights and Clinical Signs
 

High-dose animals (20,000 ppm), when compared with the controls,
 

exhibited a weight decrement of approximately 12% for males and
 

10% for females (figure 1). They had no clinical signs beyond
 

those of normal aging except for enlarged and/or hard kidneys,
 

first noted by palpation between weeks 60 and 64. By week 68,
 

13/22 males and 2/23 females had these renal abnormalities.
 

High-dose animals that died before termination were generally
 

weak, emaciated, and anemic, and had a history of the enlarged
 

and/or hard kidneys. No unusual signs were noted, however, in
 

the other groups. The mean survival time for the males fed
 

20,000 ppm Na3NTA.H2O in the diet was 92 weeks, compared with the
 

other groups, in which the mean was not reached before
 

termination at 104 weeks.
 

B. Survival
 

Figure 2 shows the estimated probabilities of survival of the
 

rats in this experiment. In the males, there was a statistically
 

significant difference (P < 0.001) among the four groups (three
 

dose groups and one control). The high-dose group had earlier
 

deaths than the other dose groups. Although 75% of the high-dose
 

11
 
TIME OF STUDY (WEEKS) 

Figure 1. Growth Curves for Rats, Na^NTA-H^O - Stanford Study
 

12
 
20 40 60 80 20 40 60 80 

TIME ON STUDY (WEEKS) TIME ON STUDY (WEEKS) 

Figure 2. Survival Curves for Rats, Na3NTA.H2O - Stanford Study
 
group died before termination of the study, only one death
 

occurred in the first year, and 12/24 (50%) of this group were
 

alive for over 90 weeks. The survival curves of the female
 

groups are comparable with each other.
 

C. Pathology
 

Histopathologic findings are tabulated in Appendix A, tables
 

A1-A2, covering neoplasms and other proliferative lesions, and in
 

Appendix D, tables D1-D2, covering nonneoplastic lesions.
 

Almost all lesions of interest occurred in the 24 male and 24
 

female rats that had received 20,000 ppm of Na3NTA.H2O. Primary
 

neoplasms of the urinary tract were seen in 14 high-dose males,
 

11 of which died before 104 weeks. Similar neoplasms were also
 

observed in 13 high-dose females; 4 of these were among the 7
 

animals that died before 104 weeks. The neoplasms of the urinary
 

tract consisted of transitional-cell carcinomas, tubular-cell
 

carcinomas, and tubular-cell adenomas of the kidney and
 

transitional-cell carcinomas of the ureter and urinary bladder.
 

Of these neoplasms, the earliest observed was a transitional-cell
 

carcinoma of the ureter in a female rat; this animal died during
 

week 42 of the study. A papilloma of the bladder was also
 

present in a mid-dose female.
 

The transitional-cell carcinomas seen in the ureter and urinary
 

14
 
bladder were generally characterized by sheets of proliferating
 

neoplastic transitional-epithelial cells which invaded the
 

basement membrane submucosa and muscular walls. Evidence of
 

vascular invasion was present in some rats, and in a few animals
 

there was penetration to the serosal surface of the urinary
 

bladder and ureter. In these rats, there was a seeding of
 

neoplastic transitional cells to the other organs of the
 

abdominal cavity. In some instances, small foci or nests of
 

neoplastic transitional-epithelial cells were present in the
 

submucosa and muscular wall of the ureter. In a few animals, the
 

transitional-cell carcinomas presented in a papillary
 

configuration with projections of proliferating transitional-


epithelial cells and their attendant stroma into the lumen of the
 

urinary bladder.
 

The tubular-cell adenomas were characterized by encapsulated
 

masses of proliferating tubules, which were generally well-


differentiated. Evidence of compression was present in the
 

surrounding normal kidney tubules. The tubular-cell carcinomas
 

were less differentiated and consisted of anaplastic tubular
 

epithelial cells, which appeared to be infiltrating the
 

surrounding kidney tissue. Numerous mitotic figures were
 

present.
 

Metastatic transitional-cell carcinomas were seen in five
 

15
 
high-dose males and five high-dose females. They appeared in the
 

lung, lymph node, pancreas, adrenal gland, and seminal vesicle.
 

The frequency of appearance at the different sites is shown in
 

table 2.
 

Table 2. Metastases of Urinary Tract Transitional-Cell Tumors
 

in Rats Treated with 20,000 ppm
 

Organ Male (24) % Female (24) %
 

Lung 4 17 5 21
 

Lymph Node 2 8 1 4
 

Pancreas 2 8 0 0
 

Adrenal Gland 1 4 0 0
 

Seminal Vesicle 1 4 0 0
 

Total animals with lesion 5 21 5 21
 

The incidence of other primary neoplasms in these rats was low,
 

with the exception of interstitial-cell tumors of the testes in
 

most of the males. Such an occurrence is not uncommon for this
 

strain of rat.
 

In most high-dose rats of both sexes, the kidneys were moderately
 

to markedly affected by a nephritis and/or hydronephrosis. These
 

microscopic alterations were characterized by varying degrees of
 

glomerulosclerosis, interstitial fibrosis, tubular dilatation,
 

protein in the lumen of the tubules, and regenerative epithelium.
 

16
 
In several of the urinary bladders, ureters, and renal pelves
 

where there were no neoplasms, there was, however, some degree of
 

transitional-epithelial dysplasia and hyperplasia.
 

The incidence of primary neoplasms of the urinary tract in these
 

high-dose rats was high, whereas primary neoplasms of the urinary
 

tract of the rat have, in general, an extremely low incidence. It
 

can only be assumed, therefore, that the administration of 20,000
 

ppm of Na3NTA.H2O was responsible for this marked increase in
 

urinary-tract neoplasia.
 

D. Statistical Analyses of Results
 

Of the 144 treated and 48 control rats entered into the study,
 

all were examined histopathologically except one that was
 

autolyzed. Tables 3 and 4 contain the analyses of the incidence
 

of tumors in both males and females as defined in (I) and (2) of
 

the preceding section. The results in table 3 suggest the
 

following conclusions for males: The incidence of total tumors
 

(excluding testicular tumors) was higher in the high-dose group
 

than in the controls. The principal cause of the elevated
 

incidence was the occurrence of primary tumors of the urinary
 

tract of the transitional-cell type. Within the urinary tract,
 

the sites of action were the kidney and the ureter. The endocrine system showed increased incidences of tumors in the low-


and mid-dose groups relative to the controls.
 

17
 
Table 3. Incidence of Tumors in Male Rats (SRI Na3NTA.H2O)
 

Controls Low Dose Mid Dose 

0 ppm 200 ppm 2,000 ppm 

Incidence of Tumors 

(excluding testicular tumors) 8/24 (33%) 15/23 (65%) 9/24 (38%) 

Statistical Testsa P - 0.007, P = 0.029 N.S. 

nonlinear 

95% Confidence Intervals 0.99 ­ 14.94 

Weeks to First Observed Tumor 82 81 104 

Incidence of Urinary Tumors
 

(including primary tumors of the
 

kidney, bladder, and ureter) 0/24 (0%) 0/23 (0%) 0/24 (0%)
 

Statistical Testsa	 P < 0.001, N.S. N.S.
 

P < 0.001
 

95% Confidence Intervals
 

Weeks to First Observed Tumor
 

High Dose
 

20,000 ppm
 

18/24 (75%)
 

P = 0.004
 

1.47 - 25.63
 

43
 

14/24 (58%)
 

P = 0.001
 

7.91 - oc
 

67
 
Table 3. Incidence of Tumors in Male Rats (SRI Na3NTA-H20)
 

(continued)
 

Incidence of Tumors of the Kidney
 

Statistical Testsa
 

95% Confidence Intervals
 

Weeks to First Observed Tumor
 

Incidence of Transitional-cell
 

Carcinoma of the Kidney
 

Statistical Testsa
 

95% Confidence Intervals
 

Weeks to First Observed Tumor
 

Controls
 

0 ppm
 

0/24 (0%)
 

P < 0,001,
 

P < 0.001
 

0/24 (0%)
 

P - 0.006,
 

P - 0.003
 

Low Dose
 

200 ppm
 

0/23 (0%)
 

N.S.
 

0/23 (0%)
 

N.S.
 

Mid Dose
 

2,000 ppm
 

0/24 (0%)
 

N.S.
 

0/24 (0%)
 

N.S.
 

High Dose
 

20,000 ppm
 

9/24 (38%)
 

P = 0.001
 

3.41 - oc
 

56
 

4/24 (17%)
 

P = 0.054
 

0.96 - oc
 

56
 
Table 3. Incidence of Tumors in Male Rats (SRI Na3NTA.H20)
 

(continued)
 

Incidence of Tumors of the Ureter
 

Statistical Testsa
 

95% Confidence Intervals
 

Weeks to First Observed Tumor
 

Incidence of Primary Tumors
 

of the Lung
 

Statistical Testsa
 

95% Confidence Intervals
 

Weeks to First Observed Tumor
 

Controls
 

0 ppm
 

0/24 (0%)
 

P < 0.001,
 

P < 0.001
 

1/24 (4%)
 

N.S.,N.S.
 

104
 

Low Dose
 

200 ppm
 

0/23 (0%)
 

N.S.
 

0/23 (0%)
 

N.S.
 

Mid Dose
 

2,000 ppm
 

0/24 (0%)
 

N.S.
 

1/24 (4%)
 

N.S.
 

104
 

High Dose
 

20,000 ppm
 

8/24 (33%)
 

P = 0.002
 

2.81 - a
 

56
 

1/24 (4%)
 

N.S.
 

98
 
Table 3. Incidence of Tumors in Male Rats (SRI Na3NTA-H20)
 

(continued) 

Incidence of Primary
 

Endocrine Tumors
 

Statistical Testsa
 

95% Confidence Intervals
 

Weeks to First Observed Tumor
 

Incidence of Primary
 

ho Hematopoietic Tumors
 

Statistical Testsa
 

95% Confidence Intervals
 

Weeks to First Observed Tumor
 

Controls
 

0 ppm
 

2/24 (8%)
 

P = 0.037,
 

nonlinear
 

103
 

2/24 (8%)
 

P = 0.044,
 

nonlinear
 

100
 

Low Dose
 

200 ppm
 

8/23 (35%)
 

P = 0.03
 

0.94 - 61.92
 

98
 

6/23 (26%)
 

N.S.
 

81
 

Mid Dose
 

2,000 ppm
 

7/24 (29%)
 

P = 0.068
 

0.71	 - 48.64
 

104
 

1/24 (4%)
 

N.S.
 

104
 

High Dose
 

20,000 ppm
 

2/24 (8%)
 

N.S.
 

78
 

1/24 (4%)
 

N.S.
 

104
 

aBeneath the untreated-control incidence are two quantities: (a) the P value for a 2x4 contingency table using
 

chisquare theory; (b) the P value for the test for linear trend if a linear trend fits the data, or the word
 

"nonlinear," which means that a linear trend on a logistic scale does not fit the data.
 

Beneath the dosed-group incidence is the Fisher-Irwin one-tailed test for comparison of that group with the
 

control group when it is below 0.10; or, otherwise N.S. - not significant.
 
Table 4. Incidence of Tumors in Female Rats (SRI Na3NTA*H20)
 

Incidence of Tumors (exclusive 

of benign uterine tumors) 

Controls 

0 ppm 

/ 

13/24 /(54%) 

Low Dose 

200 ppm 

17/24 (71%) 

Mid Dose 

2,000 ppm 

15/24 (63%) 

High Dose 

20,000 ppm 

19/24 (79%) 

Statistical Testsa N.S. 

P = 0.066 

N.S. N.S. P = 0.062 

95% Confidence Intervals 0.78 - 14.96 

Weeks to First Observed Tumor 88 83 76 42 

Incidence of Primary Urinary Tumors 

(inclusive primary tumors of the 

kidney, bladder, and ureter) 0/24 (0%) 0/24 (0%) 1/24 (4%) 13/24 (54%) 

Statistical Testsa P < 0.001, 

P < 0.001 

N.S. N.S. P = 0.001 

95% Confidence Intervals 6.73 - oc 

Weeks to First Observed Tumor 99 42 
Table 4. Incidence of Tumors in Female Rats (SRI
 

(continued)
 

Incidence of Primary Tumors
 

of the Kidney
 

Statistical Testsa
 

95% Confidence Intervals
 

Weeks to First Observed Tumor
 

Incidence of Tumors
 

of the Ureter
 

Statistical Testsa
 

95% Confidence Intervals
 

Weeks to First Observed Tumor
 

Controls
 

0 ppm
 

0/24 (0%)
 

P = 0.006,
 

P = 0.003
 

0/24 (0%)
 

P < 0.001,
 

P < 0.001
 

Low Dose
 

200 ppm
 

0/24 (0%)
 

N.S.
 

0/24 (0%)
 

N.S.
 

Mid Dose
 

2,000 ppm
 

0/24 (0%)
 

N.S.
 

0/24 (0%)
 

N.S.
 

High Dose
 

20,000 ppm
 

4/24 (17%)
 

P = 0.054
 

1.04 - <*
 

104
 

6/24 (25%)
 

P = 0.011
 

1.79 - *
 

42
 
Table 4. Incidence of Tumors in Female Rats (SRI Na3NTA.H20)
 

(continued)
 

Controls Low Dose Mid Dose High Dose
 

0 ppm 200 ppm 2,000 ppm 20,000 ppm
 

Incidence of Tumors
 

of the Bladder 0/22 (0%) 0/23 (0%) 1/22 (5%) 5/24 (21%)
 

Statistical Testsa P = 0.010, N.S. N.S. P = 0.031
 

P = 0.001
 

95% Confidence Intervals 1.23 - <*
 

Weeks to First Observed Tumor 104 96
 

Incidence of Endocrine Tumors 3/24 (13%) 8/24 (33%) 5/24 (21%) 4/24 (17%)
 

Statistical Testsa N.S.,N.S. P = 0.084 N.S. N.S.
 

95% Confidence Intervals 0.68 - 23.17
 

Weeks to First Observed Tumor 98 92 80 92
 

aBeneath the untreated-control incidence are two quantities: (a) "the P value for a 2x4 contingency table using
 

chisquare theory; (b) the P value for the test for linear trend if a linear trend fits the data, or the word
 

"nonlinear,ff

 which means that a linear trend on a logistic scale does not fit the data.
 

Beneath the dosed-group incidence is the Fisher-Irwin one-tailed test for comparison of that group with the
 

control group when it below 0.10; or, otherwise N.S. - not significant.
 
For the female rats also there was a statistically significant
 

increase of urinary tract tumors in the high-dose group. While
 

the kidney and the ureter were the sites of action in the males,
 

the bladder was an additional site in the females. Most of these
 

tumors in the females, as in the males, were transitional-cell
 

carcinomas.
 

25
 

IV. MATERIALS AND METHODS - LITTON BIONETICS, INC. (LBI)
 

A. Chemically 

The batches of NTA and Na3NTA.H2O were manufactured by the
 

Hampshire Chemical Division of W. R. Grace and Co., Nashua, New
 

Hampshire. Analyses for purity were not performed in the LBI
 

study. According to the manufacturer's specifications, commercial NTA and Na3NTA.H2O contain 99.5% active ingredients. Since
 

the samples used at LBI and samples of the trisodium salt tested
 

at SRI (see p. 3, above) were obtained from the same manufacturer, it is expected that they would be of similar purity.
 

B. Dietary Prej^ra-tion 

A 6-kg mixture of chemical in feed (WayneB^ Lab Blox Meal, Allied
 

Mills) was prepared in a hooded twin-shell blender twice a week
 

for mice and three times a week for rats. Feed preparations and
 

chemicals were refrigerated until they were used. No corn oil
 

was added to the feed.
 

Analyses that were conducted at the beginning of the study and 10
 

days later to determine the percent recovery of NTA in dosage
 

mixtures showed that NTA was stable in feed for at least 10 days.
 

Similar analyses of samples taken from three positions in the
 

blender indicated that the mixture was homogeneous. Assays of
 

27
 
the stability of Na3NTA.H2O in feed and of the homogeneity of
 

such mixtures were not performed.
 

C. Animals
 

Inbred Fischer 344 rats and hybrid B6C3F1 mice were obtained from
 

A. R. Schmidt, Madison, Wisconsin. Additional mice of the same
 

strain were procured from Charles River Breeding Laboratories,
 

Wilmington, Massachusetts. These laboratories were under
 

contract to the Division of Cancer Treatment, NCI, to provide the
 

animals used for testing.
 

In the NTA test, half of the mice were from Charles River and
 

half from A. R. Schmidt; in the Na3NTA.H2O study, 90% of the mice
 

were from Charles River and 10% were from A. R. Schmidt. No
 

information is available on how the animals from the two sources
 

were assigned to the test groups.
 

Animals were received at 28 days of age and quarantined for a
 

period of 14 days. At the end of this period, animals with no
 

clinical signs of disease were weighed individually. Mice
 

weighing 19-22 g and rats weighing 85-110 g were considered
 

acceptable for testing and were then segregated into equal weight
 

groups. Cage assignments were made by choosing one animal from
 

each such group so that the total weights of animals in different
 

cages were equal.
 

28
 
D. Animal Maintenance
 

During the chronic study, rats were housed four per cage and mice
 

five per cage in solid polycarbonate cages suspended on moveable
 

racks. All cages were lined with heat-treated hardwood-chip
 

bedding and covered witn filter paper over a wire-mesh screen.
 

Clean cages and fresh bedding were provided twice a week. Water
 

bottles were sanitized and refilled with acidulated water (pH
 

2.5) twice a week; feed was available ad libitum and supplied
 

three times a week. Air in the animal rooms was changed 15 times
 

per hour and exhausted through HEPA filters. Animal rooms were
 

negatively pressurized with respect to the clean hall, and
 

positively pressurized with respect to the dirty hall. The room
 

air was maintained at 21-25° C and 45-55% relative humidity.
 

Fluorescent lighting illuminated the rooms for 8 hours a day.
 

Rats in the NTA bioassay were maintained concurrently in a room
 

with rats that were being treated with N-9H-fluoren-2-flacetamide, 4,4'-diisocyanato-3,3'-dimethoxy-l,l'-biphenyl, and Nphenyl-l,4-benzenediamine hydrochloride. Rats on test with
 

Na3NTA.H2O were in a room with animals that were given
 

4,4'-raethylenebis(N,N'-dimethyl)benzenamine, ethenylbenzene (styrene), and 2,5-cyclohexadiene-l,4-dione, dioxime.
 

All mice in this study were in a room with mice receiving
 

N-9H-fluoren-2-ylacetamide, 2-4-dichloro-l-(4-nitrophenoxy)ben29
 
zene, 1H-1,2,4-triazol-3-amine, ethenylbenzene (styrene),
 

2-nitroethenylbenzene, and 4-nitroso-N-phenylbenzenamine.
 

Controls were housed in the same rooms as their corresponding
 

experimental groups.
 

E. Subchronic Study and Design of Chronic Study
 

In the bioassay at Litton Bionetics, both NTA and Na3NTA.H2O were
 

tested. To estimate the maximum tolerated dose for the chronic
 

study, an 8-week subchronic toxicity test was conducted in which
 

the compounds were administered in the diet to both species at
 

concentrations up to 31,600 ppm for rats and 21,600 ppm for mice.
 

At these concentrations there were no deaths or gross pathologic
 

findings at necropsy, and depression of body weight was in the
 

range of only 10-20%.
 

During the chronic study, the doses of NTA and Na3NTA.H2O were as
 

outlined below (table 5). Since equimolar quantities of these
 

compounds were not used, given concentrations of Na3NTA.H2O
 

represented 30% less NTA than did equal concentrations of the
 

free acid. Table 5 therefore expresses dosages in terms of
 

millimoles per kilogram as well as parts per million. Treatment
 

began when animals were 6 weeks of age and continued for 18
 

months. At the end of that time, rats were maintained on control
 

30
 
diets (feed only) for an additional 6 months, and mice for 3

 

months. 

Table 5. Dosage Schedule for Chronic Studies of 

NTA and Na3NTA-H20: LBI 

Concentration in Feed Treatment
 

Species No./Sex (ppm) (mM/kg) Period (months)
 

NTA
 

Fischer 344 Rat	 20/M & 20/F 0 0 18
 

50/M & 50/F 7,500 (1/2 MTD) 40 18
 

50/M & 50/F 15,000 (MTD) 80 18
 

B6C3F1 Mouse	 20/M & 20/F 0 0 18
 

50/M & 50/F 7,500 (1/2 MTD) 40 18
 

50/M & 50/F 15,000 (MTD) 80 18
 

Na3NTA-H2O
 

Fischer 344 Rat 20/M & 20/F 0 0 18
 

50/M & 50/F 7,500 (1/2 MTD) 27 18
 

50/M & 50/F 15,000 (MTD) 55 18
 

B6C3F1 Mouse 20/M & 20/F 0 0 18
 

50/M & 50/F 2,500 (1/2 MTD) 9 18
 

50/M & 50/F 5,000 (MTD) 18 18
 

F. Clinical and	 Pathologic Examinations
 

Treatment and control groups were observed twice a day, and any
 

animal that was moribund or had large masses was killed and
 

necropsied to minimize the risk of autolysis. Cage-mates were
 

31
 
weighed as a group each week for the first 6 weeks, biweekly for
 

the next 6 weeks, and monthly thereafter. Palpation for tumors
 

was performed routinely when the animals were weighed. At
 

termination animals were killed by C02-induced asphyxiation and
 

necropsied.
 

The following tissues were examined during necropsy and processed
 

for histopathologic examination: skin, trachea, lung, heart,
 

salivary gland, esophagus, stomach, duodenum, jejunum, ileum,
 

colon, liver, pancreas, kidney, urinary bladder, thyroid,
 

parathyroid, adrenal, pituitary, mandibular and mesenteric lymph
 

nodes, bone marrow, spleen, mammary gland, seminal vesicle,
 

prostate, testis, ovary, uterus, brain, and rib, as well as any
 

tissue mass. Large tissue masses were weighed, described
 

morphologically, and sectioned. All tissues were preserved in
 

10% buffered formalin, embedded in paraffin, sectioned, routinely
 

stained with hematoxylin and eosin, and examined histopathologically. An occasional section was subjected to special staining
 

techniques for more definitive diagnosis.
 

A few tissues were not examined for some animals, particularly
 

for those that showed early deaths. Also, some animals were
 

missing, cannibalized, or judged to be in an advanced state of
 

autolysis as to preclude histopathologic interpretation. Thus,
 

the number of animals for which particular organs, tissues, or
 

32
 
lesions were examined microscopically, varies and does not
 

necessarily represent the number of animals that were placed on
 

experiment in each group.
 

G. Data Recording and Statistical Analyses
 

Data obtained from Litton Bionetics were handled as described
 

earlier for data obtained from Stanford,
 

33
 

V. RESULTS - LITTON BIONETICS, INC.- NTA
 

A. RATS (NTA)
 

1. Body Weights and Clinical Signs
 

The average weights of both male and female rats were depressed
 

in a dose-related manner when compared with those of the controls
 

(figure 3). No significant signs or lesions were observed until
 

the sixth month of the study, when the first lesion occurred in a
 

high-dose male. A crusted mass appeared on the back and remained
 

until the termination of the study. In the second year of the
 

study an insignificant number of palpable tissue masses and
 

cataracts appeared in a few of the animals.
 

2. Survival
 

Estimates of the probabilities of survival of rats are shown in
 

figure 4. Survivals of treated and control groups of both sexes
 

are comparable. The Tarone test of male-rat survival did not
 

indicate a dose-related trend (P > 0.10), but in female rats the
 

dose-related statistic approached significance (P = 0.057). In
 

every group of both sexes, more than 94% of the animals survived
 

for a year or more.
 

35
 
TIME ON STUDY (WEEKS) 

Figure 3, Growth Curves for Rats, NTA - Litton Study
 

36 
TIME ON STUDY (WEEKS) 40 GO 

TIME ON STUDY (WEEKS) 

Figure 4. Survival Curves for Rats, NTA - Litton Study
 
3.
 

Histopathologic findings are tabulated in Appendix B, tables Bl
 

and B2, covering neoplasms and other proliferative lesions, and
 

in Appendix E, tables El and E2, covering nonneoplastic lesions.
 

All of the 200 treated rats and the 40 control rats that
 

comprised this study were examined histopathologically with the
 

exception of a single high-dose male that was autolyzed. Thus,
 

where the number of animals with lesions is delineated,
 

comparisons should be made to the respective group size, i. e.,
 

20 control male, 50 low-dose male, 49 high-dose male, 20 control
 

female, 50 low-dose female, and 50 hi|*h-dose female animals.
 

A significant number of tumors occurred in the urinary system of
 

the treated animals. Seven high-dose male rats showed a total of
 

eight neoplasms of the kidney and ureter; three of these
 

neoplasms were classified as tubular-cell adenomas of the kidney,
 

two as tubular-cell adenocarcinomas of the kidney (one widely
 

metastasized), one as a papillary adenoma of the ureter, and two
 

as papillomas of the ureter. A low-dose male rat showed a
 

tubular-cell adenoma. In the females, tumors of the urinary
 

bladder were more frequent than those of the kidney. Eleven
 

high-dose and two low-dose females showed transitional-cell
 

carcinomas of the bladder. An additional high-dose female showed
 

a squamous-cell carcinoma of the bladder. Tumors of the kidney
 

38
 
were found only in two high-dose females that were not included
 

among those that had shown transitional-ceLl carcinomas of the
 

bladder; one of these showed a transitional-cell papilloma of the
 

renal pelvis, the other a tubular-cell adenoma. Ho neoplasms of
 

the urinary system were observed in the controls.
 

Microscopically, the tubular-cell adenomas were discrete expanding masses of tubules of varying sizes, usually located in the
 

cortex. The cells lining and filling the tubules resemoled
 

normal renal epithelium, except that the nuclei tended to be more
 

hyperchromatic than those of the normal contiguous renal
 

epitnelial cells.
 

In the adenocarcinomas, this tubular pattern was less distinct.
 

Basement membranes were poorly defined and the cells tended to
 

pile up; in some areas they appeared to be growing in sheets
 

rather than in a tubular pattern. Rapid growth was suggested by
 

the anaplasia, nuclear pleotaorphisw, frequent mitoses, and foci
 

of necrosis.
 

The tumors of the bladder varied in appearance from benign,
 

well-differentiated squamous- or transitional-cell papillomas to
 

carcinomas. The latter were characterized by disarray in
 

cellular polarity and arrangement as well as by subepithelial
 

invasions by nests of pleomorphic epithelial cells. The mitotic
 

index was low and metastases did not occur.
 

39
 
Hepatocellular carcinomas occurred in three of the low-dose male
 

rats and neoplastic nodules appeared in three control, two
 

low-dose, and two high-dose males. However, 8 of the low-dose
 

and 22 of the high-dose females had neoplastic nodules, compared
 

with 2 in the controls. This is a strikingly higher incidence
 

than was noted with female rats receiving Na3NTA*H2O (Section VI,
 

A ).
 

Alveolar/bronchiolar adenoma and carcinoma occurred in one
 

control, five low-dose, and five high-dose males, and in three
 

low-dose and seven high-dose females. This is a frequently
 

encountered neoplasm in the respiratory system of the Fischer 344
 

rat.
 

Fourteen primary tumors of the integument of various types
 

occurred throughout all groups with a slightly higher proportion
 

among controls. Eight of these tumors arose from adnexae. Of
 

these, four were malignant, with two metastasizing. The remainder arose from connective tissue, of which two were classified as
 

malignant.
 

In the endocrine system, pituitary tumors occurred in 1 control,
 

6 low-dose, and 2 high-dose males, and in 6 control, 10 low-dose,
 

and 12 high-dose females. Among males, pheochromocytomas were
 

present in one control, nine low-dose, and five high-dose
 

animals. Fourteen pheochromocytomas were present in the
 

40
 
high-dose females, but none occurred in the low-dose. A. single
 

pheochromocytorna was present in the controls. Both of these
 

tumors are frequently found in Fischer 344 rats.
 

The number and types of thyroid tumors in the treated animals
 

were similar to those in the controls. In the male reproductive
 

system, carcinomas of the preputial gland occurred in two
 

low-dose, four high-dose, but no control animals.
 

Interstitial-cell tumors of tVie testes occurred in nearly all
 

male rats. This extremely high incidence is characteristic of
 

Fischer 344 rats.
 

A number of different types of neoplasms occurred in various
 

other organ systems, apparently not related to treatment.
 

In the urinary system, particularly the kidney, severe chronic
 

inflammatory changes in the treatment groups far exceeded those
 

seen in the controls. The lesions are those commonly associated
 

with kidneys of old rats. These consist of fibrosed glomeruli,
 

diffuse interstitial scarring, and dilated and cast-containing
 

tubules, accompanied by focal accumulation of mononuclear
 

inflammatory cells.
 

Hyperplasia of tissue in the urinary system was seen only in the
 

treated rats. Epithelial hyperplasia of the urinary bladder was
 

present in 2 low-dose males, 1 high-dose male, and 11 high-dose
 

41
 
females. One high-dose rat had a similar lesion in the ureter.
 

The gamut of lesions comprising the chronic murine pneumonia
 

syndrome is responsible for the majority of the nonneoplastic
 

lesions seen in this study. The treated males appear to have had
 

a higher incidence of disease than did the controls. The reverse
 

appears to have been the case with the females.
 

4. Statistical Analyses of Results (Rats)
 

As shown in table 6, tumors of the urinary tract in male rats
 

occurred more frequently in the high-dose group than in the
 

control group (P = 0.076), and a positive linear dose-response
 

relation existed (P = 0.006). The low-dose group produced a
 

higher incidence of endocrine tumors than the control group.
 

In female rats (table 7), there was a significant dose-related
 

trend (P < 0.001) in overall incidence of tumors (excluding
 

uterine), due mainly to differences in the incidences of the
 

high-dose group and the controls (P = 0.026). This dose-response
 

statistic was nonlinear. Further analyses revealed that the
 

overall incidence of tumors in the high-dose group is different
 

from that of the controls due to the higher incidence of
 

nonmalignant tumors of the liver (P = 0.024) and malignant tumors
 

of the urinary bladder (P = 0.014) in the high-dose group as
 

42
 
Table 6. Incidence of Tumors in Male Rats (Litton NTA)
 

Controls Low Dose High Dose
 

0 ppm 7,500 ppm 15,000 ppm
 

Incidence of Tumors of the Urinary Tract 0/20 (0%) 1/49 (2%) 7/48 (15%)
 

Statistical Testsa P - 0.021, N.S. P = 0.076
 

P = 0.006
 

95% Confidence Intervals 0.83 - oc
 

Weeks to First Observed Tumor 104 66
 

Incidence of Tumors of the Endocrine System	 2/20 (10%) 16/49 (33%) 8/49 (16%)
 

Statistical Testsa	 P - 0.054, P = 0.045 N.S.
 

nonlinear
 

95% Confidence Intervals	 0.85 - 42.62
 

Weeks to First Observed Tumor	 104 93 104
 

aBeneath the untreated-control incidence are two quantities: (a) the P values for a 2x3 contingency table
 

using chisquare theory: (b) the P value for the test for linear trend if a linear trend fits the data, or
 

the word "nonlinear,1, which means that a linear trend on a logistic scale does not fit the data.
 

Beneath the dosed-group incidence is the Fisher-Irwin one-tail test for comparison of that group with the
 

control group when it is below 0.10; or, otherwise N.S. - not significant.
 
Table 7. Incidence of Tumors in Female Rats (Litton NTA)
 

Incidence of Tumors
 

Statistical Testsa
 

95% Confidence Intervals
 

Weeks to First Observed Tumor
 

Incidence of Non-uterine Tumors
 

Statistical Testsa
 

95% Confidence Intervals
 

Weeks to First Observed Tumor
 

Controls
 

JJJIIE.1 1 !TM
 

14/20 (70%)
 

P « 0-002
 

nonlinear
 

76b/96
 

14/20 (70%)
 

P < 0.001,
 

nonlinear
 

76b/96
 

Low Dose
 

7,500 pgm
 

33/50 (66%)
 

N.S.
 

86
 

30/50 (60%)
 

N.S.
 

86
 

High Dose
 

15,000 pj>m
 

47/50 (94%)
 

P = 0.013
 

1.20 - 45.34
 

73
 

46/50 (92%)
 

P = 0.026
 

0.98 - 26.61
 

73
 
Table 7. Incidence of Tumors in Female Rats (Litton NTA)
 

(continued)
 

Incidence of Tumors of the Lung
 

(all malignant)
 

Statistical Testsa
 

95% Confidence Intervals
 

Weeks to First Observed Tumor
 

Incidence of Tumors of the Liver
 

(all nonmalignant)
 

Statistical Testsa
 

95% Confidence Intervals
 

Weeks to First Observed Tumor
 

Controls
 

0 ppm
 

0/15 (0%)
 

N.S.
 

P = 0.033
 

2/15 (13%)
 

P = 0.003,
 

P = 0.001
 

99c

/104
 

Low Dose
 

7,500 ppm
 

3/49 (6%)
 

N.S.
 

104
 

8/49 (16%)
 

N.S.
 

104
 

High Dose
 

15,000 ppm
 

7/46 (15%)
 

N.S.
 

73
 

22/49 (45%)
 

P = 0.024
 

1.00 - 52.13
 

93
 
Table 7. Incidence of Tumors in Female Rats (Litton NTA)
 

(continued)
 

Incidence of Tumors of the Urinary Tract
 

Statistical Testsa
 

95% Confidence Intervals
 

Weeks to First Observed Tumor
 

Incidence of Tumors of the Bladder
 

(all malignant)
 

Statistical Testsa
 

95% Confidence Intervals
 

Weeks to First Observed Tumor
 

Controls
 

0 ppm
 

0/20 (0%)
 

P < 0.001,
 

P < 0.001
 

0/18 (0%)
 

P = 0.002,
 

P < 0.001
 

Low Dose
 

7,500 ppm
 

2/50 (4%)
 

N.S.
 

104
 

2/45 (4%)
 

N.S.
 

104
 

High Dose
 

15,000 ppm
 

14/50 (28%)
 

P = 0.005
 

2.06	 -«
 

91
 

12/48 (25%)
 

P - 0.014
 

1.56	 -oc
 

91
 
Table 7. Incidence of Tumors in Female Rats (Litton NTA)
 

(continued)
 

Controls Low Dose High Dose 

0 ppm 7,500 ppm 15,000 ppm 

Incidence of Tumors of the 

Endocrine System 6/20 (30%) 13/50 (26%) 25/50 (50%) 

Statistical Testsa P = 0.036, N.S. N.S. 

P = 0.024 

95% Confidence Intervals 

Weeks to First Observed Tumor 99c 101 91 

Incidence of Adrenal Phenochromocytoma	 1/20 (5%) 0/50 (0%) 14/48 (29%)
 

Statistical Testsa	 P < 0.001, N.S. P = 0.024
 

nonlinear
 

95% Confidence Intervals	 1.01 - 347.90
 

Weeks to First Observed Tumor	 99c
 93
 

a

Beneath the untreated-control incidence are two quantities; (a) the P values for a 2x3 contingency
 

table using chisquare theory: (b) the P value for the test for linear trend if a linear trend fits
 

the data, or the word "nonlinear," which means that a linear trend on a logistic scale does not fit
 

the data.
 

Beneath the dosed-group incidence is the Fisher-Irwin one-tailed test for comparison of that group
 

with the control group when it is below 0.10; or, otherwise N.S. - not significant.
 

kAnimal at week 76 was accidentally killed; the first natural death with a tumor was at week 96.
 c

Animal at week 99 was a scheduled sacrifice, not a natural death.
 
compared with the controls. Also, a positive linear dose-


response relation exists for the incidence of nonmalignant tumors
 

of the liver (P = 0.001) and malignant tumors of the urinary
 

bladder (P < 0.001). The incidence of tumors in the endocrine
 

system shows a positive linear dose-response relation (P =
 

0.024). Finally, the incidence of adrenal tumors is elevated in
 

the high-dose group relative to the controls (P « 0.024).
 

B. MICE (NTA)
 

1. Body Weights and Clinical Signs
 

The average weights of the high-dose male mice and the high- and
 

low-dose female mice were depressed when compared with those of
 

the controls (figure 5). No significant gross lesion or palpable
 

mass was observed in this group of animals during the treatment
 

period.
 

2. Survival
 

The survival curves of the treated and control groups of both
 

sexes of the mice were comparable (figure 6). More than 92% of
 

the animals survived to the end of the study, and the results can
 

be evaluated over the entire time period for both the treated and
 

control groups. The Tarone test for life-table analyses showed
 

no significant difference in survival between the groups.
 

48
 
TIME ON STUDY (WEEKS) 

TIME ON STUDY (WEEKS) 

Figure 5. Growth Curves for Mice, NTA - Litton Study
 

49 
TIME ON STUDY (WEEKS) TIME ON STUDY (WEEKS) 

Figure 6. Survival Curves for Mice, NTA - Litton Study
 
3. Pathology
 

Histopathologic findings are tabulated in Appendix B, tables B3
 

and B4, covering neoplasms and other proliferative lesions, and
 

in Appendix E, tables E3 and E4, covering nonneoplastic lesions.
 

The study began with 200 treated and 40 matched-control mice; of
 

these, 222 animals received histopathologic examination.
 

Eighteen mice escaped, including 1 low-dose male, 6 high-dose
 

males, and 11 low-dose females. Thus, where the number of
 

animals with lesions is delineated, comparisons should be made
 

with regard to the respective group size, i.e., 20 control male,
 

49 low-dose male, 44 high-dose male, 20 control female, 39
 

low-dose female, and 50 high-dose female animals.
 

As with the rats, the urinary system was the site of treatment-


related neoplasia in mice. Altogether, 32 treated mice showed
 

urinary tract tumors. Tubular-cell adenocarcinomas, four of
 

which were bilateral, were found in 22 high-dose males. The same
 

tumor was also found in five low-dose males and in four high-dose
 

females. In one of the high-dose females, the tumor was
 

bilateral. A papilloma occurred in the renal pelvis of an
 

additional high-dose male. No renal neoplasms were present in
 

either the low-dose females or controls.
 

The pattern of these tubular-cell adenocarcinomas varied from
 

51
 
small cortical foci to large masses containing blood-filled
 

cystic spaces and areas of necrosis. While ductal structures
 

could usually be found in the tumor, large areas of neoplastic
 

cells grew in sheets in which cell borders and basement membranes
 

were indistinct. The individual cell types varied from fairly
 

well-differentiated renal tubule cells to highly pleomorphic,
 

often polygonal-shaped large cells with eosinophilic cytoplasm,
 

frequently containing bizarre nuclei and mitotic figures.
 

Six high-dose, four low-dose, and two control male mice developed
 

neoplasms of the hematopoietic system. These hematopoietic
 

neoplasms were represented by malignant lymphomas in three of the
 

high-dose males. Ten high-dose, seven low-dose, and four control
 

female mice developed neoplasms of the hematopoietic system.
 

These neoplasms were represented by malignant lymphomas in eight
 

high-dose, three low-dose, and two control females.
 

In males, hepatocellular adenomas were observed in one low-dose
 

and three control animals; and hepatocellular carcinomas occurred
 

in two high- and two low-dose mice, but none was found in the
 

control group. In females, only one hepatocellular carcinoma
 

occurred, this in a low-dose animal.
 

The incidences of pulmonary neoplasms in treated male and female
 

mice appear similar. In males alveolar/bronchiolar carcinomas
 

occurred in four control, two low-dose, and four high-dose
 

52
 
animals; in addition, adenomas were found in three low-dose and
 

two high-dose animals. In the females, two alveolar/bronchiolar
 

carcinomas were seen in each treatment group, but none in the
 

controls. Two high-dose females had adenomas. These neoplasms
 

are frequently encountered in both sexes of mice.
 

Additional neoplasms were found only in the skin and in the
 

circulatory and female reproductive systems. These occurred as
 

single lesions or in such small numbers as to be insignificant.
 

The kidney was the most frequent site of nonneoplastic lesions in
 

both male and female mice. The most frequently observed lesion,
 

hydronephrosis, was encountered in eight high-dose and three
 

low-dose males. Twelve high-dose females had this same lesion.
 

Hydronephrosis was not observed in low-dose females or in any of
 

the controls. Since epithelial hyperplasia of the kidney, renal
 

pelvis, and ureter was described in only one female and one male
 

of the treated mice, the cause of the hydronephrosis is obscure.
 

However, no special diagnostic procedures were employed. No
 

lesion of the urinary bladder was observed.
 

A variety of other nonneoplastic lesions occurred in both control
 

and treated animals in insignificant numbers. The most frequently occurring lesions of this type were corpora araylacea in the
 

brain, pneumonic changes in both males and females, inflammatory
 

and cystic hyperplastic changes seen in the uterus, lymphoid
 

53
 
hyperplasia, and hyperplasia of the C-cell of the thyroid.
 

4. Statistical Analyses of Results
 

Table 8 shows that in male mice the high-dose group had a higher
 

overall incidence of tumors than the control group (71% vs. 50%),
 

due mainly to the higher incidence of tumors of the kidney in the
 

high-dose group (55% vs. 0%). Thus, the important finding for
 

male mice administered NTA was the statistically significant
 

incidence (P < 0.001) of tumors of the kidney in the high-dose
 

group. No other significant findings were observed.
 

In the females, tumors again appeared only in the high—dose
 

group, but at a rate of incidence which was too low to be
 

significant (table 9).
 

54
 
Table 8. Incidence of Tumors in Male Mice (Litton NTA)
 

Controls Low Dose High Dose
 

0 ppm 7,500 ppm 15,000 ppm
 

Incidence of Tumors 10/20 (50%) 14/49 (29%) 31/44 (71%)
 

Statistical Testsa P < 0-001 N.S. P = 0.0976
 

nonlinear
 

95% Confidence Intervals 0.69 - 8.11
 

Weeks to First Observed Tumor 76 82 66
 

Incidence of Tumors of the Urinary Tract
 

(all kidney tumors) 0/20 (0%) 5/49 (10%) 24/44 (55%)
 

Statistical Testsa	 P < 0.001 N.S. P < 0.001
 

nonlinear
 

95% Confidence Intervals	 6.34 - oc
 

Weeks to First Observed Tumor 91	 88 74
 

a

Beneath the untreated-control incidence are two quantities: (a) the P value for a 2x3 contingency
 

table using chisquare theory; (b) the P value for the test for linear trend if a linear trend fits
 

the data or the word "nonlinear," which means that a linear trend on a logistic scale does not fit
 

the data.
 

Beneath the dosed-group incidence is the Fisher-Irwin one-tailed test for comparison of that group
 

with the control group when it is below 0.10; or, otherwise N.S. - not significant.
 
Table 9. Incidence of Tumors in Female Mice (Litton NTA)
 

Controls Low Dose High Dose 

0 ppm 7,500 ppm 15,000 ppm 

Incidence of Tumors of the Urinary Tract 

(all malignant kidney tumors) 0/20 (0%) 0/39 (0%) 4/50 (8%) 

Statistical Testsa P = 0.086, N.S. N.S. 

P = 0.041 

95% Confidence Intervals 

Weeks to First Observed Tumor 91 

aBeneath the untreated-control incidence are two quantities; (a) the P value for a 2x3 contingency
 

table using chisquare theory; (b) the P value for the test for linear trend if a linear trend fits
 

the data or the word "nonlinear," which means that a linear trend on a logistic scale does not fit
 

the data.
 

Beneath the dosed-group incidence is the Fisher-Irwin one-tail test for comparison of that group
 

with the control group when it is below 0.10; or, otherwise N.S. - not significant.
 
VI. RESULTS - LITTOM BIONETICS, INC. - N
 

A. RATS (Na3NTA.H2O)
 

1. Body Weights and Clinical Signs
 

The average weights of both male and female rats were depressed
 

in a dose-related manner when compared with those of the controls
 

(figure 7).
 

No significant clinical signs were observed until late in the
 

second year when a few animals began to show loss of weight and
 

discharges from the eyes and nose. Near the end of the study,
 

nearly all of the low-dose females appeared to be suffering from
 

respiratory disease.
 

2. Survival
 

Figure 8 shows the estimated probability of survival of the rats
 

in this study. The times to death of all groups of both sexes
 

are comparable. While the number of early deaths increased
 

slightly with dose, there is no statistically significant
 

dose-related trend (P > 0.10). In every group more than 85% of
 

the animals survived for 80 weeks or more. Six of the high-dose
 

male rats and five of the high-dose female rats were accidentally
 

killed during the test, and were examined histopathologically.
 

57
 
TIME ON STUDY (WFEKS)
 

Figure 7. Growth Curves for Rats, Na3NTA*H2O - Litton Study
 

58
 
TIME ON STUDY (WEEKS) TIME ON STUDY (WEEKS) 

Figure 8. Survival Curves for Rats, Na3NTA-H2O - Litton Study
 
These animals were entered as censored observations in the Tarone
 

test for dose-related survival, and they are included among the
 

number of animals examined in the analyses of tumors.
 

3. Pathology
 

Histopathologic findings are tabulated in Appendix C, tables CI
 

and C2, covering neoplasms and other proliferative lesions, and
 

in Appendix F, tables Fl and F2, covering nonneoplastic lesions.
 

Of the 200 treated and 40 control rats entered into this study,
 

all were necropsied, and 238 were examined histopathologically.
 

The tissues from one low-dose male and one high-dose female were
 

lost. Thus, where the number of animals with lesions is
 

delineated, comparisons should be made with the respective group
 

size, i.e., 20 control male, 49 low-dose male, 50 high-dose male,
 

20 control female, 50 low-dose female, and 49 high-dose female
 

animals.
 

As was noted in animals treated with NTA, a number of primary
 

neoplasms of the urinary system occurred in both the high- and
 

low-dose groups. No such neoplasm occurred in the controls. In
 

the low-dose males, a tubular-cell adenocarcinoma of the kidney
 

was found in one animal and a papilloma of the ureter in the
 

other; similarly, in the high-dose males a tubular-cell carcinoma
 

of the kidney was found in one animal and a papilloma of the
 

60
 
ureter in another. In female rats the tumors were confined to
 

the bladder. One female bearing a transitional-cell carcinoma
 

and a second bearing a papilloma were found in the high-dose
 

group; a squamous-cell carcinoma in one female and transitional-


cell carcinomas in three additional females were found in the
 

low-dose group.
 

The tubular-cell adenocarcinomas seen in this study had many of
 

the morphologic characteristics of those tumors seen in the rats
 

dosed with NTA. One of the neoplasms in Na3NTA.H2O-treated rats
 

differed somewhat from those previously described. This
 

carcinoma appeared as a discrete cortical multiloculated mass
 

comprised of centers of necrosis surrounded by proliferating
 

neoplastic cells. These varied in size from small cells
 

resembling normal renal tubule epithelium to very large cells,
 

many of which contained a single vacuole displacing the nucleus
 

to the periphery of the cell.
 

In the respiratory system, nine males and six females had
 

alveolar/bronchiolar carcinomas. This tumor is not infrequently
 

encountered in the Fischer 344 rats in this laboratory. Other
 

primary neoplasms of the lung appeared in about equal numbers in
 

both treated and control animals. In contrast to the large
 

number of neoplastic nodules of the liver found in rats receiving
 

NTA, only one neoplastic nodule was found in each of one low-dose
 

61
 
and one high-dose female, one control and one high-dose male.
 

In the female rats, chromophobe adenoma of the pituitary occurred
 

in three control, 15 low-dose, and 12 high-dose animals. Lesser
 

numbers occurred in both treated and control males. This is a
 

frequently encountered tumor in several strains of rats, particularly in females. Pheochromocytoma of the adrenal gland and
 

isletcell adenomas of the pancreas were found in small numbers in
 

both treated and control animals.
 

Interstitial-cell tumor of the testis occurred in nearly all male
 

rats. This extremely high incidence is found only in Fischer 344
 

rats.
 

Various benign and malignant tumors of the mammary gland were
 

scattered throughout both treated and control groups.
 

As is usually seen in long-term chronic studies, a large number
 

of inflammatory, degenerative, and proliferative lesions
 

occurred. The morphologic changes associated with senile rat
 

kidneys were the most prominent nonneoplastic lesions occurring
 

in the treated animals of both sexes. All the treated animals
 

had moderate to severe chronic nephritis. Fewer control animals
 

also exhibited this inflammation, but it was less severe.
 

Epithelial hyperplasia of the urinary tract occurred in the
 

62
 
treated animals only. Three high-dose males, four low-dose
 

females, and five high-dose females had this proliferative change
 

in the urinary bladder. Four low-dose males had urothelial
 

proliferation in the ureter or renal pelvis. This lesion is
 

considered noteworthy, since a substantial number of treated
 

animals had urinary tract lesions.
 

The pulmonary lesions, consisting for the most part of
 

morphologic changes associated with chronic murine pneumonia,
 

were more prevalent in both high-dose males and females than in
 

controls. However, the incidence of chronic murine pneumonia in
 

controls was similar to that in low-dose males and females; thus,
 

no compound-related effect appears to be present.
 

The treated females had a higher incidence of basophilic
 

cytoplasmic alteration of hepatocytes than did the controls.
 

This lesion was recognized in 2 controls, 15 low-dose, and 15
 

high-dose females. This change was observed in the males as
 

well, but in nearly equal proportions in all groups, including
 

the controls.
 

In the endocrine system, there were many types of lesions
 

scattered through all the treated groups and controls. The only
 

cluster of lesions that appears significant is in the 12 low-dose
 

males with C-cell hyperplasia of the thyroid. This represents a
 

24% incidence, compared with 5% in the controls. However, C-cell
 

63
 
tumors of the thyroid appeared in approximately equal numbers in
 

both treated and control groups.
 

4. Statistical Analyses of Results (Rats)
 

Tables 10 and 11 show the overall incidence of tumors as well as
 

the incidence of site-specific tumors for male and female rats,
 

respectively. In the males, the overall incidence of tumors in
 

the treated animals did not differ statistically (P > 0.10) from
 

that of the controls. Although the incidence of primary tumors
 

in the lung in the low-dose group was higher than in the
 

controls, the difference was not statistically significant.
 

In the female rats, no P values less than 0.10 occur.
 

B. MICE (Na3NTA-H20)
 

1. Body Weights and Clinical Signs
 

The average weights of male mice were depressed in a dose-related
 

manner throughout most of the study, although the differences
 

decreased toward the end of the test (figure 9). The average
 

weights of female mice were depressed in a dose-related manner
 

throughout the study.
 

No significant clinical signs were observed in the first year of
 

treatment. In the second year an occasional tissue mass was
 

observed, and a few animals appeared to lose weight.
 

64
 
Table 10. Incidence of Tumors in Male Rats (Litton - Na3NTA.H20) 

Controls Low Dose High Dose
 

0 ppm 7,500 ppm 15,000 ppm
 

Incidence of Tumors
 

(excluding testicular tumors) 10/20 (50%) 26/49 (53%) 24/50 (48%)
 

Statistical Testsa	 N.S.,N.S. N.S. N.S.
 

95% Confidence Intervals
 

Weeks to First Observed Tumor 99	 51 81
 

Incidence of Tumors of the Lung
 

(excluding metastatic) 2/19 (11%) 10/47 (21%) 3/48 (6%)
 

Statistical Testsa	 P = 0.089 N.S. N.S.
 

nonlinear
 

95% Confidence Intervals
 

Weeks to First Observed Tumor 106	 106 106
 

a .*•% ^M •* »• J ^"% ^ ^ •^•^ "^— " ^ •^*^ ^ ^ ^ ^ *" ^ ^*» ^ V
 Beneath the untreated-control incidence are two quantities: (a) the P value for a 2x3 contingency
 

table using chisquare theory; (b) the P value for the test for linear trend if a linear trend fits
 

the data, or the word "nonlinear," which means that a linear trend on a logistic scale does not fit
 

the data.
 

Beneath the dosed-group incidence is the Fisher-Irwin one-tailed test for comparison of that group
 

with the control group when it is below 0.10; or, otherwise N.S. - not significant.
 
Table 11. Incidence of Tumors in Female Rats (Litton ­

Controls Low Dose High Dose
 

0 ppm 7,500 ppm 15,000 ppm
 

Incidence of Tumors (all tumors) 13/20 (65%) 36/50 (72%) 26/49 (53%)
 

Statistical Testsa N.S.,N.S. N.S. N.S.
 

95% Confidence Intervals
 

Weeks to First Observed Tumor 96 75 84
 

Incidence of Tumors (excluding
 

uterine tumors) 12/20 (60%) 33/50 (66%) 24/49 (49%)
 

ON
 

o> Statistical Testsa N.S.,N.S. N.S. N.S.
 

95% Confidence Intervals
 

Weeks to First Observed Tumor 96 75 84
 

a

Beneath the untreated-control incidence are two quantities: (a) the P value for a 2x3 contingency
 

table using chisquare theory; (b) the P value for the test for linear trend if a linear trend fits
 

the data, or the word "nonlinear,1, which means that a linear trend on a logistic scale does not fit
 

the data.
 

Beneath the dosed-group incidence is the Fisher-Irwin one-tailed test for comparison of that group
 

with the control group when it is below 0.10; or, otherwise N.S. - not significant.
 
TIME ON STUDY (WEEKS) 

Figure 9. Growth Curves for Mice, Na3NTA*H2O - Litton Study
 

67
 
2. Survival
 

The survival curves for mice are shown in figure 10. Survival
 

was comparable between all groups of both sexes. More than 85%
 

of each group survived to the end of the study, and the results
 

can be evaluated over the entire time period for both treated and
 

control groups. There is no dose-related trend (P > 0.10).
 

3. Pathology
 

Histopathologic findings are tabulated in Appendix C, tables C3
 

and C4, covering neoplasms and other proliferative lesions, and
 

in Appendix F, tables F3 and F4, covering nonneoplastic lesions.
 

Of the 200 treated and 40 control animals that were assigned to
 

this study, all were necropsied and evaluated histopathologically
 

with the exception of 11 that were autolyzed. Thus, where the
 

number of animals with lesions is delineated, comparisons should
 

be made with the respective group size, i.e., 20 control male, 48
 

low-dose male, 50 high-dose male, 18 control female, 46 low-dose
 

female, and 47 high-dose female animals.
 

Hepatocellular carcinomas occurred in one control, five low-dose,
 

and three high-dose males. In addition, three hepatocellular
 

adenomas occurred in three male controls. In females the
 

incidence of hepatocellular carcinomas was similar in both
 

treated and control groups.
 

68
 
TIME ON STUDY (WEEKS) TIME ON STUDY (WEEKS) 

Figure 10• Survival Curves for Mice, Na3NTA*H2O - Litton Study
 
A leukemia appeared in four high-dose males and four high-dose
 

females, seven low-dose females, and three female controls.
 

Malignant lymphomas occurred only in treated animals; however,
 

the lesions were randomly dispersed throughout the hematopoietic
 

system as single entities. Their significance is not clear at
 

this time.
 

Alveolar/bronchiolar carcinomas were observed in one control and
 

six low-dose males. In addition, each of these groups displayed
 

one adenoma, a frequently encountered neoplasm in this strain of
 

mouse.
 

The only nonneoplastic lesion occurring in these mice was hydronephrosis, which was observed in I low-dose and 28 high-dose
 

males. A similar picture was seen in the females: 1 low-dose
 

and 30 high-dose animals had hydronephrosis in 1 or both kidneys.
 

Hydronephrosis was not observed in any of the controls. No
 

occlusive lesions were observed; however, no special procedures
 

were used for their detection.
 

4. Statistical Analyses of Results (Mice)
 

In the male mice (table 12), the overall incidence of tumors in
 

the treatment groups was comparable to that of the control group
 

(P > 0.10). On the other hand, tumors of the hematopoietic
 

system were statistically more frequent in the high-dose group
 

70
 
Table 12. Incidence of Tumors in Male Mice (Litton Na3NTA.H2O)
 

Incidence of Tumors
 

Statistical Testsa
 

95% Confidence Intervals
 

Weeks to First Observed Tumor
 

Incidence of Tumors of the Lung
 

Statistical Testsa
 

95% Confidence Intervals
 

Weeks to First Observed Tumor
 

Controls
 

0 ppm
 

7/20 (35%)
 

N.S.,N.S.
 

91
 

2/20 (10%)
 

P = 0.013,
 

nonlinear
 

91
 

Low Dose
 

2,500 ppm
 

18/48 (38%)
 

N.S.
 

75
 

8/48 (17%)
 

N.S.
 

76
 

High Dose
 

5,000 ppm
 

14/50 (28%)
 

N.S.
 

0/49 (0%)
 

N.S.
 

72 
Table 12. Incidence of Tumors in Male Mice (Litton
 

(continued)
 

Controls Low Dose High Dose
 

0 ppm 2,500 ppm 5,000 ppm
 

Incidence of Tumors of the Hematopoietic
 

System (all malignant) 0/20 (0%) 4/47 (9%) 9/50 (18%)
 

Statistical Testsa	 P = 0.073, N.S. P = 0.038
 

P = 0.015
 

95% Confidence Intervals	 1.11 - oc
 

Weeks to First Observed Tumor	 75 86
 

a

Beneath the untreated-control incidence are two quantities: (a) the P value for a 2x3 contingency
 

table using chisquare theory; (b) the P value for the test for linear trend if a linear trend fits
 

the data, or the word "nonlinear," which means that a linear trend on a logistic scale does not fit
 

the data.
 

Beneath the dosed-group incidence is the Fisher-Irwin one-tailed test for comparison of that group
 

with the control group when it is below 0.10; ox, otherwise N.S. - not significant.
 
(P e 0.038) than in the controls; also, a significant linear
 

positive dose-response relation was found (P * 0.015).
 

In the females, no significant findings were obtained (table 13).
 

73
 
Table 13. Incidence of Tumors in Female Mice (Litton
 

Controls Low Dose High Dose
 

0 ppm 2,500 ppm 5,000 ppm
 

Incidence of Tumors 6/18 (33%) 11/46 (24%) 16/47 (34%:
 

Statistical Testsa N.S.,N.S. N.S. N.S.
 

95% Confidence Intervals
 

Weeks to First Observed Tumor 85 68 70
 

a

Beneath the untreated-control incidence are two quantities: (a) the P value for a 2x3 contingency 

table using chisquare theory: (b) the P value for the test for linear trend if a linear trend fits 

the data, or the word "nonlinear,11 which means that a linear trend on a logistic scale does not fit 

the data. 

Beneath the dosed-group incidence is the Fisher-Irwin one-tailed test for comparison of that group 

with the control group when it is below 0.10; or, otherwise N.S. - not significant. 
VII. DISCUSSION
 

The bioassays of NTA and Na3NTA.H2O showed that a variety of
 

neoplastic and nonneoplastic lesions of the urinary tract
 

developed in rats as a result of the administration of 7,500 to
 

20,000 ppm of either one of the test compounds in the diet.
 

These same lesions were detected in mice as a result of the
 

administration of 7,500 or 15,000 ppm of the acid, but not of
 

2,500 or 5,000 ppm of the salt form of the test compound.
 

Almost all of the tumors were primary epithelial in origin.
 

Their occurrence was particularly significant because they were
 

not found in any control animals and only rarely develop
 

spontaneously among animals of the strains on test. More tumors
 

were found in the higher- than the lower-dose groups for each
 

chemical and species (tables 14 and 15). When comparing results
 

between compounds and between laboratories, it must be recognized
 

that given concentrations of Na3NTA.H2O represent 30% less NTA
 

than do equal concentrations of the free acid and that the SRI
 

animals were treated for a total of 24 months while LBI animals
 

were treated for only 18. Changing the units of concentration
 

from parts per million to millimoles made such comparisons
 

easier, and the results became more consistent. In all cases
 

there was a rapid drop from the number of tumors observed at high
 

doses to those at lower doses. This finding is consistent with
 

75
 
-

- -

-

-

-

-

-

-

-

-

- -

-

-

-

TABLE 14: SUMMARY OF PRIMARY EPITHELIAL NEOPLASMS OF THE URINARY TRACT - RATS 

Stanford Litton Bionetics
 

All Na» NTA-H2,0 Na_ NTA-H 0 Na3 NTA'H20
 

Controls 20,000 ppm 15,000 ppm 7,500 ppm
 

SPECIFIC ORGAN/ (73 mM/Kg) (55 mM/Kg) (27 mM/Kg)
 

TUMOR TYPE M F M F
 M F M F


KIDNEY
 

Transitional-cell
 

Carcinoma 0/63 0/64* 4/24(17%) - - ­

Transitional-cell
 

Papilloma 0/63 0/64 -
 1/49(2%) ­

lubular—ceXJ.
 

Adenocarcinoma
 0/63 0/64 1/24(4%) 1/24(4%) 1/49(2%) - 1/48(2%) ­

Tubular-cell
 

Adenoma 0/63 0/64 3/24(13%) 3/24(13%)
 

URETER
 

Transitional-cell
 

Carcinoma 0/64 0/64 8/24(33%) 6/24(25%) _ ­

Papilloma 0/64 0/64 - - - 1/48(2%) ­

Papillary Adenoma 0/64 0/64
 

URINARY BLADDER
 

Transitional-cell
 

Carcinoma 0/57 0/56 1/24(4%) 5/24(21%) 1/42(2%) 3/43(7%)
 

Squamous-cell
 

Carcinoma 0/57 0/56 - - - 1/43(2%)
 

Papilloma 0/57 0/56 - - 1/42(2%) ­

Total Number of Animals
 

with Tumors of the Urina ry
 

Tract 0 0 13 13 2 2 2 4
 

Percent (54) (54) (4) (4) <4j> • <9)
 

denominators equal number of tissues examined hietopathologically.
 

In female rat receiving 2,000 ppm
 

Stanford
 

Na3 NTA-H20
 

200-2,000 ppm
 

(0.7-7 mM/Kg)
 

M/F
 

-


-


-


-


-


-


-


-


-


l/23(4%)b
 

1
 

(4)
 

Litton Bionetics
 

NTA NTA
 

15,000 ppm 7,500 ppm
 

(78 mM/Kg) (39 mM/Kg)
 

M F M F
 

1/49(2%)
 -


2/49(4%) - ­

3/49(6%) 1/49(2%) 1/49(2%)
 

2/49(4%) - ­

1/49(2%) - ­

- 11/48(23*) 2/45(4%)
 

-
 1/48(2%) ­

7 14 1


(14) <28) (2) (4)
 

76
 

 2 
TABLE 15: SUMMARY OF PRIMARY EPITHELIAL NEOPLASMS OF THE URINARY TRACT - MICE

 

Litton Bionetics 

All Na3 NTA H20 Na3 NTA.H20 NTA NTA 

Controls 5,000 ppm 2,500 ppm 15,000 ppm 7,500 ppm 

(18 mM/Kg) (9 mM/Kg (78 mM/Kg) (39 mM/Kg) 

SPECIFIC ORGAN/TUMOR TYPE M F M F M F M F M F 

KIDNEY 

Tubular-cell Adenoma 0/40a

 0/39 - - ­ 1/43(2%) -

Tubular-cell Adenocarcinoma 0/40 0/39 - - ­ 22/43(50%) 4/50(8%) 5/48(10%) ­

Papilloma 0/40 0/39 - 1/43(2%) -

Total Number of Animals with 

0 0 0 0 0 0 24 4 5 0 

Percent (56) (8) (10) 

Denominators equal number of tissues examined histopathologically. 

77
 
that of Nixon et al., (1972) who reported an absence of tumors in
 

rats receiving 5,000 ppm Na3NTA.H2O in the diet for 2 years.
 

Most of the tumors of the urinary tract in rats were of transitional-cell origin and were found mainly in the kidneys of males,
 

bladders of females, and ureters of both sexes. In addition to
 

the transitional-cell tumors at the different sites, tubular-cell
 

adenomas or carcinomas developed in highly significant
 

frequencies in the kidney of the rat (table 14). The various
 

lesions of the urinary tract may have been even more significant
 

had a greater number of kidney and bladder sections been examined
 

and had the ureters been routinely saved at necropsy; the tumors
 

of the ureter that were actually examined were those recognized
 

grossly or fortuitously sectioned with the kidneys. In mice, the
 

tumors of the urinary tract consisted primarily of tubular-cell
 

carcinomas of the kidney.
 

Transitional epithelial dysplasia and hyerplasia occurred in many
 

of the treated animals that displayed no urinary neoplams, but
 

not in untreated animals (Appendixes D, E, and F)•
 

Hydronephrosis and/or nephritis was observed in rats given high
 

doses of the test compounds. In mice hydronephrosis was induced
 

by low, nontumorigenic doses (5,000 ppm Na3NTA.H2O) as well as by
 

high, tumorigenic doses (15,000 ppm NTA).
 

Numerous other neoplastic lesions occurred, some of which
 

78
 
appeared statistically significant in certain tests. For
 

example, hematopoietic tumors showed a dose-related trend in male
 

rats given Na3NTA.H2O at SRI (P - 0.044), tumors of the liver in
 

female rats given NTA at LBI (P » 0.003), and endocrine tumors in
 

both male rats given Na3NTA.H2O at SRI (P « 0.037) and female
 

rats given NTA at LBI (P • 0.036). However, in other instances
 

the levels of incidence or of statistical significance were
 

comparatively low, the data were not reproducible in the
 

different tests, or the tumors were known to be variable in rate
 

of spontaneous occurrence. Therefore, no conclusions are drawn
 

as to the meaning of occurrence of tumors other than those of the
 

urinary tract.
 

In all tests body weights of high-dose rats and mice were lower
 

during the greater part of the test period than were respective
 

controls. The lower-dose groups generally showed lesser changes.
 

Decrease in survival also was related to the size of the dose,
 

but only in rats, for which levels of significance of the trend
 

were P « 0.001 in males given Na3NTA.H2O and P e 0.057 in females
 

given NTA.
 

The pathologic findings in the kidney were not unexpected, since
 

nephrotoxicity has been noted in several previous studies. Renal
 

adenomas were reported in treated rats that died before
 

termination of 2-year tests in which Na3NTA was administered in
 

79
 
the drinking water (Chernoff and Courtney, 1970). Rats receiving
 

diets containing up to 20,000 ppm Na3NTA for 90 days developed
 

hydronephrosis and renal tubule-cell damage at concentrations of
 

7,500 ppm and over (Nixon, 1971). The incidence and severity of
 

nephritis and nephrosis increased in rats receiving 1,500 or
 

5,000 ppm of the compound in the diet for 2 years, but no
 

compound-related increase in tumors was reported at these levels
 

Oixon et al., 1972). Rats administered 0.01, 0.1, or 1.0%
 

Na3NTA in the drinking water for 10 weeks showed marked
 

vacuolization of renal tubules and high mortality at the 1%
 

level, but no effect on weight gain and no histopathologic
 

changes in the kidney, brain, liver, or pancreas at the two lower
 

concentrations (Mahaffey and Goyer, 1972). When 20 ml of a 5%
 

NTA solution, disodium salt, was administered to rats in place of
 

drinking water 5 nights/week for 84 weeks, no significant
 

increase in the incidence of tumors was noted (Lijinski et al.,
 

1973); similarly treated mice also showed no significant increase
 

in the incidence of tumors (Greenblatt and Lijinsky, 1974).
 

Metabolic studies with several species indicate that NTA and
 

Na3NTA.H2O are either poorly absorbed, or if absorbed are rapidly
 

excreted and that metabolic transformation does not occur.
 

Michael and Wakim (1971) reported that NTA was readily absorbed
 

from the gastrointestinal tract in rats and dogs, but was poorly
 

absorbed in rabbits and monkeys. In either case, most of the
 

80
 
dose that remained in the body after 72 hours was found in the
 

skeleton, or, in the case of rabbits, still in the gastrointestinal tract. Absorption studies have not been reported with
 

mice; however, Tjalve (1972) observed that radioactivity
 

accumulated in the skeleton, kidney, and urinary bladder of mice
 

given 14C-labeled NTA. Metabolic data for NTA in humans
 

resembled data for rats and dogs except that the extent of
 

absorption for rats and dogs is four times greater than in humans
 

(Budney and Arnold, 1973). Little or no biotransformation was
 

observed in humans, judging by the finding that the radioactivity
 

present in urine of humans given the labeled NTA was represented
 

by unchanged NTA.
 

Since NTA and Na3NTA.H2O are eliminated rapidly through the
 

kidneys, lesions of the urinary tract which arise as a result of
 

administration of the compounds may be due to a local effect
 

which can be brought about only by high concentrations. The
 

occurrence of treatment-related neoplasms at high doses among
 

animals of this bioassay should be taken into account in
 

evaluations of hazards posed by use of the compounds.
 

81
 

VIII. BIBLIOGRAPHY
 

Berenblum, I., ed. Carcinogenicity Testing. UICC Technical
 

Report Series, Vol. 2. International Union Against Cancer,
 

Geneva, 1969.
 

Budney, J. A. and Arnold, J. D. Nitrilotriacetate (NTA): Human
 

metabolism and its importance in the total safety evaluation
 

program. Toxicol. Appl. Pharmacol. 25:48-53, 1973.
 

Chau, Y. K. and Fox, M. E. A GC method for the determination of
 

nitriloacetic acid in lake water. J. Chromatog. Sci.
 

9:271-275, 1971.
 

Chernoff, N. and Courtney, K. D. Maternal and fetal effects of
 

NTA, NTA and cadmium, NTA and mercury, NTA and nutritional
 

imbalance in mice and rats. Unpublished progress report.
 

NIEHS, dated 1 Dec. 1970.
 

Cochran, W.G. Soire methods of strengthening the common X^ tests.
 

Biometrics 10:417-451,1954.
 

Cox, D. R. Analysis of Binary Data, Methuen, London, 1970, pp.
 

61-65.
 

Cox, D. R. Regresion models and life tables. J. Roy. Statist.
 

Soc. B 34:187-220,1972.
 

Cureton, G. L. Aminopolycarboxylic acid chelating agents.
 

Chemical Econmomics Handbook, February, :512.5020A512~5020U, 1967."
 

Gart, J. J. Point and interval estimation of the common odds
 

ratio in the combination of 2x2 tables with fixed marginals.
 

Biometrika 57:471-475, 1970.
 

Greenblatt, M. and Lijinsky, W. Carcinogenesis and chronic
 

toxicity of nitrilotriacetic acid in Swiss mice. J. Natl.
 

Cancer Inst. 52:1123-1126, 1974.
 

Kaplan, E. L. and Meier, P. Nonparametric estimation from
 

incomplete observations. J. Amer. Statist. Assn. 53:457-481,
 

1958.
 

83
 
Lijinsky, W., Greenblatt, M., and Konmineni, C. Feeding studies
 

of nitrilotriacetic acid and derivatives in rats. J. Natl.
 

Cancer Inst. 50: 1061-1063, 1973.
 

Linhart, M. S., Cooper, J. A., Martin, R. L., Page, N. P., and
 

Peters, J. A. Carcinogenesis bioassay data system. J. Comp.
 

Biomed. Res. 7:230-248, 1974.
 

Mahaffey, K. R. and Goyer, R. A. Trisodium nitrilotriacetate in
 

drinking water: Metabolic and renal effects in rats. Arctu
 

Environ. Health 25:271-275, 1972.
 

Michael, W. R. and Wakim, J. M. Metabolism of nitrilotriacetic
 

acid (NTA). Toxicol. Appl. Pharmacol. 18:407-416, 1971.
 

Miller, R. G., Jr. Simultaneous Statistical Inference,
 

McGraw-Hill, New York, 1966.
 

Nixon, G. A. Toxicity evaluation of trisodium nitrilotriacetate.
 

Toxicol. Appl. Pharmacol. 18:398-406, 1971.
 

Nixon, G. A., Buehler, E. V., and Niewenhuis, R. J. Two-year rat
 

feeding study with trisodium nitrilotriacetate and its
 

calcium chelate. Toxicol. Appl. Pharmacol. 21:244-252, 1972.
 

Saffiotti, U., Cooper, J. A., and Page, N. P. Report on the
 

evaluation of the carcinogenesis bioassay of
 

nitrilotriacetic acid (NTA). Unpublished manuscript, dated,
 

1971.
 

Tarone, R. E. Tests for trend in life-table analysis. Biometrika
 

62:679-682, 1975.
 

Thayer, P. S. and Kensler, C. J. Current status of the
 

environmental and human safety aspects of nitrilotriacetic
 

acid. CRC Crit. Rev. Environ. Control 3:375-404, 1973.
 

Thomas, 0. Exact and asymptotic methods for the combination of
 

2x2 tables. J. Comp. Biomed. Res. 8:423-446, 1975.
 

Thomas, D., Breslow, N. and Gart, J.J. Regression analyses of 

proportions and life-table data. Unpublished manuscript, 

dated 1976. 

Tjalve, H. A study of the distribution and teratogenicity of
 

nitrilotriacetic acid (NTA) in mice. Toxicol. Appl.
 

Pharmacol. 23:216-221, 1972. 

84
 
APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS AND OTHER
 

PROLIFERATIVE LESIONS IN RATS FED NA3NTA-H20
 

IN THE DIET (SRI)
 

85
 

Table A1. Male Rats 

NUMBER OP MALE RATS WITH TUMOPS BY ANATOMIC SITE (NTA. NA3, H2O): SRI
 

(PERCENTAGES BY SYSTEM ARE BASED ON THE EFFECTIVE NUMBER OF ANIMALS)
 

CONTROL (VEH) LOW DOSE MID DOSE 

EFFECTIVE NUMBER OF ANIMALS 24(100%) 24(100%) 24 (100%) 

ANIMALS WITH TUMORS 22 (92%) 23 (96%) 24 (100%) 

INTEGUMENTARY SYSTEM* U (17%)
 

SKIN 1 2
 

PAPILLOMA 1
 

SQUAMOUS CELL CARCINOMA 1 1
 

SUBCUT TISSUE 2
 

FIBROMA 1
 

LIPOMA 1
 

RESPIRATORY SYSTEM 1 ("%) 1 (4%)
 

LUNG 1 1
 

TRANSIT-CELL CARCINOMA METASTAT
 

ALVEOLAR/BRONCHIOLAR ADENOMA 1
 

ALVEOLAR/BRONCHIOLAR CARCINOMA 1
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM 2 (8%) 1 (4%)
 

LIVER 2 1
 

NEOPLASTIC NODULE 2 1
 

PANCREAS
 

TRANSIT-CELL CARCINOMA METASTAT
 

URINARY SYSTEM
 

KIDNEY
 

TRANSITIONAL-CELL CARCINOMA
 HEMANGIOMA
 

TUBULAR-CELL ADENOMA
 TUBULAR-CELL ADENOCARCINOMA
 

URETER
 TRANSITIONAL-CELL CARCINOMA
 

URINARY BLADDER
 

TRANSITIONAL-CELL CARCINOMA
 

• COLUMNS APE OFFSET ACCORDING TO ORGAN SYSTEM, SPECIFIC ORGAN AND TUMOR TYPE
 

HIGH DDSE
 

24(100%)
 

24 (100%)
 

5 (2]%)
 

5
 

4
 

1
 

5 (21%)
 

3
 

3
 

2
 

2
 

14 (58%)
 

9
 

4
 

1
 

3
 

1
 

8
 

8
 

1
 

1
 

87
 
Table Al Male Rats (Continued)
 

NUMBER OF MALE RATS WITH TUMORS BY ANATOMIC SITE (NT A-NA3. H2O)2 

(PERCENTAGES BY SYSTEM ABE BASED ON THE EFFECTIVE NUMBER OF ANIMALS)
 

____________ ________ ________ ______.__­

ENDOCRINE SYSTEM* 2 (8%) 8 (33%) 7 (29*) 3 (13%) 

PITUITARY 2 1 

CHROMOPHOBE ADENOMA 2 1 

ADRENAL 2 1 2 3 

TRANSIT-CELL CARCINOMA METASTAT 1 

PHEOCHROMOCYTOMA 1 1 2 2 

CORTICAL CARCINOMA 1 

THYROID 4 4 

C-CELL CARCINOMA 3 

C-CELL ADENOMA 1 4 

PANCREATIC ISLETS 1 2 

ISLET-CELL ADENOMA 1 2 

HEMATOPOIETIC SYSTEM 2 (8%) 6 (25%) 1 (4%) 3 (13* ) 

MULTIPLE ORGANS 1 6 1 1 

LEUKEMIA 1 1 1 

LEUKEMIA GRANULOCYTIC 6 

MESENTERIC LYMPHNODE 2 

TRANSIT-CELL CARCINOMA METASTAT 2 

THYMUS 1 

MESOTHELIOMA MALIGNANT 1 

REPRODUCTIVE SYSTEM 22 (92%) 23 (96%) 24 (100%) 23 (96%) 

MAMMARY GLAND 1 

FIBROADENOMA 1 

SEMINAL VESICLE 1 

TRANSIT-CELL CARCINOMA METASTAT 1 

TESTIS 22 23 24 23 

INTERSTITIAL-CELL TUMOR 22 23 24 23 

MESOTHELIOMA 1 

* COLUMNS ARE OFFSET ACCORDING TO ORGAN SYSTEM, SPECIFIC ORGAtf AND TUMOR TYPE 
Table Al. Male Rats (Continued)
 

NUMBER OF MALE RATS WITH TUMORS 3Y ANATOMIC SITE (NT A. NA3. H2O)^ ST?T
 

(PERCENTAGES BY SYSTEM APE BASED ON THE EFFECTIVE NUMBER OF ANIMALS)
 

CONTROL(VEH) LOW DOSE MID DOSE ~ HiGH~55si 

NERVOUS SYSTEM 

NOFE 

MUSCULOSKELETAL SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

NONE 

ALL OTHE? SYSTEMS*

DIAPHRAGM

MESOTHELIOMA MALIGNANT

PLEHRA

MESOTHELIOMA

 2 (8%) 

1 

1 

1 

1 

TUMOR SUMMARY 

TOTAL ANIMALS WITH BENIGN TUMORS

TOTAL BENIGN TUMORS

 22 (92%)

 24

 23 (96%)

 31

 24 (100%)

 33

 23 (96%) 

 31 

TOTAL ANIMALS WITH MALIGNANT TUMORS 4 (17%) 8 (33%) 2 (8%)

TOTAL MALIGNANT TUMORS 5 10 2

TOTAL ANIMALS WITH METASTATIC TUMORS

TOTAL METASTATIC TUMORS

• COLUMNS ARE OFFSET ACCORDING TO ORGAN SYSTEM, SPECIFIC ORGAN AND TUMOR TYPE 

 11 (46%) 

 15 

 5 -(21%) 

 10 

89
 
Table A2. Female Rats
 

NUMBER OF FEMALE RATS WITH TUMORS
 

(PERCENTAGES BY SYSTEM ARE BASED ON THE EFFECTIVE NUMBER OF ANIMALS)
 

CONTROL (VEH) LOW DOSE MID DOSE
 

EFFECTIVE NUMBER OF ANIMALS 2U (100%) 24(100*) 24 (100%)
 

ANIMALS WITH TUMORS 18 (75%) 19 {19%) 17 (71%)
 

INTEGUMENTARY SYSTEM* 2 (8%) 1 (4%) 

SKIN 2 1 

SQUAMOUS CELL CARCINOMA 2 

SEBACEOUS ADENOCARCINOMA 1 

SEBACEOUS ADENOMA 

RESPIRATORY SYSTEM 1 ("%) 1 (4%) 3 (13%)
 

NASAL CAVITY 1
 

UNDIFFERENTIATED CARCINOMA 1
 

LUNG 1 1 3
 

UNDIFFERENTIATED CARCINOMA METAS 2
 

TRANSIT-CELL CARCINOMA METASTAT
 

ALVEOLAR/BRONCHIOLAR ADENOMA 1
 

ALVEOLAP/BRONCHIOLAR CARCINOMA 1
 

ADENOCARCINOMA METASTATIC 1
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM 2 (8%) 3 (13%) 2 (8%)
 

SALIVARY GLAND 1
 

UNDIFFERENTIATED CARCINOMA METAS 1
 

LIVER 1 3 1
 

NEOPLASTIC NODULE 1 3 1
 

SMALL INTESTINE 1
 

ADENOCARCINOMA 1
 

1
 

UNDIFFERENTIATED CARCINOMA 1
 

CECUM
 

• COLUMNS ARE OFFSET ACCORDING TO OPSAN SYSTEM, SPECIFIC ORGAN AND TUMOR TYPE
 

HIGH D3SE
 

24(100%)
 

20 (83%)
 

1 (4%)
 

1
 

1
 

8 (33*)
 

8
 

5
 

3
 

2 (8%)
 

1
 

1
 

1
 

1
 

90
 
Table A2. Female Rats (Continued)
 

NUMBER OF FEMALE PATS WITH TUMORS BY ANATOMIC SITE (NTA. NA3. H2O)f SRI
 

(PERCENTAGES BY SYSTEM ARE BASED ON THE EFFECTIVE NUMBER OF ANIMALS)
 

CONTROL (VEH) LOW DOSE MID DOSE HIGH DOSE
 

URINARY SYSTEM* 1 (4%) 2 (9%) 13 (5U%)
 

KIDNEY 1 1 U
 

UNDIFFERENTIATED CARCINOMA METAS 1
 

ADENOCARCINOMA METASTATIC 1
 

TUBULAR-CELL ADENOCARCINOMA 1
 

TUBULAR-CELL ADENOMA 3
 

URETEP 6
 

TPANSITIONAL-CELL CARCINOMA 6
 

URINARY BLADDER 1 5
 

PAPILLOMA 1
 

TRANSITIONAL-CELL CARCINOMA 5
 

ENDOCRINE SYSTEM 3 (13%) 8 (33%) 5 (21%) 4 (17*)
 

PITUITARY 2 U 3 2
 

CHROMOPHOBE ADENOMA 2 4 3 2
 

ADRENAL ' 1 1 2 1
 

PHEOCHROMOCYTOMA 1 1 2 1
 

THYROID 3
 

C-CELL CARCINOMA 2
 

C-CELL ADENOMA 1
 

PANCREATIC ISLETS 1
 

ISLET-CSLL ADENOMA 1
 

HEMATOPOIETtC SYSTEM 5 (21%) 4 (1*7%) 7 (29%) «* (17*)
 

MULTIPLE DRGANS a U "» 3
 

MALIGNANT LYMPHOMA 3 1 4 3
 

MALIG.LYMPHOMA LYMPHOCYTIC TYPE 1 1
 

LEUKEMIA GRANULOCYTIC 1 2
 

LEUKEMIA 2
 

LYMPH NODE 1
 

TRANSIT-CELL CARCINOMA METASTAT 1
 

LIVER 1
 

MALIGNANT LYMPHOMA 1
 

• COLUMNS ARE OFFSET ACCORDING TO ORGAN SYSTEM, SPECIFIC ORGAN AND TUMOR TYPE
 

91
 
Table A2. Female Rats (Continued)
 

NUMBER OF FEMALE RATS WITH TUMORS BY ANATOMIC SITE fNTA. NA3. H2O): SRI
 

(PERCENTAGES BY SYSTEM APE BASED ON THE EPFECTIVE NUMBER OF ANIMALS)
 

REPRODUCTIVE SYSTEM*
 

MAMMARY GLAND
 

ADENOMA
 

FIBROADENOMA
 

CLITOPAL GLAND
 

SEBACEOUS ADENOCARCINOMA
 

UTERUS
 

ADENOCARCINOMA
 

ENDOMETRIAL STROMAL POLYP
 

LEIOMYOMA
 

CERVIX UTERI
 

ENDOMETRIAL STROMAL POLYP
 

NERVOUS SYSTEM
 

BRATN
 

ASTROCYTOMA
 MENINGIOMA
 

MUSCULOSKELETAL SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

NONE
 

ALL OTHER SYSTEMS
 

NONE
 

TU1OR SUMMARY
 

TOTAL ANIMALS WITH BENIGN TUMOPS
 

TOTAL BENIGN TUMORS
 

TOTAL ANIMALS WITH MALIGNANT TUMORS
 

*OTAI MALIGNANT TUMORS
 

TOTAL ANIMALS WITH METASTATIC TUMORS
 

TOTAL METASTATIC TUMORS
 

* COL'IMMS ARE OFFSET ACCOPDING TO ORGAN

CONTROL (V EH) LOW DOSE
 

11 (46%) 11 (46%)
 

3 2
 

1
 

2 2
 

1
 

1
 

8 11
 

2 2
 

6 10
 

1
 

1
 

1
 

1 (4%) 1 (4%)
 

1 1
 

1
 

1
 

11 (46%) 13 (54%)
 

13 19
 

9 (38%) 8 (33%)
 

10 12
 

1 (4%)
 

2
 

 SYSTEM, SPECIFIC ORGiS AND

MID DOSE
 

7 (29%)
 

2
 

1
 

1
 

6
 

6
 

12 (50%)
 

15
 

10 (42%)
 

10
 

2 (8%)
 

4
 

 TUMOR TYPE 

HIGH DOSE
 

3 (13%)
 

1
 

1
 

2
 

2
 

1 (4%)
 

1
 

1
 

9 (33%)
 

11
 

16 (67%)
 

20
 

5 (21%)
 

6
 

92 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS AND OTHER
 

PROLIFERATIVE LESIONS IN RATS AND MICE FED NTA
 

IN THE DIET (LBI)
 

93
 

Table B1. Male Rats 

NUMBER OP MALE PATS WITH TUMORS BY ANATOMIC SITE1NTA); LBI
 

(PERCENTAGES BY SYSTEM ARE BASED ON THE EFFECTIVE NUMBER OF ANIMALS)
 

CONTROL(UNTP) LOW DOSE HIGH DOSE
 

EFFECTIVE NU1BER OF ANIMALS 20(100%) 50(100%) 50(100%)
 

ANIMALS WITH TU1ORS 19 (95%) 43 (86%) hi (94%)
 

INTEGUMENTARY SYSTEM* 2 (10%) U (8%) * (3%)
 

SKIN 1 2 3
 

PAPILLOMA 1
 

BASAL CELL CARCINOMA 1
 

ADNEXAL ADENOMA 1
 

ADNEXAL CARCINOMA 1
 

KERATOACANTHOMA 1
 

SARCOMA,NOS 1
 

SUBCUT TISSUE 1 2 1
 

FIBROMA 1
 

FIBROSARCOMA 1
 

HEMANGIOMA 1
 

KERATOACANTHO1A 1
 

RESPIRATORY SYSTEM 1 (5%) 5 (10%) 6 (12%)
 

LUNG 1 5 6
 

ALVEOLAF/BRONCHIOLAR ADENOMA 1 1
 

ALVEDLAP/BRONCHIOLAP CARCINOMA 1 a a
 

SARCOMA METASTATIC 1
 

CIRCULATORY SYSTEM 1 (2%)
 

MYOCARDIUM 1
 

SARCOMA METASTATIC 1
 

DIGESTIVE SYSTEM 3 (15%) 5 (10%) 3 (6%)
 

LIVER 3 5 2
 

NEOPLASTIC NODULE 3 2 2
 

HEPATOCELLULAR CAPCINO1A 3
 

STOMACH 1
 

PAPILLOMA 1
 

URINARY SYSTEM 1 (2%) 7 (14%)
 

KIDNEY 1 5
 

TUBULAR-CELL ADENOMA 1 3
 

TUBULAR-CELL ADENOCARCINOMA 2
 

URETER 3
 

PAPILLOMA 2
 

PAPILLARY ADENCMA 1
 

• COLUMNS ABE OFFSET ACCORDING TO ORGAN SYSTEM, SPECIFIC °> KG AN AND TUMOR TYPE 

95 
Table Bl. Male Rats (Continued)
 

NUMBER OP MALE RATS WITH TUMORS BY ANATOMIC SITE(NTA): LBI
 

(PERCENTAGES BY SYSTEM ARE BASED ON THE EFFECTIVE NOMBER OF ANIMALS)
 

ENDOCRINE SYSTEM*
 

PITUITARY
 

CHROMOPHOBE ADENOMA
 

BASOPHIL ADENOMA
 

ADRENAL
 

PHEOCHROMOCYTOMA
 

THYROID
 

FOLLICULAR-CELL ADENOMA
 

FOLLICULAR-CELL CARCINO1A
 

PANCREATIC ISLETS
 

ISLET-CELL ADENOMA
 

HEMATOPOIETIC SYSTEM
 

MULTIPLE ORGANS
 

LEUKEMIA
 

ERYTHROCYTIC LEUKEMIA
 

LIVER
 

ERYTHROCYTIC LEUKEMIA
 

REPRODUCTIVE SYSTEM
 

PREPUTIAL GLAND
 

* ADENOCARCIN3MA
 

TESTIS
 

INTERSTITIAL-CELL TUMOR
 

EPIDIDYMIS
 

HEMANGIOMA
 

NERVOUS SYSTEM
 

NONE
 

MUSCULOSKELETAL SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

EYELID
 

SARCOMA,NOS
 

CONTROL(UNTR) LOW DOSE
 

2 (10%) 16 (32%)
 

1 6
 

1 5
 

1
 

1 9
 

1 9
 

2
 

1
 

1
 

1 (5%) 3 (6%)
 

1 3
 

1 3
 

17 (85%) 42 (8U%)
 

2
 

2
 

16 42
 

16 42
 

1
 

1
 

1 (5%)
 

1
 

1
 

HIGH DOSE
 

8 (16%)
 

2
 

2
 

5
 

5
 

1
 

1
 

1
 

1
 

2 (4%)
 

1
 

1
 

1
 

45 (90%)
 

4
 

4
 

45
 

45
 

• COLUMNS APE OFFSET ACCORDING TO ORGAN SYSTEM, SPECIFIC ORGAN AND TUMOR TYPE
 

96
 
Table Bl. Male Rats (Continued)
 

NUMBER OF MALE RATS witH TUMORS BY ANATOMIC SITE(NTA): LBI
 

(PERCENTAGES BY SYSTEM ARE BASED ON THE EFFECTIVE NUMBER OF ANIMALS)
 

ALL OTHER SYSTEMS* 1 (2%) 

MULTIPLE ORGANS 1 

TUBULAR-CELL ADENOCARCINOMA META 1 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH BENIGN TUMORS 18 (90%) 43 (86%) 46 (92%).
 TOTAL BENIGN TUMORS 19 63 65
 

TOTAL ANIMALS WITH MALIGNANT TUMOFS 5 (25%) 13 (26%) 12 (24%)
 

TOTAL MALIGNANT TUMORS 5 14 13
 

TOTAL ANIMALS WITH METASTATIC TUMORS 2 (4%)
 

TOTAL METASTATIC TUMOFS 3
 

• COLUMN? ARE OFFSET ACCORDING TO ORGAN SYSTEM, SPECIFIC ORGAN AND TUMOR TYPE
 

97
 
Table B2. Female Rats
 

NUMBER OF FEMALE RATS WITH TUMORS BY ANATOMIC SITE(NTA): LBI 

(PERCENTAGES BY SYSTEM APE BASED ON THE
 

EFFECTIVE NUMBER OF ANIMALS
 

ANIMALS WITH TUMORS
 

INTESUMENTARY SYSTEM*
 

SKIN
 

BASAL CELL CARCINOMA
 

CARCINOSARCOMA
 

SUBCUT TISSUE
 

SQUAMOUS CELL CARCINOMA
 

CARCINOSARCOMA
 

RESPIRATORY SYSTEM
 

LUNG
 

SQUAMOUS CELL CARCINOMA METASTAT
 

ALVEOLAR/BPONCHIOLAP CARCINOMA
 

CARCINOSARCOMA PETASTAT
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

LIVER
 

NEOPLASTIC NODULE
 

PANCREAS
 

ACINAR-CELL ADENOMA
 

JPINARY SYSTEM
 

KIDNEY
 

TRANSIT-CELL PAPILLOMA
 

1UJBULAP-CELL ADENOMA
 

URINAPY BLADDEP
 

SQUAMOUS CELL CARCINOMA
 

TPANSITIOMAL-CELL CARCINOMA
 

ENDOCRINE SYSTEM
 

PITUITARY
 

CHROMOPHOBE ADENOMA
 

BASOPHIL AD2NOMA
 * COLUMNS ARE OFFSET ACCOPDING TO ORGAN

EFFECTIVE NUMBER OF ANIMALS) 

CONTPOL (UNT P) LOW DOSE HIGH DDSE 

20(100%) 

14 (70%) 

50(100%) 

33 (66%) 

50(100%) 

47 (94%) 

2 (10%) 1 (2%)
 

2
 

1
 

1
 

1 1
 

1
 

1
 

1 (c

%) 4 (8%) 7 (14%)
 

1 u 7
 

1
 

3 7
 

1
 

2 (10%) 8 (16%) 22 (44%)
 

2 8 22
 

2 8 22
 

1
 

1
 

2 (4%) 14 (28%)
 

2
 

1
 

1
 

2 12
 

1
 

2 11
 

6 (30%) 13 (26%) 25 (50%)
 

6 10 12
 

6 9 11
 

1
 1
 

 SYSTEM, SPECIFIc'OEG AN AND~TUMOR '"YPE " 

98 
Table B2. Female Rats (Continued)
 

NUMBEP_OF_FEMALE-RATS_WirH-.TUMORS_BY_ANATOHIC_SITE1£TA) r LBI 

(PERCENTAGES BY SYSTE1 APE BASED ON THE

ADRENAL


PHEOCHROMOCYTOMA


THYROID


C-CELL ADENOMA


C-CELL CARCINOMA


HEMATOPOIETIC SYSTEM*


MULTIPLE ORGANS


MALIGNANT LYMPHOMA


MALIG.LYMPHOMA LYMPHOCYTIC TYPE


MALICLYMPHOMA HISTIOCYTIC TYPE


LEUKEMIA


ERYTHROCYTIC LEUKEMIA


SUBMANDIBULAR L.NODE

SQUAMOUS CELL CARCINOMA METASTAT

PANCREATIC L-tfODE


MALIG.LYMPHOMA HISTIOCYTIC TYPE


LUNG


MALIG.LYMPHOMA HISTIOCYTIC TYPE


PEPRODUCTIVE SYSTEM


MAMMAFY GLAND


ADENOCARCINOMA


CYSTADENOMA


FIBROADENOMA


MAMMARY DUCT


CARCINOMA


CYSTADENOCARCINOMA


FIBROADENOMA


CLITORAL GLAND


ADENOCARCINOMA


UTERUS


ADENOCARCINOMA


MULTIPLE POLYPOSIS


ENDOMETRIAL STROMAL POLYP


ENDOMETRIAL STROMAL SARCOMA


OVARY


FIBROSAPCOMA


 EFFECTIVE NUMBER OF ANIMALS) 

„„„„„„_ „___„ „„-_-„­

1 1U
 

1 1 <4
 

3 3 3
 

2 2 2
 

1 1 1
 

 3 (15%) 6 (12%) 6 (12%)
 

3 U 6
 

1
 

1
 

1
 

3 2 3
 

2
 

1 

1 

1
 

1
 

1
 

1
 

 7 (35%) 1S (30%) 7 (1U%)
 

6 U 2
 

1
 

3 1
 

3 3 1
 

3
 

1
 

1
 

1
 

1 2
 

1 2
 

2 7 «
 2
 

2
 

2 4 2
 

1
 

1
 

1
 

* COLUMNS ARE OPFSE^ ACCORDING TO ORGAN SYSTEM, SPECIFIC *>RGAN AND TUKOE TYPE
 

99
 
Table B2. Female Rats (Continued)
 

NUMBER 0F FEMALE RATS WITH TUMORS BY ANATOMIC SJTE(NTA): LBI
 

(PERCENTAGES BY SYSTEM ARE BASED CN THE EFFECTIVE NUMBER OF ANIMALS)
 

NERVOUS SYSTEM*
 

CEPEBRUM
 

ASTPOCYTOMA
 

BRAIN
 

GLIOMA
 

MUSCULOSKELETAL SYSTEM
 

nNE
 

SPECIAL SENSE ORGANS
 

EAR CANAL
 

SQUAMOUS CELL CARCINOMA
 

ALL OTHEP SYSTEMS
 

NONE
 

TUrOP SUMMARY
 

TO^AL ANIMALS WITH BENIGN TUMORS
 

TOTAL BENIGN TUMORS
 

T?TAL ANIMALS WITH MALIGNANT TUMOPS
 

TOTAL MALIGNANT TUMORS
 

TOTAL ANIMALS WITH METASTATIC TUMOPS
 

TOTAL METASTATIC TUMOPS
 

CONTFOLfUN^E) LOW DOSE HIGH DOSS 

1 (2%) 2 (UX) 

1 

1 

1 1 

1 1 

1 (5%) 

1 

1 

9 (45%) 18 (3f%) 27 (5U%) 

17 23 35 

7 (35%) 16 (32%) 25 (50*) 

8 20 31 

1 (5%) 

1 

1 (2%) 

2 

• COLUMNS AFE OFFSET ACCOPDING TO OPGAN SYSTEM, SPECIFIC ORGAN AND TUMOR TYPE
 

100
 
Table B3. Male Mice 

NUMBER OF MALE MICE WITH TUMORS BY ANATOMIC SITE(NTA):
 

BY SYSTEM APE BASED CN THE EFFECTIVE NUMBER ^F ANIMALS)
 

CONTROL (UNTF) LOW DOSE HIGH DDSE 

EFFECTIVE NUMBER OF ANIMALS 20(100%) U9(100%) tttt(100%) 

ANIMALS WITH TUMORS 10 (50%) 1U (29%) 31 (10%) 

INTEGUMENTARY SYSTEM* 2 (5%) 

SUBCUT TISSUE 2 

HEMANGIOMA 2 

RESPIRATORY SYSTEM U (20%) 5 (10%) 6 (1U%)
 

IUNG U F 6
 

ALVEOLAR/BRONCHIOLAR ADENOMA 3 2
 

ALVEOLAP/BRONCHIOLAP CARCINOMA U 2 tt
 

CIFCULATOP.Y SYSTEM
 

NONE
 

DIGESTIVE SYSTEM 3 (15%) U (8%) 2 (5%) 

LIVER

HEPATOCELLULAR

HEPATOCELLULAR

HAMARTOMA

 ADENOMA

 CARCINOMA

 3 

3 1 

2

 1 

^ 2 

2 

URINAPY SYSTEM 5 (10%) 25 (55%)
 

KIDNEY 5 23
 

TUBULAR-CELL ADENOMA 1
 

TUBULAR-CELL ADENOCARCINOMA 5 22
 

KIDNEY/PELVIS 1
 

PAPILLOMA 1
 

ENDOCRINE SYSTEM
 

NCNE
 

HEMATOPOIETIC SYSTEM 2 (10%) tt (8%) 6 (1tt%)
 

MULTIPLE ORGANS 1 4 U
 

MALIGNANT LYMPHOMA 1
 

LEUKEMIA 1 2 3
 

LEUKEMIA LYMPHOCYTIC 2
 

SPLEEN 1
 

HEMANGIOMA 1
 

* COLUMNS ARE OFFSET ACCORDING TO ORGAN SYSTFM, SPECIFIC OFGAN AND TUMOR TYPE
 

101
 
Table B3. Male Mice (Continued)
 

NUMBEP 0F MALE MICE WITH TUMOPS BY ANATOMIC SITE(NTA): LBI 

(PEPCFNTAGES 3Y SYSTEM ARE BASED ON THE EFFECTIVE NUMBEP OF ANIMALS) 

CONTROL (fJNTF) LOW DOSE HIGH DOSS 

PANCPEATIC L.rODE


MALIGNANT LYMPHOMA


MESENTERIC LYMPHNODE


MALIGNANT LYMPHOMA


REPRODUCTIVE SYSTEM
 

NONE
 

NERVOUS SYSTEM
 

NONE
 

MUSCULOSKELETAL SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

NONE
 

ALL OTHEP SYSTEMS*


PELVIS


HEMANGIOMA


TUMOR SUMMARY
 

TOTAL ANIMALS WITH BENIGN TUMORS


TOTAL BENIGN TUMORS


TOTAL ANIMALS WITH MALIGNANT TUMOPS


TOTAL MALIGNANT TUMORS


TOTAI ANIMALS WITH METASTATIC TUMORS
 

TOTAI METASTATIC TUMORS
 

1
 

1
 

1
 

1
 

 1 (5%)
 

1
 

1
 

5 (25%) 5 (10%) 5 (112)
 

5 5 6
 

 5 (25%) 11 (22%) 28 (6U£)
 

5 13 34
 

* CCLURNS APE OFFSET ACCORDING TO OP1AN SYSTEM, SPECIFIC ORGAN AND TUMOP TYPE
 

102
 
Table B4. Female Mice 

NUMBER OF FEMALE MICE WITH TUMORS BY ANATOMIC SIT E (NTA): LBI 

(PERCENTAGES BY SYSTEM ARE BASED ON THE EFFECTIVE NUMBER OF ANIMALS) 

CONTROL (UNTR) LOW DOSE HIGH DOS 12
 

EFFECTIVE NUMBER OF ANIMALS 20(100%) 39(100%) 50(10054)
 

ANIMALS WITH TUMORS U (20%) 13 (33%) 19 (38%)
 

INTEGUMEKTARY SYSTEM* 1 (2%)
 

SKIN 1
 

HEMANGIOMA 1
 

RBSPIPATORY SYSTEM 2 (5%) U (8%)
 

LUNG 2 tt 

ALVEOLAR/BRONCHIOLAP ADENOMA 2 

ALVEOLAR/BRONCHIOLAR CARCINOMA 2 2 

CIRCULATORY SYSTEM 1 (3%)
 

HEART 1
 

NONCHROMAFFIN PARAGANGLIOMA 1
 

DIGESTIVE SYSTEM 2 (5%) 1 (2%)
 

LIVER 2
 

HEPATOCELLULAR CARCINOMA 1
 

HEMANGIOMA 1
 

ESOPHAGUS 1
 

SQUAMOUS CELL CARCINOMA 1
 

URINARY SYSTEM U (8%)
 

KIDNEY U
 

TUBULAR-CELL ADENOCARCINOMA <*
 

ENDOCRINE SYSTEM 1 (3%) 

PITUITARY 1 

CHROMOPHOBE ADENOMA 1 

HEMATOPOIETIC SYSTEM 4 (20%) 7 (18%) 10 (20%) 

MULTIPLE ORGANS 3 1 7
 

MALIGNANT LYMPHOMA 1 3 5
 

LEUKEMIA 2 3 1
 

LEUKEMIA LYMPHOCYTIC 1 1
 

COLUMNS APE OFFSET ACCORDING TO ORGAN SYSTEM, SPECIFIC ORGAN AND TUMOR TYPE
 

103 
Table B4. Female Mice (Continued)
 

NUMBEP OP FEMALE MICE WITH TUMORS BY ANATOMIC SITE (NTA):
 LBI
 

(PERCENTAGES BY SYSTEM ARE BASED ON THE EFFECTIVE NUMBER OF ANIMALS)
 

CONTROL(UNTR) LOW DOSE HIGH DOSE
 

SPLEEN 1
 

MALIGNANT LYMPHOMA 1
 

MEDIASTINAL L.NODE 1
 

MALIGNANT LYMPHOMA 1
 

MESENTERIC LYMPHNODE 2
 

MALIGNANT LYMPHOMA 2
 

REPRODUCTIVE SYSTEM* 1 (3%) 1 (2%)
 

UTERUS 1 1
 

ADENOMA 1
 

LEIOMYOMA 1
 

NERVOUS SYSTEM
 

NONE
 

MUSCULOSKELETAL SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

NONE
 

ALL OTHER SYSTEMS 1 (3%)
 

UTERINE LIGAMENT 1
 

HEMANGIOMA 1
 

TUMOR SUMMARY 

TOTAL ANIMALS WITH BENIGN TUMORS

TOTAI BENIGN TUMORS

 U (10%)

 5

 k (8%) 

U 

TOTAL ANIMALS WITH MALIGNANT TUMORS

TOTAL MALIGNANT TUMORS

 U (20%)

 U

 10 (26%)

 10

 16 (32%) 

 17 

TOTAL ANIMALS WITH METASTATIC 'TUMORS 

TOTAL METASTATIC TUMORS
 

• COLUMNS ARE OFFSET ACCORDING TO 0R3AN SYSTEM, SPECIFIC OP.SAN AND TUMOR TYPE
 

104
 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS AND OTHER
 

PROLIFERATIVE LESIONS IN RATS AND MICE FED
 

Na3NTA-H20 IN THE DIET (LBI)
 

105
 

Table CI. Male Rats 

NUMBER OF MALE RATS WITH TUMORS BY ANATOMIC SITE(NTA,NA3.H2O) LBI 

(PERCENTAGES BY SYSTEM ARE BASED ON THE EFFECTIVE NUMBER OF ANIMALS) 

CONTPOL(UNTR) LOW DOSE HIGH D0S2
 

EFFECTIVE NUMBER OF ANIMALS 20(100%) 49(100%) 50(100%)
 

ANIMALS WITH TUMORS 20(100%) 48 (98ft) 44 (88%)
 

INTEGUMENTARY SYSTEM * 2 (4%) 2 (4%)
 

SKIN 1
 

SQUAMOUS CELL CARCINOMA 1
 

SUBCUT TISSUE 1 2
 

FTBPOMA 1 1
 

SARCOMA,NOS 1
 

RESPIRATORY SYSTEM 2 (10%) 12 (24%) 3 (6%)
 

LUNG 2 12 3
 

SQUAMOUS CELL CARCINOMA METASTAT 1
 

ALVEOLAR/BRONCHIOLAP ADENOMA 1 2 2
 

ALVEOLAF/BRONCHIOLAR CARCINOMA 1 8 1
 

SARCOMA METASTATIC 1
 

CIFCULATORY SYSTEM 1 (2%)
 

HEART 1
 

SARCCMA,NOS 1
 

DIGESTIVE SYSTEM 2 (10%) 1 (2%) 3 (6%)
 

SALIVARY GLAND 1
 

MIXED TUMOR MALIGNANT 1
 

LIVER 1 1
 

NEOPLASTIC NODULS 1 1
 

STOMACH 1
 

PAPILLOMA 1
 

SMALL TNTESTIFS 1 1
 

ADENOCARCINOMA 1 1
 

URINARY SYSTEM U (8%) 2 {*%)
 

KIDNEY 3 1
 

TUBULAR-CELL ADENOCARCINOMA 1 1
 

SARCOMA METASTATIC 1
 

HAMARTOMA 1
 

KIDNEY/PELVIS 1
 

"RANSITiCELL_PAPILLOMA _ 1
 * COLUMNS AFE OFFSET ACCORDING TO ORGAN SYSTEM, SPECIFIC ORGAN AND TUMOR TYP£
 

107 
Table CI. Male Rats (Continued)
 

NUMBEP. OF MALE PATS WITH TUTORS BY ANATOMIC ST E (NTA. NA3. H2O) I LBI 

(PERCENTAGES BY SYSTEM APE BASED ON THE EFFECTIVE NUMBEP OF ANIMALS)
 

CONTROL (UNTF) LOW DOSE HIGH DOSE
 

UPETER 1
 

PAPILLOMA 1
 

ENDOCRINE SYSTEM* 8 (40%) 11 (2255) 11 (22%)
 

PITUITARY 2 3 1
 

CHROMOPHOBE ADENOMA 2 3 1
 

ADRENAL 4 3 5
 

PHEOCHROMOCYTOMA 4 3 5
 

THYROID 2 2 5
 

C-CELL ADENDMA 2 1 2
 

C-CELL CARCINOMA 1 2
 

FIBROSARCOMA 1
 

PANCREATIC ISLETS 1
 

ISLET-CELL ADENOMA 3 1
 

HEMATOPOIETIC SYSTEM 3 (15%) 1 (2%) 6 (12£)
 

MULTIPLE ORGANS 2 5
 

LEUKEMIA 2 4
 

ERYTHROCYTIC LEUKEMIA 1
 

MALIG.LYMPHOMA HISTIOCYTIC TYPE 1
 

SPLEEN 1 1
 

SARCOMA 1
 

MALIG.LYMPHOMA HISTIOCYTIC TYPE 1
 

REPRODUCTIVE SYSTEM 20 (100%) U5 (92%) 41 (92%)
 

MAMMARY GLAND 1
 

FIBROADENOMA 1
 

PREPUTIAL SLAND 1 1
 

AD2N0CARCIN)MA 1 1
 

TESTIS 20 US 41
 

INTERSTITIAL-CELL TUMOR 20 U5 41
 

EPIDIDYMIS 1
 

HEMANGIOMA 1
 

NEPVOUS SYSTEM 1 (2%) 1 (2%)
 

BRAIN 1
 1
 

ASTROCYTOMA 1
 

OLIGODENDROGLIOMA 1
 

* COLUMNS APE OFFSET ACCORDING TO ORGAN SYSTEM, SPECIFIC OPGAN AND TUMOP TYPE 

108 
Table CI. Male Rats (Continued)
 

NUMBER OF MALE PATS WITH TUMORS BY ANATOMIC SIT E (NT A. NA 3 . H2O); LBI 

(PERCENTAGES BY SYSTEM ARE BASED Otf THE EFFECTIVE NUMBER OF ANIMALS) 

CONTROL(UNTR) LOW DOSE HIGH DOSE 

MUSCULOSKELETAL SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

NONE 

ALL OTHEP SYSTE1S*	 2 (4%) 1 (2%) 

MULTIPLE ORGANS 1 1 

MESOTHELIOMA 1 1 

PERITONEUM 1 

MESOTHELIOMA 1 

TUMOR SUMFAFY 

TOTAL ANIMALS WITH BENIGN TUMORS 20 (100%) 47 (96%) 42 (84%) 

TOTAL BENI3N TUMORS 30 6 1 55 

TOTAL ANir.ALS WITH MALIGNANT TUTORS 5 (25%) 15 (31%)* * 13 (26%) 

TOTAI MAIIGNANT TUMORS 5 16 16 

TOTAL ANIKALS WITH METASTATIC TUMORS 2 (H%) 

TOTAI METASTATIC TUMORS 3 

• COLUMNS APE OFFSET ACCORDING TO ORGAK SYSTEM, SPECIFIC OPGAN AND TUMOR TYPE 

••	 ANTMAL #1O OF GROUP 11-120 3 HAS A PRIKARY SARCOMA WHICH THE LITTON BIONETICS 

LABORATORY WAS UFABLE TO FIND, 

109
 
Table C2. Female Rats
 

HUMBEF OF FEMALE RATS HITK TUMORS BY ANATOFIC SITE(NTA.NA3.H2O): LBI
 

(PERCENTAGES BY SYSTEM ARE BASED ON THE EFFECTIVE NUMBER OF ANIMALS)
 

controL(unTR) LOW DOSE HIGH DOSE 

EFFECTIVE NUMBER OF ANIMALS 20(100%) 50(100%) 49 (100%) 

ANIMALS WITH TUMORS 13 (65%) 36 (72%) 26 (53%) 

INTEGUMENTAPY SYSTEM * 2 (4%)
 

SUBCUT TISSUE 2
 

FIBROMA 1
 

LIPOMA 1
 

RESPIRATORY SYSTEM 1 (5%) 5 (10%) 3 (6%)
 

LUNG 1 5 3
 

ALVEOLAR/BRONCHIOLAP ADENOMA 1 1 1
 

ALVEOLAR/BRONCHIOLAF CARCINOMA 4 2
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM 2 (4%) 2 (4%)
 

LIVER 1 1
 

NEOPLASTIC NODULE 1 1
 

ESOPHAGUS 1
 

SQUAMOUS CELL CARCINOMA 1
 

COLON 1
 

ADENOMATOUS POLYP 1
 

URINAPY SYSTEM 4 (8%) 2 (4%)
 

URINARY BLADDER 4 2
 

PAPILLOMA 1
 

SQUAMOUS CELL CARCINOMA 1
 

TRANSITIONAL-CELL CAFCISOSA 3 1
 

ENDOCRINE SYSTEM 7 (35%) 19 (38%) 13 (21%)
 

PITUITARY 3 16 13
 

ADENOMA 1
 

CHROMOPHOBE ADENOMA 3 15 12
 

BASOPHIL ADENOMA 1
 

ADRENAL 1
 

PHEOCHROMOCYTOMA 1
 

• COLUMNS ARE OFFSET ACCORDING TO ORGAN SYSTEM, SPECIFIC ORGAN AND TUMOR TYPE
 

110
 
Table C2. Female Rats (Continued)
 

NUMBER OP FEMALE RATS WITH TUMOPS BY ANATOMIC SITE (NT A. NA3^H2O): LBI 

(PERCENTAGES BY SYSTEM APE BASED ON THE EFFECTIVE NUMBER OF ANIMALS)
 

CONTROL(UNTR) LOW DOSE HIGH D0S 3 

THYROID 2 4 2

 

FOLLICULAR-CELL ADENOMA 1

 

C-CEIL ADENOMA 1 4 1 

C-CELL CARCINOMA 1 1

 

PANCREATIC ISLETS 1

 

ISLET-CELL ADENOMA 1

 

HEMATOPOIETIC SYSTEM* 2 (10%) 1 (2%) 5 (10%) 

MULTIPL 5 E ORGANS 2 1

 

MALIGNANT LYMPHOMA 1 

LEUKEMIA 2 1

 4 

REPRODUCTIVE SYSTEM 8 (40%) 18 (36%) 8 (16%) 

MAMMARY GIAND 4 10 3

 

SQUAMOUS CELL CARCINOMA 1

 

ADENOMA 1

 

ADENOCARCINOMA 1

 

CYST ADENOMA 1

 

FIBROMA 1

 

FIBROADENOMA 4 7 1

 

CLITORAL GLAND 1 2 1

 

ADENOCARCINOMA 1 2 1

 

4 

ENDOMETRIAL STROMAL POLYP 7 

UTERUS 7 

NERVOUS SYSTEM 

NOFE 

MUSCULOSKELETAL SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

NONE 

ALL OTHER SYSTEMS 

NONE 

* COLUMNS ARE OFFSET ACCORDING TO ORGAN SYSTEM, SPECIFIC ORGAN AND TUMOR TYPE
 

111
 
Table C2. Female Rats (Continued)
 

NUMBER OF FEMALE RATS WITH TUMORS BY ANATOMIC SITE (NTA.NA3-H2O), : LBI
 

(PERCENTAGES BY SYSTEM ARE BASED ON THE EFFECTIVE NUMBEP OF ANIFALS)
 

CONTROL(UNTR) LOW DOSE HIGH DOSE
 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH BENIGN "UMORS 10 (50%) 29 (58%) 17 (35*)
 

TOTAL BENIGN TUMORS 15 42 21
 

TOTAL ANIMALS WITH MALIGNANT TUTORS 4 (20%) 11 (22%) 12 (2U%)
 

TOTAL MALIGNANT TUMOFS 4 11 13
 

TOTAL ANIMALS WITH METASTATIC TUMORS
 

TOTAL METASTATIC TUMORS
 

* COLUMNS APE OFFSET ACCORDING TO ORGAN SYSTEM, SPECIFIC OFGAN AND TUMOR TYPE
 

112
 
Table C3. Male Mice 

NUMBER OF MALE MICE HUH TUMORS BY ANATOMIC SITE (NT A. NA3. H2O): LBI 

(PERCENTAGES BY SYSTEM APE BASED ON THE EFFECTIVE NUMBER OF ANIMALS) 

CONTROL (UNTR) LOW DOSE HIGH DOSE 

EFFECTIVE NU

ANIMAIS WITH

MBER OF

 TUMORS 

ANIMALS 20(100%) 

7 (35%) 

U8(100%) 

18 (38%) 

50(100%) 

U (28%) 

INTEGUMENTAPY SYSTEM * 1 (2%) 1 (2%) 

SKIN 1 1 

SEBACEOUS ADENOMA 1 

FIBROSARCOMA 1 

RESPIRATORY SYSTEM 3 (15%) 9 (19%) 

1UNG 3 q 

HEPATOCELLULAR CARCINOMA METASTA 1 

ALVEOLAR/BRONCHIOLAR ADENOMA 1 1 

ALVEOLAR/BRONCHIOLAR CARCINOMA 1 6 

NONCHROMAFFIN PARAGANGLIOMA 1 

PHEOCHROBOCYTOMA METASTATIC 1 

CIPCULATORY SYSTEM 

NONE 

DIGESTIVE SYSTEM 5 (25%) 6 (13%) U (8%) 

LIVEfi 4 6 a 

HEPATOCELLULAR ADENOMA 3 

HEPATOCELLULAR CARCINOMA 1 5 3 

NONCHROMAFFIN PARAGANGLIOMA 1 

SARCOMA,UNDIFFERENTIATED 1 

SMALL INTESTINE 1 

ADENOCARCINOMA 1 

URTNAFY SYSTEM 1 (2%) 

KIDNEY 1 

NONCHROMAFFIN PARAGANGLIOMA 1 

ENDOCPINE SYSTEM 1 (5%) a (8%) 

ADRENAL 3 

CORTICAL CARCINOMA 1 

FONCHROMAFFIN PARAGANGLIOMA 1 

PHEOCHFOHCCY^OHA WALIGNAN* 1 

THYROID 1 1 

FOLLICUtAR-CELL ADENOfA 1 1 

• COLUMNS ARE OFFSET ACCORDING TO ORGAN SYSTEM, SPECIFIC OBGA^ AND TUMOR TYPE 

113
 
Table C3. Male Mice (Continued)
 

NUMBER OF HALE MICE WITH TUMORS BY ANATOMIC SITE (NTA. NA3. H20L* LBI
 

(PERCENTAGES BY SYSTEM ARE BASED ON THE EFFECTIVE NUMBER OF ANIHALS)
 

CONTROL(UNTR) LOW DOSE HIGH DOSE 

HEMATOPOIETIC SYSTEM* 4 (8%) 9 (18%) 

MULTIPLE ORGANS 1 5 

MALIGNANT LYMPHOMA 1 1 

LEUKEMIA 4 

SPLEEN 1 1 

HEMANGIOMA 1 1 

MESENTERIC LYHPHNODE 1 3 

MALIGNANT LYMPHOMA 1 3 

LUNG 1 

MALIGNANT LYMPHOMA 1 

KIDNEY 1 

MALIGNANT LYMPHOMA 1 

REPRODUCTIVE SYSTEM 1 (2%) 

TESTIS 1 

INTERSTITIAL-CELL TUMOR 1 

NERVOUS SYSTEM 1 (2%) 

BRAIN STEM 1 

SARCOMA,UNDIFFERENTIATED 1 

MUSCULOSKELETAL SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

NONE 

ALL OTHER SYSTEMS 1 (2%)
 

MESENTERY 1
 

HEMANGIOMA 1
 

AUTOLYSIS/NO NECROPSY PERFORMED 2
 

* COLUMNS APE OFFSET ACCORDING TO OBGAN SYSTEK, SPECIFIC ORGAN AND TUMOR TYPE
 

114
 
Table C3. Male dice (Continued) 

NUMBER OF MALE MICE WITH TUMORS BY ANATOMIC SITE (NT A. NA3. H2O):LBI
 

(PERCENTAGES BY SYSTEM ARE BASED ON THE EFFECTIVE NUMBER OF ANIMALS)
 

CONTPOI (UNTR) LOW DOSE Hloh DOSE 

TUMOR SUMMARY 

TOTAL ANIMALS WITH BENIGN TUMORS 4 (20%) 4 (8%) 4 (8%) 

TOTAL BSNI3N TUMORS 5 4 4 

TOTAL ANIPALS WITH MALIGNANT TUMORS 3 (15%) 15 (31%) 12 (24%) 

TOTAL MALIGNANT TUMORS 3 17 13 

TOTAL ANIMALS WITH METASTATIC TUMORS 1 (5%) 1 (2%) 

TOTAL METASTATIC TUMORS 1 1 

115 
Table C4. Female Mice 

NUMBER OF FEMALE MICE WITH TUMORS BY ANATOMIC SITE (NT A.NA3. H2O): LBI
 

(PERCENTAGES BY SYSTEM ARE BASED ON THE EFFECTIVE NUMBEP OF ANIMALS) 

CONTROL(UNTR) LOW DOSE HIGH DOSE 

EFFECTIVE NUMBER OF ANIMALS 

ANIMALS WITH TUMORS 

1R(100#) 

6 (33%) 

46(100%) 

11 (24%) 

47(100%) 

16 (34%) 

INTEGUMENTARY SYSTEM * 1 (2%) 

SKIN 

FIBROSARCOMA 

1 

1 

RESPIRATORY SYSTEM 1 (6%) 2 (4%)
 

LUNG 1 2
 

ALVEOLAP/BRONCHIOLAF ADENOMA 1
 

ALVEOLAR/BRONCHIOLAR CARCINOMA 1 1
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM 2 (11%) 3 (6%) 3 (6%)
 

LIVER 2 3 3
 

HEPATOCELLULAR ADENOMA 1
 

HEPATOCELLULAR CARCINOMA 2 2 2
 

SARCOMA METASTATIC 1
 

URINARY SYSTE*
 

NONE
 

ENDOCRINE SYSTEM 1 (2%) 3 (6%)
 

PITUITARY 1 1
 

CHROMOPHOBE ADENOMA 1 1
 

ADRENAL 1
 

PHEOCHROMOCYTOMA 1
 

THYROID 1
 

FOLLICULAR-CSLL CARCINOMA 1
 

HEMATOPOIETIC SYSTEM 3 (17%) 8 (17%) 9 (19%)
 

MULTIPLE ORGANS 3 8 8
 

MALIGNANT LYMPHOMA 1 4
 

LEUKEMIA 3 7 4
 

SPLEEN 1
 

MALTGNANT LYMPHOMA 1
 

* COLUMNS ARE OFFSET ACCORDING TO ORGAN SYSTEM, SPECIFIC ORGAN AND TUMOR TYPE 

116
 
Table C4. Female Mice (Continued)
 

NUMBER OF FEMALE MICE WITH TUMORS BY ANATOMIC SITE(NTA.NA3^H2O): LBI 

(PERCENTAGES BY SYSTEM ARE BASED ON THE EFFECTIVE NUMBER OF ANIMALS) 

CONTROL (ONTP) LOW DOSE HIGH DOSE
 

REPRODUCTIVE SYSTEM* 1 (2%)
 

UTERUS
 1
 

SARCOMA,NOS
 1
 

NERVOUS SYSTEM
 

NONE
 

MUSCULOSKELETAL SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

NOME
 

ALL OTHER SYSTEMS
 

_NONE
 

TUMOR SUMMARY 

TOTAL ANIMALS WITH BENIGN TUMORS 2 (4%) 3 (6%) 

TOTAL BENIGN TUMORS 2 3 

TOTAL ANIMALS WITH MALIGNANT TUMORS 6 (33%) 11 (24%) 13 (28%) 

TOTAL MALIGNANT TUMORS 6 11 14 

TOTAL ANIMALS WITH METASTATIC TUMORS 1 (2%) 

TOTAL METASTATIC TUMORS 1 

* COLUMNS ARE OFFSET ACCORDING TO ORGAN SYSTEM, SPECIFIC ORGAN AND TUMOR TYPE 

117
 

APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN RATS FED NA3NTA-H20 IN THE DIET (SRI)
 

119
 

TABLE D1

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS 

IN MALE RATS FED NA3NTAH2O IN THE DIET

 

CONTROL LOW DOSE SID DOSS RICH DOST
 

AlIBALS INITIALLY IN STUDY
 AHIBALS NECROPSIED
 

A11MALS EXAMINED HISTOPATHOLOGICALLY
 AHIBALS WITH NON-TUMOR PATHOLOGY
 

INTEGUMENTARY SYSTBB
 

1OVE
 

BESPIRATORY SYSTEM •
 

TRACHEA
 INFLAMMATION
 

LUNG/BRONCHUS
 

BRONCHIECTASIS
 

LYMPHOID HYPERPLASIA
 

LUNG
 

MINERALIZATION
 

EMPHYSEMA
 

ATELECTASIS
 

CONGESTION
 EDEMA
 HEMORRHAGE
 

INFLAMMATION
 

INFLAMMATION INTERSTITIAL
 ABSCESS
 

HYPERPLASIA ADENOMATOUS
 

HYPERPLASIA ALVEOLAR-CELL
 

CIRCULATORY SYSTEM
 

EPlCARDlUr?
 

INFLAMMATION
 

HEART
 

FIBROSIS
 

24
 

2a (ioo%)
 

24 .
 24(100%)
 

15 (63%)
 

7
 

7
 

4
 

4
 

1
 

11
 

1
 

2
 

1
 

2
 

2
 

24 (100%)
 

1
 

1
 

24
 

24(100%)
 

23
 

23 (96%)
 

17 (74%)
 

5
 

5
 

10
 

2
 

9
 

8
 

1
 

1
 

1
 

1
 

3
 

1
 

23 (100%)
 

24
 

24(100%)
 

24
 

24(100%)
 

15 (63%)
 

8
 

8
 

7
 

3
 

5
 

10
 

2
 

1
 

5
 

2
 

24 (100%)
 

24
 

24(100%)
 

24
 

18 f75*>
 

4
 

4
 

t
 

1
 

15
 

13
 

t
 

4
 

22 (92%)
 

3
 

3
 

121 
3 

TABLE D1 MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MYOCARDIUM
 

INFLAMMA1ION
 FIBROSIS DIFFUSE
 

PULHONARY ARTEFY
 

THROMBOSIS
 

DIGESTIVE SYSTLH
 

LIVER
 GRANULOMA
 FIBROSIS FOCAL
 PELIOSIS HEPATIS
 

NECROSIS FOCAL
 BASOPHILIC CYTOPLASM ALTERATION
 HEMATOPOIESIS
 

LIVER/HEPATOCYTES
 

CYTOPLASMIC VACUOLIZATION
 

BILE DUCT
 INFLAMMATION
 HYPERPLASIA
 

PANCREAS
 

INFLAMMATION
 INFLAMMATION CHRONIC
 

FIBROSIS
 

FIBROSIS DIFFUSE
 

STOMACH
 INFLAMMATION
 ULCER
 INFLAMMATION CHROMIC
 

GASTRIC MUCOSA
 

EROSIVE INFLAMMATION
 

CONTROL
 

24
 

2
 

23
 

17 (71%)
 

5
 

4
 

1
 

12
 

3
 

11
 

3
 

3
 

4
 

2
 

1
 

1
 

LOW DOSE
 

?3
 

23
 

1
 

1
 

18 (78%)
 

8
 

6
 

1
 

1
 

12
 

6
 

11
 

4
 

4
 

1
 

1
 

MID DOSE
 

24
 

24
 

22 (92%)
 

8
 

6
 

1
 

1
 

1
 

1
 

19
 

7
 

15
 

4
 

1
 

3
 

3
 

1
 

3
 

2
 

2
 

HIGH DOSE
 

19
 

17
 

10 (42%)
 

5
 

5
 

6
 

3
 

3
 

1
 

1
 

122
 
TABLE D1 MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

 

URINARY SYSTEH
 

KIDNEY
 

HYDRONEPHROSIS
 

INFLAMMATION
 

INFLABHATION CHRONIC
 

NEPHROSIS
 

HYPERPLASIA
 

HYPERPLASIA TOBULAR-CELL
 

KIDNEY/PELVIS
 

HYPERPLASIA EPITHELIAL
 

ORETER
 HYPERPLASIA EPITHELIAL
 

URINARY BLADDER
 

HYPERPLASIA EPITHELIAL
 DYSPLASIA EPITHELIAL
 

ENDOCRINE SYSTEM
 

ADRENAL CORTEX
 CYTOPLASMIC VACUOLIZATION
 

HYPERPLASTIC NODULE
 

ADRENAL MEDULLA
 HYPERPLASIA FOCAL
 

THYROID
 

COLLOID CYST
 HYPERPLASIA C-CELL
 HYPERPLASIA FOLLICULAR-CELL
 

HEMATOPOIETIC SYSTEH
 

SPLEEN
 

HEMOSIDEROSIS
 

CONTROL
 

17 (71%)
 

17
 

17
 

1
 

1
 

* (17%)
 

2
 

2
 

1
 

1
 

1
 

1
 

22 (92%)
 

22
 

20
 

LOW DOSE
 

19 (83%)
 

19
 

1
 

18
 

1
 

1
 

3
 

3
 

1
 

5 (22%)
 

5
 

4
 

1
 

1
 

1
 

17 (71%>
 

17
 

17
 

BID DOSE
 

24 (100%)
 

24
 

2
 

22
 

3
 

3
 

4
 

4
 

5
 

3
 

4
 

6 (25%y
 

6
 

6
 

23 (96%)
 

23
 

23
 

HIGH DOSE
 

24 (100%)
 

24
 

18
 

4
 

2
 

1
 

20
 

7
 

7
 

3
 

3
 

8
 

8
 

1 (*%>
 

1
 

t
 

23 (96%)
 

23
 

16
 

123
 
TABLE D1 MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HID DOSE HIGV DOSE 

SPLEEN (CONT.) 

LYMPHOID HYPERPLASIA

HEMATOPOIESIS 21 15 22

 X 

 20 

MANDIBULAR L. NODE

GRANULOMA

MESENTERIC LYHPHNODE

ATROPHY

 1 

1 

1 

1 

REPRODUCTIVE SYSTEM

MAMMARY GLAND

HYPERPLASIA

PROSTATE

INFLAMMATION

SEMINAL VESICLE

DISTENTION

 3 (13%)

 1 

t 

1 

1 

1 

1 

2

2

 2 (9%) 3 (13%) 

3 

3 

NERVOUS SYSTEM

BRAIN

HEMORRHAGE

 1 (4%) 

1 

1 

MUSCULOSKELETAL SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

NONE 

124
 
TABLE D1 MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

COWTROL LOW DOSE RID DOSE HIGH DOSE 

ALL OTHER SYSTEMS 10 (42%) 4 (17%) 10 (42%) 8 (33%> 

ADIPOSE TISSUE NECROSIS FA1 10 

10 

4 

4 

10 

to 

8 

8 

AOTOLYSIS/NECROPSY PEHF/NO HISTO PERP 1 

ANIKAL DISPOSITION SUMMARY 

ANIMALS INITIALLY IN STUDY NATURAL DEATH* 

MORIBUND SACRIFICE SCHEDULED SACRIFICE ACCIDENTALLY KILLED TERMINAL SACRIFICE ANIMAL HISSING 

24 

6 

18 

24 

16 

24 

24 

24 

18 

• INCLUDES AUTOLYZED ANIBALS 

* SYSTEM PERCENTAGES ARE BASED ON NUMBER OF ANIMALS NECROPSIEP125
 
TABLE D2

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS 

IN FEMALE RATS FED NA3NTA'H20 IN THE DIET 

ANIMALS INITIALLY IN STDDY 

ANIMALS KECROPSIED
 

ANIBALS EXAMINED HISTOPATHOLOGICALLT
 ANIMALS WITH NON-TUMOR PATHOLOGY
 

INTEGOMENTAFY SYSTEM
 

NONE
 

RESPIRATORY SYSTEM •
 

TRACHEA
 

INFLAMMATION
 

LUNG/BBONCBUS
 

BRONCHIECTASIS
 

LYMPHOID HYPERPLASIA
 

LUNG
 

EDEBA
 

INFLAMMATION
 

INFLAMMATION INTERSTITIAL
 ABSCESS
 

LOMG/ALYEOLI
 

PHAGOCYTIC CELL
 

CIRCULATORY SYSTEM
 

HEART
 

MINERALIZATION
 

FIBROSIS
 

FIBROSIS FOCAL
 

MYOCARDIUM
 

INFLAMMATION
 

FIBROSIS DIFFUSE
 

CONTROL
 

2U
 

2U (100%)
 

24
 

23 (96%)
 

11 (46%)
 

6
 

23 (96%)
 

23
 

1
 

23
 

LOW DOSB
 

2a
 

24(100%)
 

24
 

23 (96%)
 

11 (46%)
 

23 (96%)
 

23
 

1
 

23
 

HID DOSE
 

24
 

24(100%)
 

24
 

24(100%)
 

7 (29%)
 

5
 

24 (100%)
 

1
 

1
 

23
 

23
 

HIGH DOSE
 

24
 

24(100%)
 

24
 

23 (96%)
 

13 (54%)
 

9
 

9
 

10 *
 

10
 

1
 

1
 

6 (25%)
 

4
 

1
 

3
 

2
 

2 ___
 

126
 
TABLE 02 FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

 

DIGESTIVE SYSTLK
 

LIVFR
 

INFL^'AIION GRANULOMATOUS
 

GRANULOMA
 FIBROSIS POCAL
 NECROSIS FOCAL
 BASOPHILIC CYTOPLASM ALTERATION
 

BILE DDC1
 

iNPLAMKATION
 HYPERPLASIA
 

PANCREAS
 

INFLAKWA1IOF
 INFLAMMATION CHRONIC
 

S?GRACK
 INFLAMMATION
 

COLON
 

INFLAMfAllOF
 

CECUM
 

INFLAMMATION
 

UPIhARY SYSTEM 

KIDNEY
 

HYDRONEPHROSIS
 

INFLAMMATION
 INFLAMMATION CHRONIC
 

HYPERPLASIA TUBULAR-CELL
 HEMOSIDEROSIS
 

KIDNEY/CORTEX
 FIBROSIS
 

R3NAL TUBULE
 HYPERPLASIA
 

KIDNEY/PELVIS
 

HYPERPLASIA EPITHELIAL
 

URETER
 

HYPERPLASIA EPITHELIAL
 

CONTROL
 

19 (79%)
 

11
 

9
 

2
 

1b
 

13
 

10
 

8
 

2
 

6
 

1
 

1
 

2
 

2
 

(21%) 

LOi DOSE
 

17 (71%)
 

8
 

7
 

1
 

11
 

9
 

6
 

3
 

3
 

1
 

1
 

1
 

1
 

5 (21%)
 

3
 

2
 

1
 

HID DOSE
 

19 (79%)
 

12
 

1
 

8
 

1
 

1
 

3
 

11
 

6
 

6
 

1
 

1
 

16 (67%)
 

5
 

1
 

1
 

HIGH uOSB
 

17 (71*)
 

8
 

1
 

6
 

1
 

T5
 

14
 

11
 

1
 

1
 

24 (100%)
 

21
 

20
 

11 

11 

11 

10 

10 

127 
TABLE D2 FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

 

CONTROL LOW DOSE HID DOSE HIGH DOSE 

URET2R (CONT.) 

DYSPLASIA 

URINARY BLADDER EDEMA HYPERPLASIA EPITHELIAL HYPERPLASIA FOCAL DYSPLASIA EPITHELIAL 

15 

13 

1 

3 

18 

1 

14 

8 

BNDOCEINE SYSTEH 3 (13%) U (17%) 6 (25%) 6 (25%) 

CYST 

1 

ADRENAL 

FIBROSIS FOCAL AN6IECTASIS 

ADRENAL CORTEX 

CYST FIBROSIS FOCAL NECROSIS FOCAL CYTOPLASMIC VACUOLIZATION 

HYPERPLASIA FOCAL 

THYROID 

HTPERPLASIA C-CELL 

THYROID FOLLICLE HYPERPLASIA 

HEMATOPOIETIC SYSTEH 18 (75%) 19 (79%) 16 (67%) 18 C 

SPLEEN 

HEBOSIDEROS1S 

HEMATOPOIESIS 

17 

17 

17 

18 

17 

18 

16 

16 

13 

18 

12 

17 

BANDIBOLAR L. NODE ATROPHY 

128
 
TABLE 02 FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

 

COUTHOL LOW DOSE BID DOSE HIGH DOSE 

MESENTERIC LYUPHNODE 1 

LYMPHANGIECTASIA 1 

REPRODUCTIVE SYSTEM 7 (29%) 7 (29%) 7 (29%)
 

MAMMARY GLAND 1 t
 

INFLAMMATION "YSTIC
 

HYPERPLASIA
 

MAMMARY GLAND/LOBDLE
 HYPERPLASIA
 

UTERUS
 

HYDROMETFA
 CYST
 INFLAMMATION
 

METAPLASIA SQUAMOUS
 

UTEPUS/ENDOMETRIUH
 INFLAMMATION
 

INFLAMMATION SUPPURATIVE
 INFLAMMATION CYSTIC
 

HYPERPLASIA
 

FALLOPIAN TUBE
 INFLAMMATION
 

OVARY
 CYST
 INFLAMMATION
 

INFLAMMATION GRANULOMATOUS
 

•ERVOUS SYSTEM
 

NONE
 

MUSCULOSKELETAL SYSTEM
 

NO IE
 

SPECIAL SENSE ORGANS
 

NONE
 

129
 
TABLE D2 FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

 

ALL OTHER SYSTEMS
 

ADIPOSE TISSUE
 

NECROSIS PAT
 

OMENTUM
 

CYST
 

HO LESION REPORTED
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY
 NATURAL DEATH*
 

MORIBUND SACRIFICE
 SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 TERMINAL SACRIFICE
 ANIMAL HISSING
 

• INCLUDES AUTOLYZBD ANIMALS
 

* SYSTEM PERCENTAGES ARE BASED ON
 

CONTROL
 

2 (8%)
 

1
 

1
 

1
 

1
 

24
 

3
 

3
 

18
 

NUMBER OF NAIMALS
 

130 

LOW DOSE MID DOSE HIGH DOSE
 

1 

2U 

1 

4 

19 

24 

2 

6 

16 

24 

1 

6 

17 

NECROPSIED. 
APPENDIX E
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN RATS AND MICE FED NTA IN THE DIET (LBI)
 

131
 

TABLE E1

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS 

IN MALE RATS FED NTA IN THE DIET 

CONTROL LOW DOSB HIGH DOSE
 

ANIMALS INITIALLY III STODT 

AHIHALS NECROPSIED 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 

ANIMALS WITH NON-TUBOR PATHOLOGY 

20 

20 

20 

19 

(100%) 

(95%) 

50 

50 

50 

49 

(100%) 

(98%) 

50 

50 

49 

49 

(100%) 

(98%) 

INTEGUMENTARY SYSTEM 1 (5%) 

SKIN 

EPIDERMAL INCLUSION CYST 

RESPIRATORY SYSTEM 6 (30%) 34 (68%) 34 (68%) 

TRACHEA 

INFLAMMATION 

INFLAMMATION CHRONIC 

LONG/BRONCHOS 

INFLAMMATION 

133
 
TABLE El MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

LUNG/BRONCHIOLE 

HYPERPLASIA ADEHOHATOOS 

LONG 

BRONCHOPNEUMONIA SUPPURATIVE 

ABSCESS 

PNEUttONIA CHRONIC ftURINE 

INFLAHHATION CHRONIC 

33 

31 

1 

32 

1 

2 

29 

1 

CIRCULATOR! STSTEH 5 (25%) 18 (36%) (28%) 

HYOCARDIUB 

INFLAMMATION FOCAL 

FIBROSIS 

FIBROSIS FOCAL 

FIBROSIS DIFFUSE 

DEGENERATION 

18 

13 

1 

4 

9 

1 

DIGESTIVE SYSTEM 13 13 f26%) 13 (26%) _ 

134 
TABLE El MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE
 

SALIVARY GLAND 

FIBROSIS DIFFUSE 

HYPERPLASIA 

LIVER 

METAMORPHOSIS FATTY 

BASOPHILIC CYTOPLASM

LEUKBMOID REACTION 

 ALTERATION 

3 

1 

PANCREAS 

FIBROSIS 

FIBROSIS FOCAL 

FIBROSIS DIFFUSE 

HYPERPLASIA INTRADUCTAL 

1 

2 

1 

2 

2 

PANCREATIC ACINUS 

ATROPHY 

SHALL INTESTINE 

INFLAMMATION 

ULCER 

INFLAMMATION CHRONIC 

LYMPHOID HYPERPLASIA 

135 
TABLE El MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE
 

COLON 

LYMPHOID HYPERPLASIA 

URINARY SYSTEM 10 (S0%) 46 (92%) 43 (86%) 

KIDNEY 

INFLAMMATION CHRONIC 

TOXIC NEPHROPATHY 

HYPERPLASIA BENAL-CELL 

HYPERPLASIA FOCAL 

10 

9 

46 

46 

43 

43 

URETER 

HYPERPLASIA EPITHELIAL 

URINARY BLADDER 

HYPERPLASIA EPITHELIAL 

136
 
TABLE E1 MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

 

CONTROL LOW DOSE HIGH DOSE 

ENDOCRINE SYSTEM 4 (8%) 

PITUITARY 

HEBATOCYST 

ADRENAL 

LYMPHANGIECTASIS 

ADRENAL CORTEX 

HYPERPLASIA NODULAR 

THYROID 

HYPERPLASIA C-CELL 

HEMATOPOIETIC SYSTEM 8 (40%) 6 (12%) 17 

BONE HARROW 

HYPERPLASIA 

SPLEEN 

CONGESTION 

LEDREHOID REACTION 

HYPERPLASIA RETICULUH-CELL 

137
 
TABLE E1 MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOU DOSE HIGH DOSE
 

LYMPHOID HYPERPLASIA
 

LYMPH NODE
 

HYPERPLASIA RETICULUM-CELL
 

LYMPHOID HYPERPLASIA
 

SUBMANDIBULAR LYMPH
 

INFLAMMATION HEMORRHAGIC
 

INFLAMMATION CHRONIC
 

INFLAMMATION CHRONIC SUPPURATIVE
 

LYMPHOID HYPERPLASIA
 

CERVICAL LYMPH NODE
 

HYPERPLASIA RETICULUM-CELL
 

MESENTERIC LYMPHNODE
 

EDEMA
 

INFLAMMATION HEMORRHAGIC
 

INFLAMMATION CHRONIC
 

HYPERPLASIA RETICULUM-CELL
 

LYMPHOID HYPERPLASIA
 

138
 
TABLE El MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

COHTROL LOW DOSE HIGH DOSE
 

REPRODUCTIVE SYSTEM 3 (15%) 3 (6%) 2 (4%)
 

PREPUTIAL GLAND 1
 

HYPERPLASIA EPITHELIAL 2
 

PROSTATE 2 2 2
 

INFLAHHATIOS CYSTIC 1
 

HYPERPLASIA 1 1
 

HYPERPLASIA EPITHELIAL 1
 

HYPERPLASIA CYSTIC 1
 

HYPERPLASIA IHTRADUCTAL 1
 

SBHIHAL VESICLE 1 1
 

INFLAMMATION SUPPURATIVE 1
 

HYPERPLASIA CYSTIC 1
 

TESTIS 1
 

ATROPHY 1
 

NBRVOUS SYSTER 1 (2%)
 

BRAIN 1
 

HYDROCEPHALUS INTERNAL 1
 

139
 
TABLE E1 MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE
 

MUSCULOSKELETAL SYSTEM
 

HONE
 

SPECIAL SENSE ORGANS
 

NONE
 

ILL OTHER SYSTEMS
 

MO ASSOCIATED ORGAN
 

AOTOLYSlS/NBCROPSY PEEP/HO HISTO
 

MESENTERY
 

NECROSIS PAT 

140 
TABLE El MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

AH IHAL DISPOSITION SUMMARY 

ANIMALS INITIALLY IN STODY

NATURAL DEATH*

MORIBUND SACRIFICE 

SCHEDULED SACRIFICE

ACCIDENTALLY KILLED

TERMINAL SACRIFICE

ANIHAL HISSING 

• INCLUDES AUTOLYZED ANIMALS 

 20

 18

 50

 39

 50 

1 

6 

1 

 41 

141
 
TABLE E2

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS 

IN FEMALE RATS FED NTA IN THE DIET 

CONTROL (UHTR) LOW DOSE HIGH DOSE 

1HIH1LS

ANIHALS

AHIHALS

ANMALS

 1M1TIALLY IV STUDY

 HECHOPSIED

 EXAMINED BISTOPATHOLOGICALLY

 WITH NON-TUHOB PATHOLOGY

 20 

 20(100%) 

 2 0 

 19 (95%) 

50 

50(100%) 

50 

4b (92%) 

50 

50 

50 

48 

(100%) 

(96%) 

IHTEGUHBHTARY

BO1E 

 SYSTEM 

RESPIRATORY SYSTEB 12 (60%) 18 (36%) 24 (48%) 

TRACHEA 

INFLAHHATION

IHPLABBATION

 NECROTIZING 

 CHROMIC SUPPURATIfB 

LONG 

PNEUHOIIA CHRONIC RURINE 

INPLAHMATION CHRONIC 

INFARCT 

11 

10 

1 

18 

18 

23 

22 

1 

142 
TABLE E2 FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL(UHTR) LOW DOSE HIGH DOSE 

CIRCULATORY SYSTEM 3 (15%) 9 (18%) 13 (26%) 

EPICARDIDH 

IVPLARBATIOH 

HEART 

PERIARTERITIS 

MYOCARDIUM 

PIBROSIS 

PIBROSIS DIPPUSE 

DEGEHERATIOB 

3 

1 

U 

13 

DIGESTIVE SYSTBB 6 (30%) 11 (22%) 7 (1*%) 

LIVER 

HECROSIS FOCAL 

HETAflORPHOSIS PATTY 

HEMOSIDEROSIS 

143 
TABLE E2 FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

 

CONTROL(UHTF) LOW DOSE HIGH DOSE 

LIVER (CONT.) 

BASOPHILIC CYTOPLASM ALTERATION 

BILE DOCT 

LYMPHOCYTIC INFLAH INFILTRATE 

PANCREAS 

INFLAMMATION FOCAL 

LYMPHOCYTIC INFLAM INFILTRATE 

FIBROSIS DIFFUSE 

HYPERPLASIA INTRADUCTAL 

1 

1 

1 

3 

SHALL INTESTINE 

INFLAMMATION 

LYMPHOID HYPERPLASIA 

1 

6 

URINARY SYSTEM 4 (20%) 26 (52%) 40 (80%) 

KIDNEY 

HYDRONEPHROSIS 

PYELONEPHRITIS 

INFLAMMATION SUBACUTE 

INFLAMMATION CHRONIC 

NEPHROPATHY 

2 

2 

25 

2 

24 

36 

1 

1 

1 

33 

144
 
TABLE E2 FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

COHTEOL LOW DOSE HIGH DOSE 

GLOMERULOSCLEROSIS 

REIAL TUBULE 

REGENERATION 

UR III ART BLADDER 2 11 

HYPERPLASIA EPITHELIAL 2 11 

ENDOCRINE SYSTEM 6 (30%) 8 (16%) 12 (24%) 

PITUITARY 

CYST 2 

HEHATOCYST 1 

XIPLAβRATIOS CYSTIC 

145
 
TABLE E2 FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

corrBOL LOW DOSE HIGH DOSE 

PITUITARY (COHT.) 

PIGHEHTATIOB 

HEROSIDEROSIS 

HYPERPLASIA POCAL 

ADEBHAL COFTEI 

HEHATOCYST 

IWPLAHSATIOH SUPPURATIVE 

LIPOIDOSIS 

HYPERPLASIA MODULAR 

HYPERPLASIA POCAL 

ADREHAL HEDULLA 

HEHATOCYST 

THYROID 

CYSTIC POLLICLBS 

HYPERPLASIA C-CBLL 

a 

1 

7 

HEBATOPOIETIC SYSTEH 2 (10%) 8 (16%) 

BO1E HARROW 

FIBROSIS FOCAL 

HYPERPLASIA HEHATOPOIETIC 

146
 
TABLE E2 FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL(UNTR) LOW DOSS HIGH DOSE
 

SPLEEN
 

INFLAMMATION CHRONIC
 

INFLAMMATION CHRONIC NECROTIZING
 

NECROSIS DIFFUSE
 

HEMOSIDEROSIS
 

BRYTHROPHAGOCYTOSIS
 

HYPERPLASIA ERTTHROID
 

HYPERPLASIA RBTICULUM-CBLL
 

LTRPH NODE
 

HYPERPLASIA RETICULUM-CELL 2
 

LYMPHOID HYPERPLASIA 1
 

SUBMANDIBULAR LYMPH
 

INFLAMMATION SUPPURATIVE
 

147
 
TABLE E2 FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

COHTROL LOW DOSE HIGH DOSE 

SUBBAWDIBOLAB LYBPH NODE (CONT.) 

HYPEBPLASIA BETICOLOB-CELL 3 

HESBVTEBIC LYBPHHODE

HYPEBPLASIA BBTICULOB-CELL

LYBPHO1D HYPERPLASIA

 1 

1 

1 

1 

3 

2 

1 

REPRODUCTIVE SYSTEB 2 (10%) 18 (36%) 11 (22%) 

BABBABI

CYST

 GLAND 1 

1 

OTBBOS

HEBATOBA

lKPLABBATIOH SOPPORATIVE

PYOHETRA

ABSCESS

FIBROSIS

HYPERPLASIA CYSTIC

 8

 1 

1 

3

 2 

1 

1 

2 

2 

OTBBOS/ENDOBBTBIOB

III PL ABB AT IOV

IHFLABBATION SOPPORATIVE

INPLABBATION ACUTE

 1 

1 

7

5

 1 

6 

2 

1 

148 
TABLE E2 FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)
 

CONTROL LOH DOSE HIGH DOSE
 

1NFLAHHATION ACUTE SUPPURATIVE
 1
 

HYPERPLASIA 2
 

HYPERPLASIA CYSTIC 2
 3
 

FALLOPIAN TUBE 1
 

INFLAMMATION SUPPURATIVE 1
 

OVARY 2 4 5
 

CYST 4 4
 

INFLAMMATION CYSTIC 1
 

INFLAMMATION CHRONIC 1
 

NECROSIS FAT 1
 

NERVOUS SYSTEg 1 (5%)
 

149
 
TABLE E2 FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LO* DOSE HIGH DOSE 

BRAIN 

HYDROCEPHALUS INTERNAL 

MUSCULOSKELETAL SYSTBH (2%) 

SPLENIUS HUSCLE 

INFLAMMATION 

SPECIAL SENSE ORGANS 

HOME 

ALL OTHER SYSTEMS 1 (5%) 2 (4%) 1 (2%) 

PERITONEUM 

INFLAMMATION 

NO ASSOCIATED ORGAN 

NO LESION REPORTED 

ANIMAL DISPOSITION SUMMARY 

ANIMALS INITIALLY IN STUDY 

NATURAL DEATH* 

20 SO 50 

150
 
TABLE E2 FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

MORIBUWD SACRIFICE 

SCHEDULED SACRIFICE 

ACCIDEHTALLY KILLED

TERHINAL SACRIFICE

ANIMAL HISSING 

INCLUDES AOTOLTZED ANIttALS 

1

 6

 1 

 39 39 

151
 
TABLE E3
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN MALE MICE FED NTA IN THE DIET
 

CONTROL LOW DOSE HIGH DOSE 

AN1BALS

ANIBALS

AN1BALS

ANIBALS

AN1HALS

 IβITIALLY IN STUDY

 HISSING 

 NECROPSIED

 EXAMINED HISTOPATHOLOGIC ALLY

 WITH NON-TOHOR PATHOLOGY

 2 0 

 20(100% ) 

 2 0 

 12 (60%) 

50 

1 

48 

48 

30 

(100%) 

(63%) 

SO 

6 

44 

44 

33 

(100%) 

(75%) 

INTEGUBENTARY

HONE 

 SYSTBB 

RESPIRATORY SYSTEfi 1 (2%) 3 (7%) 

LUNG 

CONGESTION 

PHEOHONIA CHRONIC HORINE 

CIRCULATORY

NONE 

 SYSTEB 

152
 
TABLE E3 MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOU DOSE HIGH DOSE 

DIGESTIVE SYSTEH 2 (10%) 9 (19%) 2 (5%) 

LITER 

HETABORPHOSIS FATTY 

HYPERPLASIA WODULAR 

ANGIECTASIS 

2 

1 

1 

BILE DUCT 

1HFLAHHATIOW 

SHALL IHTESTIME 

LYHPHOID HYPERPLASIA 

PETERS PATCH 

LYBPHOID HYPERPLASIA 

LARGE IHTESTIRE 

LYBPHOID HYPERPLASIA 

153
 
TABLE E3 MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED)

 

COWTBOL LOW DOSS 

URINARY STSTBn 2 (10%) 5 (10%) 

KIDNEY 

HYDRONEPHROSIS 

HEHATOCYST 

IHFLAHHATIOH 

INFLABHATION CHROMIC 

IWFAKCT 

CALCIKOSIS 

BETAPLASIA OSSEOUS 

KIDVEY/CORTEX 

CYST 

DEGENERATION 

RENAL TUBULE 

DEGEHERATION 

NEPHROSIS 

URETER 

INFLAHHAT1ON CHRONIC 

HYPERPLASIA EPITHELIAL 

URETHRA 

154
 

HIGH DOSE
 

16 (36%)
 

13
 

8
 

1
 

1
 

4
 

1
 

1
 

2
 
TABLE E3 MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

HYPERPLASIA EPITHELIAL 

EHDOCRIBB SYSTEM 3 (6%) (9%) 

ADREHAL HEDOLLA 

HYPERPLASIA 

THYROID 

CYSTIC FOLLICLES 

GOITER COLLOID 

HYPERPLASIA C-CELL 

1 

1 

3 

HEHAT0P0I2TIC SYST2H 2 (10%) (8%) 9 (20%) 

SPLEEN 

LYMPHOID HYPERPLASIA 

LYMPH MODE 

LYMPHOID HYPERPLASIA 

MANDIBULAR L. NODE 

LYMPHOID HYPERPLASIA 

155
 
TABU MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED)

 

COHTROL LOU DOSE HIGH DOSE
 

MESENTERIC LTHPB1ODE 

HYPERPLASIA 

LYMPHOID HYPERPLASIA 

HEHATOPOIESIS 

3 

1 

1 

5 

REPRGDUCTIfE SYSTEH 3 (15%) 10 (21%) 19 (43%) 

PROSTATE 

IHFLAflBATIOH CHROI1C 

HYPERPLASIA CYSTIC 

10 

1 

9 

19 

19 

NERVOUS SYSTEH 7 (35%) 1 (15%) 5 (11%) 

BRAIR 

CORPORA AHYLACEA 

HUSCOLOSKELETAL SYSTEH 

HOME 

SPECIAL SEISE OSGA1S 

156
 
TABLE E3 MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE
 

NONE 

ALL OTHER SYSTEMS 5 (25%) 12 (25%) 8 (18%) 

PERITONBUH 

INFLAHIiATION NECPOTIZING 

NO ASSOCIATED ORGAN 

NO LESION REPORTED 

ANIRAL HISSING 

10 

9 

1 

2 

6 

HESENTERY 

NECROSIS FAT 

157 
TABLE E3 MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

COWTEOL LOW DOSE HIGH DOSE 

A»IHAL DISPOSITIOl SUBBART 

AW1HALS II1TIALLY IH STODI

HATOBAL DEATH*

ROBIBOffD SACBIFICE

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED

TEBH1HAL SACRIFICE

AHIBAL SISSIMG

* IHCLDDBS AUTOLTZED AilBALS 

 20

 1 

 19

4

 50

 44

 1

 50 

2 

2 

1 

 39 

6 

158
 
TABLE E4

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS 

IN FEMALE MICE FED NTA IN THE DIET 

COWTROL LOW DOSE HIGH DOSE 

AH MIL S

AHIHALS

ANIHALS

ANIRALS

ANIMALS

 IH1TIALLT IN STUDY 

 HISSING 

 HECROPSIED 

 EXAHIWED HISTOPATHOLOGICALLY 

 WITH HOH-TltoOR PATHOLOGY 

20 

20(100%) 

20 

15 (75%) 

50 

11 

38(100%) 

38 

3 1 (82%) 

50 

49(100%) 

49 

4 4 (90%) 

IHTEGUHEHTARY

BONE 

 SYSTEtt 

RESPIRATORY SYSTEH 4 (20%) 2 (5%) 3 (6%) 

LONG 

PNEOHOMIA CHROMIC rtDRlHE 

HYPERPLASIA ALVEOLAR-CELL 

LUHG/ALVEOLI 

EMPHYSEMA 

159
 
TABLE E4 FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED)

 

CONTROL LOW DOSE HIGH DOSE 

CIRCULATORY SYSTEM 1 (2%) 

MYOCARDIUM

INFLAMMATION NECROTIZING

 1 

1 

DIGESTIVE SYSTEM 6 (30%) 4 (11%) 

SALIVARY GLAND

LYMPHOCYTIC 1NFLAM INFILTRATE

 1 

1 

LIVER

NECROSIS FOCAL

INFARCT

HYPERPLASIA NODULAR

HEMATOPOIESIS

 2

 1 

1 

2 

1 

1 

GALLBLADDER

CALCULUS

INFLAMMATION CHROMIC

 1 

1 

1 

BILE DUCT

INFLAMMATION

 1 

1 

160
 
TABLE E4 FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED)

 

CONTROL LOW DOSE HIGH DOSE
 

PETERS PATCH
 

LYMPHOID HYPERPLASIA
 

COLON
 

CYST
 

URINARY SYSTEH
 

KIDNEY 

HYDRONEPHROSIS 

INFLAMMATION 

INPLAMftATION CHRONIC 

NEPHROSIS 

INPARCT 

KIDNEY/CORTEX 

NEPHROSIS 

RENAL TUBULE 

DEGENERATION 

KIDNEY/PEL?IS
 

HYPERPLASIA EPITHELIAL
 

22 (45%) 

19 

12 

2 

5 

1 

161
 
TABLE E4 FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

COHTFOL LOW DOSE HIGH DOSS 

ENDOCRINE SYSTEM 5 (13%) 4 (8%) 

THYROID 

GOITER COLLOID 

HYPERPLASIA OCELL 

2 

3 

1 

3 

PARATHYROID 

HYPERPLASIA 

HEMATOPOIETIC SYSTEB 1 (5%) 5 (13%) 

SPLEEN 

LYHPHOID HTPEPPLASIA 

162
 
TABLE E4 FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED)

 

COHTROL LOW DOSE HIGH DOSE 

LIRPR BODE 

LYMPHOID HYPERPLASIA 

BABDIBULAF L. BODE 

LYBPHOID HYPERPLASIA 

LYHPH BODE OP THORAI 

LYHPHOID HYPERPLASIA 

HESE1TERIC LYHPHBODE 

EDEHA 

IIFLAHHAT10I CYSTIC 

LYHPHOID HYPERPLASIA 

BEliL LYBPH BODE 

LYBPHOID HYPERPLASIA 

REPRODUCTIVE SYSTEH 11 (55%) 23 (61%) 30 (61%) 

UTERUS 

1BPLAHHATI0M 

PYOHETRA 

1BFLAHHATI0B CYSTIC 

163
 
TABLE E4 FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED)

 

COHTHOL LOW DOSE HIGH DOSE 

HYPERPLASIA CYSTIC 

CIFTIX BTEFI 

IIPLABBATIOI 2 

1IFLAHHATIOB SUPPURATIVE 1 

IiFLAflflATlOl ACOTE 

0TERUS/E1DOEETRIUB 15 19
 

leFLAHBATIOI 1
 

IIPLABHATIOi CTSTIC 1
 

HYPERPLASIA CTSTIC m 19
 

OYiBY
 

CIST
 

PBBORBHAGE
 

IHFLABBATIQH CYSTIC
 

164
 
TABLE E4 FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED)

 

CONTROL LOW DOSE HIGH DOSE 

NERVOUS SYSTEM 

BR HI H 

CORPORA AMILACEA 

3 (15%) (11%) 5 (10%) 

MUSCULOSKELETAL

NOME 

 STSTEH 

SPECIAL SEISE ORGANS 

NOHB 

ALL OTHER SYSTEMS 

TAIL 

IMFLAnaATlOH POCAL GRA90LOHATO0S 

3 (15%) 13 (34%) (8%) 

HO ASSOCIATED ORGAN 

HO LESION REPORTED 

ANIRAL HISSING 

13 

2 

11 

HESEHTERT 

165
 
TABLE E4 FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTFOL LOW DOSE HIGH DOSE 

CIST 1 

HECKOSIS FAT 1 

AIIHAL DISPOSITION SUHBARY 

AHIRALS INITIALLY IV STUDY 20 50 50 

HATUBAL DEATH* 1 2 1 

BOBIBUMD SACRIFICE 1 1 

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TERHI1IAL SACRIFICE 19 35 47 

ANIHAL flISSIMG 11 

• IKCiUDSS AUTOLY2ED AWIHALS 

166 
APPENDIX F
 

SUMMARY OF THE INCIDENCE OF NONNEOPASTIC LESIONS
 

IN RATS AND MICE FED NA3NTA-H20 IN THE DIET (LBI)
 

167
 

TABLE F1

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS 

IN MALE RATS FED NA3NTAH20 IN THEDIET

 

CONTROL LOW DOSE HIGH DOSE
 

ANIMALS INITIALLY IN STUDY


ANIMALS HISSING
 

ANIMALS NECROPS1ED


AN1RALS EXAMINED HISTOPATHOLOGICALLY


ANIMALS WITH NON-TUMOF PATHOLOGY


1NTEGUHEWTARY SYSTEM *
 

SCBCOT TISSUE
 

EPIDERMAL INCLUSION CYST
 

RESPIRATORY SYSTEM 

TtACHEA 

INFLAMMATION 

INPLAMHATION CHRONIC 

LUNG 

HEMORRHAGE 

AbSCESS 

PNEUMONIA CHRONIC MURINE 

HYPERPLASIA ALVEOLAP-CELL 

LUNG/ALVEOLI 

CALCIFICATION 

CIRCULATOPY SYSTEM
 

MYOCARDIUM
 

INFLAMKATION FOCAL 

FIBROSIS 

DEGEN ERA TION 

 20
 

 20<10U%)
 

 20
 

 16 (60%)
 

2 (10%)
 

5 (25%) 

5 

1 

4 

50 50
 

1
 

49 (100%) 50(100%)
 

49 50
 

43 (88%) 50 (100%)
 

1 (2%) 1 (2%) 

1 1 

1 

4 (8%) 26 (52%)
 

3
 

2
 

1
 

25
 

6
 

1
 

20
 

1
 

18 (37%) 19 (38%) 

18 19 

1 

6 2

 

12 16

 

169 
TABLE F1 MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

 

DIGESTIVE SYSTEM
 

LIVER
 

NECROSIS
 

NECROSIS FOCAL
 

METAMORPHOSIS PATTY
 

CYTOPLASMIC ALTERATION
 

CYTOPLASMIC VACUOLIZATION
 

BASOPHILIC CYTOPLASM ALTERATION
 

HEMATOPOIESIS
 

PANCREAS
 

FIBROSIS
 

FIBROSIS DIFFUSE
 

PERIARTERITIS
 

HYPERPLASIA INTRADUCTAL 

PANCREATIC ACINUS 

HYPERPLASIA NODULAR 

STOKACH 

HYPERPLASIA EPITHELIAL
 

SKALL INTESTINE
 

LYMPHOID HYPERPLASIA
 

COLON
 

NEHATODIAS1S
 

URINARY SYSTEM
 

KIDNEY
 

HEMORRHAGE 

INFLAMMATION CHRONIC 

NEPHROSIS CHOLLMIC 

PIGMENTATION 

HYPERPLASIA TUBULAR-CELL 

KIDNEY/PELVIS 

HYPERPLASIA EPITHELIAL 

URETER 

HYPERPLASIA EPITHELIAL
 

URINARY BLADDER
 

HYPERPLASIA EPITHELIAL
 

CONTROL
 

7 (35%)
 

4
 

1
 

4
 

9 (45%)
 

9
 

9
 

LOW DOSE
 

13 (27%)
 

10
 

3
 

7
 

1
 

2
 

1
 

1
 

36 (73%)
 

36
 

36
 

1
 

HIGH DOSE
 

13 (26%)
 

10
 

1
 

1
 

2
 

2
 

4
 

4
 

2
 

2
 

1
 

49 (98%)
 

47
 

5
 

39
 

2
 

1
 

1
 

170
 
TABLE F1 MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

 

ENDOCRINE SYSTEM 

PITUITARY 

CYST 

HYPERPLASIA FOCAL 

HYPERPLASIA BASOPHILIC 

ADRENAL CORTEX 

HYPERPLASIA NODULAR 

ADRENAL MEDULLA 

CYST 

THYROID 

HYPERPLASIA C-CELL 

HYPERPLASIA FOLLICULAR-CELL 

PARATHYROID 

HYPERPLASIA 

HEMATOPOIETIC SYSTEM 

SPLEEN 

FIBROSIS FOCAL 

HEMOSIDEROSIS 

LYMPHOID DEPLETION 

LYMPHOID HYPERPLASIA 

LYMPH NODE 

INFLAMMATION 

HYPERPLASIA RETICULUM-CELL 

LYMPHOID HYPERPLASIA 

SUBMANDIBULAR L.NODE 

INFLAMMATION SUPPURATIVE 

HYPERPLASIA CYSTIC 

MESENTERIC LYMPHNODE 

CYST 

INFLAMMATION 

HYPERPLASIA RETICULUM-CELL 

CONTROL LOW DOSE HIGH DOSE 

2 (10%) 16 (33%) 5 (10%) 

3 

1 

1 

1 

2 

2 

1 

1 

1 12 5

 

1 12

 

1 

1 

1 

5 (25%) 8 (16%) 5 (10%) 

1 1 3

 

1 

1 

1 

1 1 

2 5

 

1 

3 

2 1 

1 1 

1 

1 

1 2 2 

1 

2 

1 1 

171 
TABLE F1 MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

 

CONTROL 

REPRODUCTIVE SYSTEM 

PREPUTIAL GLAND 

ABSCESS 

HYPERPLASIA 

PROSTATE 

METAPLASIA SQUAMOUS 

SEMINAL VESICLE 

INFLAMMATION SUPPURATIVE 

EPIDIDYMIS 

NECROSIS PAT 

NERVOUS SYSTEM 

BRAIN/MENINGLS 

HEMORRHAGE 

1 (5%) 

BRAIN 

HYDROCEPHALUS INTERNAL 

1 

1 

MUSCULOSKELETAL SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

NONE 

ALL OTHEP SYSTEMS 

MESENTERY 

PBRIARTSRIIIS 

NECROSIS FAT 

172
 

LOW DOSE
 

3 (6%)
 

1
 

1
 

1
 

1
 

1
 

1
 

1
 

2 {<*%)
 

2
 

1
 

1
 

HIGH DOSE
 

2 (4%)
 

1
 

1
 

1
 

1
 

5 (10%)
 

5
 

5
 
TABLE F1 MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

 

CONTROL LOW DOSE HIGH DOSE 

ANIMAL DISPOSITION SUMMARY 

ANIMALS INITIALLY IN STUDY 

NATURAL DEATH* 

MORIBUND SACRIFICE 

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE 

AfllHAL MISSING 

20 

1 

1 

18 

50 

3 

1 

us 

1 

50 

4 

3 

7 

36 

• INCLUDES AUTOLYZED ANIMALS 

* SYSTEM PERCENTAGES ARE BASED ON NUMBER OP ANIMALS NECROPSIED. 

173
 
TABLE F2

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS 

IN FEMALE RATS FED NA3NTAH20 IN THE DIET 

CONTROL LOW DOSE HIGH DOSE 

ANIMALS INITIALLY IN STUDY 

ANIMALS NECROPS1ED 

ANIMALS EXAMINED HISTOPATHOLOG1CALLY 

ANIMALS WITH NON-TUMOR PATHOLOGY 

INTEGUMENTARY SYSTEM * 

SKIN 

HYPERKERATOSIS 

PARAKERATOSIS 

RESPIRATORY SYSTEM 

LUNG 

HEMORRHAGE 

PNEUMOfclA CHRONIC MURIfcK 

NECROSIS FOCAL 

HYPERPLASIA ALVEOLAt-CELL 

CIRCULATORY SYSTEM 

MYOCARDIUM 

INFLAMMATION FOCAL 

FIBROSIS 

DEGENERATION 

DIGESTIVE SYSTEM 

LIVi,R 

IlETAMiORPHOSIS FATTY 

CYTOPLASMIC VACUOLIZATION 

BASOPHILIC CYTOPLASM ALTERATION 

HEMATOPOIESIS 

PANCREAS 

FIBROSIS 

FIBROSIS FOCAL 

FIBROSIS DIFFUSE 

20 

20(100%) 

20 

12 (60%) 

3 (15%) 

3 

3 

2 (10%) 

2 

1 

1 

5 (25%) 

4 

2 

2 

50 

50(100%) 

50 

46 (92%) 

3 (6%) 

3 

2 

1 

9 (18%) 

9 

4 

5 

22 (44%) 

17 

2 

15 

1 

4 

1 

3 

50 

49(100%) 

49 

46 (94%) 

1 (2%) 

1 

1 

1 

14 (29%) 

14 

5 

8 

1 

4 (8%) 

4 

1 

2 

1 

19 (39%) 

17 

2 

15 

1 

1 

174 
TABLE F2 FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

 

PANCREATIC ACINUS
 

ATROPHY
 

SMALL INTESTINE
 

LYMPHOID HYPERPLASIA
 

URINARY SYSTEM
 

KIDNEY
 

HYDRONEPHROSIS
 

HEMORRHAGE
 

PYELONEPHRITIS
 

INFLAMMATION
 

INFLAMMATION CbRONIC
 

TOXIC NEPHROPATHY
 

NEPHROSIS ChOLEMIC
 

HYPERPLASIA TUHJLAR-CLLL
 

KIDNEY/PELVIS
 

INFLAMHAIION SUPPURATIVE
 

NECROSIS
 

URINARY BLADDER
 

HYPERPLASIA EPITHELIAL
 

ENDOCRINE SYSTEM
 

PITUITAPY
 

CYST
 

HEBATOCYST
 

HYPERPLASIA BASOPHILIC
 

CONTROL
 

1
 

1
 

2 (10%)
 

2
 

3 (15%)
 

1
 

1
 

LOW DOSE
 

2
 

2
 

32 (64%)
 

29
 

1
 

1
 

27
 

1
 

1
 

1
 

a
 

13 (26%)
 

a
 

4
 

HIGH DOSE
 

1
 

1
 

1
 

1
 

39 (80%)
 

37
 

2
 

1
 

3a
 

2
 

1
 

1
 

1
 

5
 

5
 

13 (27%)
 

6
 

3
 

2
 

1
 

175 
TABLE F2 FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

 

CONTROL LOW DOSE HIGH DOSE 

ADfrENAL 4 3 

CYST 1 

BEMATOCYST 2 2 

LIPOIDOSIS 1 1 

ADPENAL COPTEX 1 1 1

 

I1EMATOCYST 1

 

METAMORPHOSIS PATTY 1

 

HYPERPLASIA FOCAL 1 

ADRENAL MEDULLA 1

 

CYST 1

 

THYROID 2 5 5

 

HYPERPLASIA C-CELL 2 5 5

 

PARATHYROID 1

 

HEMATOCYST 1

 

HEMATOPOIETIC SYSTEM 2 (1054) 6 (12%) 5 (10%) 

SPLEEN 2 2 2

 

INFARCT 1

 

LYMPHOID HYPERPLASIA 1 2 2

 

LYMPH NODE 1 1

 

HYPERPLASIA PET1CULUS-CELL 1 1 

SUBMANDIBULAR L.NODL 2

 

INFLAMMATION 1

 

LYMPHOID HYPERPLASIA 1

 

CEIVICAL LYMPH NODE 1

 

HEMORRHAGE 1

 

MESENTERIC LYMPHNODE 1 1

 

EDEMA 1

 

HYPERPLASIA PETICULUM-CELL 1

 

176
 
TABLE F2 FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

 

REPRODUCTIVE SYSTEM 

MAHHARY GLAND 

CYST 

UTERUS 

HEMATOME3KA 

UTEEUS/ENDOMETElim 

INFLAMMATION CYSTIC 

HYPERPLASIA 

HYPERPLASIA CYSTIC 

OVARY 

CYST 

MERVOUS SYSTEM 

BFAIN/MENINGES 

HEMORRHAGE 

INFLAMMATION 

CHOROID PLEXUS 

CORPORA ABYLACEA 

CEREBRUM 

HEMORRHAGE 

bRAIN 

HYDROCEPHALUS 

HYDROCEPHALUS INTERNAL 

HEMORRHAGE 

HYPOTHALAMUS 

ATROPHY 

MIDBRAIN 

ATROPHY 

CONTROL LOW DOSE HIGH DOSE 

1 (5%) 3 (6%) 5 (10%) 

2 

2 

1 

1 

1 1 2

 

1 

1 1

 

1 

1 1 

1 1 

1 (5%) 3 (6%) 6 (12%) 

1 1 

1 

1 

1 

1 

1 

1 

4 

1 

2 

1 

1 1 

1 1 

1 

1 

177
 
TABLE F2 FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

 

MUSCULOSKELETAL SYSTEM
 

NONE
 

SPECIAL SENSE OPGANS
 

NONE
 

ALL OTHER SYSTEMS
 

PELVIS
 

HEMORRHAGE
 

NECROSIS FAT
 

ADIPOSE TISSUE
 

INFLAMMATION
 

NECROSIS FAT
 

MESENTERY
 

NECROSIS FAT
 

•0 LESION REPORTED
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY
 

NATURAL DEATH*
 

MORIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE
 

ANIMAL MISSING
 

• INCLUDES AUTOLYZED ANIMALS
 

CONTROL
 

3 (15%)
 

2
 

1
 

1
 

1
 

1
 

2
 

20
 

2
 

18
 

LOW DOSE
 

1
 

50
 

3
 

1
 

46
 

* SYSTEM PERCENTAGES ARE BASED ON NUMBER OF ANIMALS NECROPSIED,
 

178
 

HIGH DOSE
 

2 (4%)
 

1
 

1
 

1
 

1
 

1
 

2
 

50
 

5
 

5
 

39
 
TABLE F3

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS 

IN MALE MICE FED NA3NTAH20 IN THE DIET 

CONTROL LOW DOSE HIGH DOSE 

ANIMALS INITIALLY III STUDY 

ANIMALS NECEOPSIED 

ANIMALS EXAMINED H1ST0PATH0L0G1CALLY 

ANIMALS WITH NON-TUMOR PATHOLOGY 

INTEGUMENTARY SYSTEM * 

SKIN 

CYST 

SEBACEOUS CYST 

SUBCUT TISSUE 

ABSCESS 

FBbPIRATOKY SYSTEM 

LUNG 

PNEUMONIA CnfOKIC MURINE 

CIRCULATORY LYSTJCM 

KOKE 

DIGESTIVE SYSTEM 

L1VSR 

HEMATOMA 

INFLAMMATION GRANULOMATOUS 

NKCPOSIS 

METAMORPHOSIS PATTY 

LIPOIDOSIS 

HYPERPLASIA NODULAR 

HYPERPLASTIC NODULE 

ANGIECTASIS 

L1VER/PER1POSTAL 

METAMORPHOSIS PATTY 

LI?ER/CENTRILO3ULAR 

NECROSIS 

BILE DUCT 

INFLAMMATION POCAL 

20 

20(100%) 

20 

11 (b5%) 

a (20%) 

3 

1 

1 

1 

1 

1 

50 

48(100%) 

48 

18 (38%) 

2 (4%) 

1 

1 

1 

1 

2 (4%) 

2 

2 

5 (10%) 

4 

1 

1 

1 

1 

1 

1 

50 

50(100%) 

50 

34 (68%) 

2 (4%) 

2 

1 

1 

1 (2%) 

1 

1 

a (8%) 

2 

1 

1 

1 

1 

179 
TABLE F3 MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED)

 

CONTROL LOW DOSE HIGH DOSE
 

SMALL INTESTINE
 

LYMPHOID HYPEFPLASIA
 

URINARY SYSTEM
 

KIDHEY
 

HYDRONEPHROSIS
 

ENDOCRINE SYSTEM
 

THYROID
 

CYSTIC FOLLICLES
 

PANCREATIC ISLETS
 

HYPERPLASIA
 

HYPERPLASIA FOCAL
 

HEBATOPOIETIC SYSTEM
 

BONE MAFPOW
 

HYPERPLASIA
 

SPLEEN
 

HYPERPLASIA NODULAR
 

LYMPHOID HYPERPLASIA
 

MANDIBULAR L. NODE
 

HYPERPLASIA EETICULUM-CELL
 

LYMPHOID HYPERPLASIA
 

MESENTERIC LYMKINODi,
 CYST
 

HEMORRHAGE
 

INPLAMBATION GRANULOMATOUS
 

HYPERPLASIA
 

HEMATOPOIESIS
 

REPRODUCTIVE SYSTEM
 

PROSTATE
 

HYPERPLASIA CYSTIC
 

LYMPHOID HYPEPPLASIA
 

2 (10%)
 

1
 

1
 

1
 

1
 

1
 

1
 

1
 

1
 

1
 

1 (5%)
 

1
 

1
 

1
 

1
 

1 (2%)
 

1
 

1
 

1 (2%)
 

1
 

1
 

4 (8%)
 

2
 

2
 

2
 

1
 

1
 

1
 

1
 

28 (56%)
 

28
 

28
 

2 (4%)
 

1
 

1
 

1
 

1
 

5 (10%)
 

3
 

2
 

1
 

2
 

1
 

1
 

1 (2%)
 

1
 

1
 

180
 
TABLE F3 MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED)

 

CONTROL
 

NERVOUS SYSTtfl 1 (5%) 

BRAIN 1 

COPPORA AMYLACEA 1 

MUSCULOSKELETAL SYSTEI*
 

NONE
 

SPECIAL SENSE ORGANS
 

NONE
 

ALL OTHER SYSTEMS a (20*)
 

ADIPOSE TISSOL 2
 

INFLAMMATION 2
 

MESENTERY 2
 

INFLAMMATION 2
 

HO LESION REPORTED 7
 

AUTOLYSIS/NO NECROPSY PEBFORHSD
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY 20
 

NATURAL DEATH*
 

MORIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE 20
 

ANIMAL MISSING
 

* INCLUDES AUTOLYZED ANIMALS
 

* SYSTEM PERCENTAGES ARE BASED ON NUMBER OF ANIMALS

181 

LOW DOSE
 

5 (10%)
 

5
 

5
 

1 (2%)
 

1
 

1
 

1b
 

2
 

50
 

6
 

1
 

 NECROPSIEDHIGH DOSE
 

2 (4%)
 

2
 

2
 

11
 

50
 

4
 

46
 
TABLE F4 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS 

IN FEMALE MICE FED NA3NTAH20 IN THE DIET

 

CONTROL LOW DOSE HIGH DOSE
 

ANIMALS INITIALLY IN STUDY
 

ANIMALS NECBOPSIBD
 

ANIMALS EXAMINED H1STOPATHGLOGICALLY
 

1NIMALS WITH NON-TUMOfc PATHOLOGY
 

INTEGUMENTARY SYST2H
 

NONE
 

RESPIRATORY SYSTEM •
 

LUNG
 

PNEUMONIA CHRONIC MURINE
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

LIVER
 

NECROSIS FOCAL
 

LYMPHOID HYPERPLASIA
 

GALLBLADDER
 

CALCULUS
 

BIL£ DUCT
 

LYMPHOCYTIC 1NFLAB INFILTRATE
 

LARGE INTESTINE
 

ABSCESS
 

UkINAPY SYSTEM
 

KIDNEY
 

HYDRONEPHROSIS
 

20 50 50 

18(100%) 46(100%) 47(100%) 

18 46 47 

12 (67%) 35 (76%) 41 (87%) 

3 (7%) 

3 

3 

4 (9*) 1 (2%) 

2 

1 

1 

1 

1 

1 

1 

1 

1 

1 (6%) 1 (2%) 31 (66%) 

1 30 

1 30 

182 
TABLE F4 FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

KIDNEY (COHT.) 

INFLAMMATION CHRONIC 1 

LYMPHOID HYPERPLASIA 1 

URETER 1

 

mPLAMRATlON 1

 

URINARY BLADDER 1 

IHPLA MM A TIOK 1

 

EUDOCPINE SYbTBM 2 (4%) 

THYROID 2

 

HYPERPLASIA C-CELL 2

 

HEMATOPOIETIC SYSTEM 4 (22%) 5 (11%) 3 (6%) 

bPLEEN 3 U 3

 

HYPERPLASIA 1

 

HYPERPLASIA RET1CULUM-CELL 1

 

LYMPHOID HYPERPLASIA 3 3 2

 

MANDIBULAR L . NODE 1

 

UMPHOID HYPERPLASIA 1

 

BRONCHIAL LYMPH NODE 1

 

LYMPHOID HYPERPLASIA 1

 

PANCREATIC L.NODL 1

 

CYST 1

 

MESENTERIC LYMPHNODE 2 1

 

EDEMA 1

 

INFLAMMATION 1

 

HYPERPLASIA RET1CULUM-CLLL 1

 

183 
TABLE F4 FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED)

 

MESENTERIC LYMPHNODE (CONT.)
 

LYMPHOID HYPERPLASIA
 

TBYBUS
 

LYMPHOID HYPERPLASIA
 

BEPKODOCTIVE SYSTEM
 

UTERUS
 

HEMORRHAGE
 

PYOMETKA
 

CERVIX UTERI
 

INFLAMMATION
 

CTERUS/ENDOMBTR1UM
 

INFLAMMATION SUPPURATIVE
 

HYPERPLASIA
 

HYPERPLASIA CYSTIC
 

OVARY
 

CYST
 

HEMATOMA
 

ABSCESS
 

NERVOUS SYSTEM
 

BRAIN
 

ARTERIOSCLEROSIS
 

NECROSIS FOCAL
 

CORPORA AMYLACEA
 

CHOLESTEATOMA
 

MUSCULOSKELETAL SYSTEM
 

NONE
 

CONTROL
 

11 (61%)
 

1
 

1
 

5
 

1
 

a
 

6
 

6
 

2 (11%)
 

2
 

1
 

1
 

1
 

1
 

LOW DOSE
 

28 (61%)
 

3
 

1
 

2
 

1
 

1
 

22
 

1
 

1
 

20
 

8
 

7
 

1
 

3 (7%)
 

3
 

3
 

HIGH DOSE
 

1
 

1
 

1
 

23 (49%)
 

20
 

20
 

5
 

4
 

1
 

5 (11%)
 

5
 

5
 

184 
TABLE F4 FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED)

 

SPECIAL SEBSE ORGANS
 

NOVE
 

ILL OTHER SYSTEMS
 

MESENTERY
 

ABSCESS
 

GRANULOMA
 

•0 LBSIOH REPORTED
 

AUTOLYSIS/HO BECROPSY PERFORMED
 

ANIfcAL DISPOSITION SUMMARY
 

ANIMALS IHITIALLY IN STUDY
 

NATURAL DEATH*
 

MORIBUND SACPIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE
 

ANIMAL MISSING
 

• INCLUDES AUTOLYZED ANIMALS
 

COMTBOL
 

1 (5%)
 

1
 

1
 

4
 

2
 

20
 

3
 

17
 

LOW DOSE HIGH DOSE
 

12 (26%)
 

8
 

U
 

50
 

6
 

1
 

43
 

1 (2%)
 

1
 

1
 

3
 

3
 

50
 

t>
 

44
 

* SYSTEM PERCENTAGES ARE BASED ON NUMBER OF ANIMALS BECROPSIED.
 

U. S. GOVERNMENT PRINTING OFFICE : 1977 241-161/3049 

185 


DHEW Publication (NIH) No. 77-806