National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 5 

January, 1977 

BIOASSAY OF 

PROFLAVINE 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 952-23-8 

NCI-CG-TR-5 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 

PROFLAVINE
 

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) 77-805
 

BIOASSAY OF
 

PROFLAVINE
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Program, Division of Cancer Cause and Prevention
 

CONTRIBUTORS: This report presents the results of the carcino­ genesis bioassay of proflavine (CO4137) conducted under the
 

direction of the Carcinogen Bioassay and Program Resources
 

Branch, Carcinogenesis Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute (NCI), Bethesda, Maryland.
 Tests were conducted by the Dow Chemical Company, Indianapolis,
 

Indiana, initially under direct contract with the NCI, and
 

currently under a subcontract with Tracor Jitco, Inc., the prime
 

contractor for the Carcinogen Bioassay Program.
 

The principal investigator for this study was Dr. J. L.
 Emerson1

»2. Dr. C. G. Gergig1 supervised animal care and the
 

laboratory procedures involved in the preparation of test diets.
 The protocols for the chronic test were established by Dr. Ε. Κ.
 Weisburger^.
 

Histopathologic examinations were performed by Drs. J. L.
 1 1 / ^
 

Emerson, J. A. Molello1, S. D. Warner1 and R. A. Renne^»3; the
 

diagnoses included in this report represent the interpretation of
 

these pathologists. NCI and Tracor Jitco pathologists have
 

reviewed selected slides and concur with the overall pathologic
 

evaluation of the study. All chemicals were analyzed under the
 

direction of Dr. E. Murrill?, and chemical analyses were reviewed
 

by Dr. S. S. Olin6

. Statistical analyses were made by Dr. J. R.
 Joiner^, using procedures selected by Dr. J. J. Gart^. Pathology
 

tables in this report were generated from data in the Carcino­ genesis Bioassay Data System under the supervision of Mr. D.
 Tidwell9»lO# This report was written under the direction of Dr.
 J. F. Robens^ with the assistance of L. A. Waitz^. The results
 

iii
 
are discussed by Dr. G. L. Miller6. Dr. E. W. Gunberg6

 edited
 

the final version.
 

*The Dow Chemical Company, Post Office Box 68511, Indianapolis,
 

Indiana.
 

^Abbott Laboratories, North Chicago, Illinois.
 

^Carcinogenesis Program, Division of Cancer Cause and Prevention
 

National Cancer Institute, National Institutes of Health,
 

Bethesda, Maryland.
 

^Experimental Pathology Laboratories, 17 Pine Street, Herndon,
 

Virginia.
 

5Now with Battelle Pacific Northwest Laboratories, Battelle
 

Boulevard, Richland, Washington.
 

6

Tracor Jitco, Inc., 1776 E. Jefferson Street, Rockville,
 

Maryland.
 

^Midwest Research Institute, 425 Volker Boulevard, Kansas City,
 

Missouri.
 

^Field Studies and Statistics, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

9EG&G Mason Research Institute, 1530 East Jefferson Street,
 

Rockville, Maryland.
 

10Bio-Med Systems, Inc., P.O. Box 603, Herndon, Virginia.
 

iv
 
SUMMARY
 

A bioassay of the carcinogenicity of proflavine monohydrochloride
 

hemihydrate was conducted using Fischer 344/CR rats and B6C3F1
 

mice. The compound was administered in the diet at concentrations of 300 and 600 ppm to groups of 50 rats for 109 weeks and
 

at concentrations of 200 and 400 ppm to groups of 50 mice for 104
 

weeks. The animals were subjected to necropsy and histopathologic evaluation as they died or at the end of their periods of
 

treatment.
 

Average weights attained by high-dose groups were consistently
 

lower than those of control groups; weights of low-dose groups
 

showed essentially no differences from those of the controls.
 

Survival rates of the treated rats and mice did not differ from
 

those of the controls except for a lower rate among the female
 

mice.
 

Five malignant neoplasms of the intestinal tract consisting of
 

three leiomyosarcomas of the small intestine, a sarcoma near the
 

colon area, and an adenocarcinoma of the small intestine were
 

observed in five of the high-dose male rats. None were observed
 

in other treatment or control groups. If these five intestinal
 

neoplasms are considered together, they are signficiant at the
 

 • 0.026 level using the Fisher exact test. A positive dose-


related trend (P « 0.034) was also present for the three leiomyosarcomas.
 

The observed incidence of hepatocellular carcinoma in female mice
 

was 4/50 (8%) in the control group, 20/49 (41%) in the low-dose
 

group, and 22/50 (44%) in the high-dose group. The test for
 

dose-related trend showed a level of significance of Ρ < 0.001.
 

In male mice, the observed incidence of hepatocellular carcinoma
 

was 20/49 (41%) in the control group 28/49 (57%) in the low-dose
 

group, and 30/50 (60%) in the high-dose group. The dose-related
 

trend was significant at Ρ β 0.057, and the high dose was
 

significant at Ρ = 0.044.
 

The unusually high incidence of hepatocellular carcinomas and
 

hemangiosarcomas in control male mice and the unusually high
 
incidence of malignant lymphomas in all groups of female mice in
 

conjunction with the fact that a positive-control carcinogen was
 

tested in the same room with these animals, raises a question of
 

the validity of these bioassay results.
 

vi
 
TABLE OF CONTENTS
 

PAGE
 

I· Introduction · ·. 1
 

II. Materials and Methods 3
 

A. Chemicals 3
 

B· Dietary Preparation · 4
 

C. Animals 4
 

0. Animal Maintenance · 5
 

E. Subchronic Toxicity Tests 6
 

F. Design of the Chronic Studies 8
 

G. Clinical and Pathologic Examinations 8
 

H. Data Recording and Statistical Analyses 10
 

III. Results - Rats 15
 

A· Body Weights and Clinical Signs 15
 

B. Survival 15
 

C · Pathology 17
 

D· Statistical Analyses of Results 28
 

IV. Results - Mice 31
 

A. Body Weights and Clinical Signs 31
 

. Surv ival 31
 

C. Pathology 34
 

D. Statistical Analyses of Results 44
 

V· Discussion 49
 

VI· Bibliography 53
 

APPENDICES
 

Appendix A Summary of the Incidence of Neoplasms and
 

Proliferative Lesions in Rats Fed Proflavine
 

in the Diet 55
 

Table Al Proliferative Endocrine Lesions 57
 

Table A2 Proliferative Lesions: Digestive System 58
 

Table A3 Hematopoietic Neoplasms 59
 

Table A4 Proliferative Lesions: Urinary Tract 60
 

Table A5 Proliferative Lesions: Respiratory Tract 61
 

Table A6 Proliferative Lesions: Reproductive System
 

and Mammary Gland 62
 

Table A7 Proliferative Lesions: Skin and Subcutis 63
 

vii
 
PAGE
 

Table A Miscellaneous Proliferative Lesions 64
 

Table A9 Analyses of the Incidence of Primary Tumors
 

at Specific Sites in Rats, Proflavine 65
 

Appendix Β Summary of the Incidence of Neoplasms and
 

Proliferative Lesions in Mice Fed Proflavine
 

in the Diet 67
 

Table BI Proliferative Endocrine Lesions 69
 

Table B2 Proliferative Lesions: Digestive System 70
 

Table B3 Hematopoietic Neoplasms 71
 

Table B4 Proliferative Lesions: Urinary Tract 71
 

Table B5 Cardiovascular Neoplasms 72
 

Table B6 Genital and Mammary Neoplasms ············· 73
 

Table B7 Proliferative Lesions: Respiratory System 73
 

Table B8 Miscellaneous Neoplasms · · 74
 

Table B9 Analyses of the Incidence of Primary Tumors
 

at Specific Sites in Mice, Proflavine 75
 

Appendix C Summary of the Incidence of Nontumor Pathology
 

in Rats Fed Proflavine in the Diet 77
 

Appendix D Summary of the Incidence of Nontumor Pathology
 

in Mice Fed Proflavine in the Diet 91
 

TABLES
 

Table 1 Experimental Design - Proflavine 9
 

FIGURES
 

Figure 1 Growth Curves for Rats - Proflavine ···· 16
 

Figure 2 Survival Curves for Rats - Proflavine 18
 

Figure 3 Growth Curves for Mice - Proflavine·. ····· 32
 

Figure 4 Survival Curves for Mice - Proflavine 33
 

Figure 5 Kaplan and Meier Curves of the Proportion
 

of Mice Surviving Without Observed
 

Hepatocellular Adenoma or Carcinoma
 

(Proflavine Test) 46
 

viii
 
I. INTRODUCTION
 

Proflavine (3,6-diaminoacridine) is a synthetic acridine dye
 

which early in this century was found to have bacteriostatic and
 

bacteriocidal properties when administered topically (Goodman and
 

Gilman, 1965). During World War II it was widely used as a wound
 

antiseptic (Mitchell et al., 1942). With the advent of more
 

specific and less toxic antibiotics, its clinical importance
 

declined (Giarman, 1958), until it was reintroduced recently in
 

combination with ultraviolet light for the treatment of psoriasis
 

(Weisburger, 1976) and type-II herpesvirus infection (Amstey,
 

1973).
 

II. MATERIALS AND METHODS
 

A. Chemicals
 

The proflavine used for the prechronic toxicity tests was
 

purchased as Lot No. V1571 from Schwarz/Mann (Orangeburg, Ν.Υ.)
 

in the form of proflavine dihydrochloride. The chronic phase,
 

however, was conducted with proflavine monohydrochloride
 

hemihydrate because of its availability. Unless otherwise noted,
 

the term "proflavine" in this report refers to proflavine mono-


hydrochloride hemihydrate. The following analyses were performed
 

on the monohydrochloride hemihydrate of proflavine, which was
 

manufactured and supplied as Lot No. 032937 by Aldrich Chemical
 

Company (Milwaukee, Wis.). Elemental analyses were in agreement
 

with theoretical values for the monohydrochloride hemihydrate of
 

proflavine. The percentage of amine, as determined by nonaqueous
 

titration of the free amine group, was 103.4 +0.3 % of theoretical for the monohydrochloride hemihydrate. High-pressure liquid
 

chromatography indicated one impurity (0.6% of total peak area,
 

uv detector). Infrared, ultraviolet, visible, and nuclear
 

magnetic resonance spectra also conformed to expectations for
 

this structure. Thin-layer chromatography in a 1,4-dioxane :
 

ammonium hydroxide solvent system, as visualized by ultraviolet
 

light and furfural, showed three trace impurities. A second
 

analysis in a pyridine : ammonium hydroxide solvent system showed
 
a single trace impurity. No attempt was made to further
 

characterize or identify these minor impurities.
 

B. Dietary Preparation
 

Test diets were prepared every 2 weeks and used within a 2-week
 

period. A 10% premix of proflavine in Wayne** Lab Blox reground
 

meal was prepared first and then blended in a Patterson-Kelly
 

Twin Shell blender with additional feed to obtain the appropriate
 

dose concentration. All dietary preparations were stored in
 

plastic-lined fiber drums and refrigerated at 40° F.
 

Analysis of two batches of the 10% premix several weeks after
 

mixing gave 10.6 + 0.21% and 10.93 + 0.27% proflavine, corrected
 

for recovery losses, indicating adequate stability of the
 

compound in feed.
 

C. Animals
 

Fischer 344/CR rats were obtained from A. R. Schmidt/Sprague
 

Dawley, Madison, Wisconsin and Harlan Industries, Cumberland,
 

Indiana. Hybrid B6C3F1 mice were obtained from Charles River
 

Breeding Laboratories, Wilmington, Massachusetts. Animal
 

suppliers were under contract with the Division of Cancer
 

Treatment, NCI, to provide the animals used for testing. Rats
 

and mice were received at approximately 28 days of age and
 

quarantined for 7 to 14 days. Those determined to be free from
 

4
 
observable disease or parasites were assigned to treatment
 

groups. Because the rats were received from two different
 

suppliers, they were distributed so that 3/4 of the rats in each
 

treatment and control group were from A. R. Schmidt and 1/4 were
 

from Harlan Industries.
 

D. Animal Maintenance
 

All animals were housed in temperature- and humidity-controlled
 

rooms. The temperature was maintained at 23° C with variations
 

from 22°-25° C, and the humidity ranged from 45-55%. The rooms
 

had 15 complete air changes per hour. All rooms were equipped
 

with automatic timers which controlled lighting and provided a
 

14-hour-per-day light cycle. Wayne© Lab Blox ground meal (Allied
 

Mills, Inc., Chicago, II.) ^nd water (deionized chlorinated well
 

water) were consumed jic[ libitum.
 

Rats in the chronic study were first housed individually in
 

suspended cages made of stainless-steel wire mesh (Ford Fence
 

Co., Indianapolis, Ind.). At week 45 all rats were transferred
 

to suspended polycarbonate cages (Maryland Plastics,
 

Federalsburg, Md.) equipped with filters and automatic waterers
 

and lined with autoclaved Ab-Sorb-Drj^ Bedding (Lab Products,
 

Inc., Garfield, N.J.). The cages were changed, washed, and
 

sanitized at 82° C twice weekly. The feeders were changed,
 
washed, and sterilized weekly, and the filters were changed every
 

2 weeks.
 

Mice were housed five per cage in filtered prebedded cages made
 

of disposable polypropylene (Lab Products, Inc., Garfield, N.J.).
 

The cages were changed twice weekly and the used cages were
 

incinerated. Feeders, water bottles, and cage lids also were
 

changed twice weekly, and filters were changed weekly. Feeders
 

and sipper tubes were washed and sterilized prior to use. Water
 

bottles and cage lids were sanitized at 82° C.
 

Rats and mice were housed in separate rooms. The racks were
 

rotated weekly and the cages were kept in fixed positions on the
 

racks. The rats being fed proflavine were in the same room as
 

rats being fed Ν,Ν'-dicyclohexylthiourea, l,3-dichloro-5,5-dimethylhydantoin, and the positive control, N-2-fluorenylacetamide. Mice treated with proflavine were housed in a room in
 

which 2-amino-5-nitrothiazole, 3-nitropropionic acid, N,N'-dicyclohexylthiourea, 1,3-dichloro 5,5-dimethylhydantoin, and
 

N-2-fluorenylacetamide were also on test. Untreated controls
 

were housed in the same room with their respective test animals.
 

E. Subchronic Toxicity Tests
 

In the subchronic toxicity tests, proflavine dihydrochloride was
 

administered to rats in the diet at dose concentrations ranging
 
from 500 to 2,000 ppm for six weeks. Following the treatment
 

period, there was a 2-week observation period. The gain in body
 

weight of female rats was 95% of control values at a dose of 500
 

ppm, 82% at 750 ppm, 69% at 1,000 ppm, 53% at 1,500 ppm, and 50%
 

at 2,000 ppm. The gain in body weight of male rats followed the
 

same trend: 90% at 500 ppm, 97% at 750 ppm, 82% at 1,000 ppm, 57%
 

at 1,500 ppm, and 35% at 2,000 ppm. Two males and one female rat
 

died at the 2,000 ppm concentration. At concentrations above 750
 

ppm, animals displayed rough hair coats; yellow discoloration in
 

the skin, hair, and urine; and diarrhea. Pneumonia was diagnosed
 

in one animal, and males receiving a dose of 2,000 ppm had small
 

testes, although spermatogenesis was normal. The results indicated a level of 600 ppm as the MTD for male and female rats.
 

Proflavine dihydrochloride was administered to mice in the diet
 

at five dose concentrations ranging from 200 to 1,400 ppm on the
 

same schedule. The gain in body weight of females was unaffected
 

at doses of 200 and 400 ppm; the gain in weight at 600 ppm was
 

73% of that of controls. The gain in weight of males was
 

unaffected at doses below 1.400 ppm, while at 1,400 the gain in
 

weight was 81% of that of controls. Only one mouse died during
 

the subchronic tests — a control animal. Necropsy findings
 

included hydronephrosis in two mice (at 400 ppm), pyelonephritis
 

in one (at 600 ppm), and a hyperplastic nodule in the liver of
 
one mouse (at 1,400 ppm). On the basis of these findings, the
 

MTD for mice of both sexes was set at 400 ppm.
 

F. Design of the Chronic Studies
 

During the chronic study, proflavine was administered to Fischer
 

344/CR rats and B6C3F1 mice at either of two concentrations in
 

the diet (see table 1). Rats and mice were killed 2 days after
 

the treatments were concluded.
 

G. Clinical and Pathologic Examinations
 

Body weights were recorded every 14 days for the first 3 months
 

and every 28 days thereafter. Animals were inspected twice
 

daily, 7 days a week, for clinical signs and mortality. The
 

general physical condition of the animals and the nature, extent,
 

and location of any gross abnormalities were noted and recorded
 

at weekly intervals. Animals appearing moribund when examined
 

were killed and immediately necropsied, although some moribund
 

animals were isolated from their cage-mates for a few days prior
 

to killing. All animals, regardless of whether they were killed
 

early or survived to termination, were subjected to a complete
 

gross necropsy. Animals were killed by inhalation of carbon
 

dioxide, exsanguinated, and immediately necropsied.
 

Tissues were preserved in 10% buffered formalin, embedded in
 

paraffin, sectioned, stained with hematoxylin and eosin, and
 

8
 
Table 1.

RATS
 

Male
 

Matched Control
 

Low Dose
 

High Dose
 

Female
 

Matched Control
 

Low Dose
 

High Dose
 

MICE
 

Male
 

Matched Control
 

Low Dose
 

High Dose
 

Female
 

Matched Control
 

Low Dose
 

High Dose
 

aNo observation period,
 

 Experimental Design - Proflavine 

Dietary
 

No. of Concentrations Treatment Period
 

Animals (ppm) (weeks)a
 

50 0 109 

50 300 109 

50 600 109 

50 0 109 

50 300 109 

50 600 109 

50 0 104 

50 200 104 

50 400 104 

50 0 104 

50 200 104 

50 400 104 
examined microscopically. Histopathologic evaluation consisted
 

of examination of the following: gross lesions, tissue masses or
 

suspect tumors and regional lymph node(s), blood smear (if
 

anemia, enlarged thymus, lymphadenopathy, or hepatosplenomegaly
 

were present), mandibular lymph node, mammary gland, salivary
 

gland, sternebrae including marrow, thymus, trachea, lungs and
 

mainstem bronchi, heart, thyroids, parathyroids, esophagus,
 

stomach, small intestine (one section), colon, liver, gallbladder, pancreas, spleen, kidney, adrenals, bladder, prostate,
 

testes, ovaries, uterus, brain (three sections including frontal
 

cortex and basal ganglia, parietal cortex and thalamus, and
 

cerebellum and pons), pituitary, eyes (if grossly abnormal), and
 

spinal cord (if neurologic signs were present)·
 

The intent was to evaluate all organs, tissues, and gross lesions
 

for every animal as specified in the pathology protocol for the
 

Bioassay Program. However, a few tissues (especially small
 

organs) were lost during the necropsy and the process of histologic preparation; therefore, the denominator used for a particular organ, tissue, or lesion in Appendixes A and Β may not
 

necessarily equal the number of animals placed on experiment in
 

each group.
 

H. Data Recording and Statistical Analyses
 

Pertinent data on this experiment have been recorded in an
 

10
 
automatic data processing system, the Carcinogenesis Bioassay
 

Data System (Linhart et al., 1974). The data elements included
 

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.
 

Probabilities of survival were estimated by the product limit
 

procedure of Kaplan and Meier (1958) and presented in this report
 

in the form of graphs. Deaths due to accident or scheduled
 

deaths are treated as censored observations and all other deaths
 

are uncensored. Statistical tests of differences in survival
 

between groups are compared using the method of Cox (1972) for
 

two groups and an extension of this method by Tarone (1975) for
 

more than two groups.
 

The number of animals with tumors was analyzed as a percentage of
 

the number of animals pathologically examined. For specific
 

anatomic sites, the animal is not included in the denominator if
 

that particular site was not histologically examined. For tumors
 

which required gross detection, e.g. skin tumors, the denominator
 

included all animals necropsied. For tumors that may appear at
 

several sites, e.g. lymphoma, any animal that had at least one
 

11
 
involved site histologically examined is entered in the
 

denominator of the proportions given for that tumor.
 

Statistical analysis of the incidence of tumors was made using
 

the Fisher exact test (Cox, 1970) to compare a control group to a
 

group of treated animals at each dose level. In addition, the
 

Armitage and Cochran test for linear trend in proportions, with
 

continuity correction (Armitage, 1971), was used. This test,
 

assuming a linear trend, determines if the slope of the
 

dose-response curve is different from zero, at the 0.05 level of
 

significance. The method also calculates the level of
 

probability of a departure from linear trend.
 

A conservative adjustment, the Bonferroni inequality (Miller,
 

1966), was used for simultaneous comparison of several treated
 

groups with a control group. For the comparison of results
 

obtained with k different test doses with those for a control,
 

this correction requires a level of significance less than or
 

equal to 0.05/k for the overall comparison to be significant at
 

the 0.05 level. This adjustment was not made in the tables where
 

the Fisher exact test results are shown but is discussed in the
 

analysis when appropriate.
 

As an additional analysis, the exact 95% confidence interval for
 

the odds ratio (Gart, 1970) between each of the dose groups and
 

its control was calculated. The odds ratio is
 

12
 
where p^ is the true binomial probability of tumor in a dosed
 

animal and pc is the true spontaneous tumor probability in the
 

controls. The hypothesis of equality between the true proportion
 

of a specific tumor in a dosed group and that in a control is
 

expressed by an odds ratio of 1 (one). Values in excess of 1
 

(one) represent the condition of a larger proportion in the dosed
 

group than in the control. The confidence interval entries in
 

the statistical tables of this report represent the conversion of
 

each odds ratio to the difference in probabilities, Pt~Pc> where
 

Pt~Pc = 0 implies an odds ratio of 1 (one).
 

13
 

III. RESULTS - RATS
 

A · Body Weights and Clinical Signs (Rats)
 

Gains in body weight in both male and female tested rats were not
 

decreased greatly at the 3,000 ppm concentration and were not
 

less than 85% of control values at the 6,000 ppm concentration
 

(see figure 1).
 

During the 2-year period of the study, the appearance and behavior of the treated and control rats were generally comparable.
 

The first palpable mass recorded for a control male rat was on
 

day 377 and for a control female rat on day 580. The first
 

palpable masses in the treated rats were recorded for a low-dose
 

male and a high-dose female on day 491. Palpable masses were
 

recorded on day 519 for a low-dose female and on day 659 for a
 

high-dose male. During the second year of the study the control
 

group and the treated groups began to exhibit similar incidences
 

of masses. Unilateral and occasionally bilateral cataracts were
 

observed at the end of the first year and through the second year
 

in both controls and treated rats.
 

B. Survival (Rats)
 

The survival rate of the dosed male rats was similar to that of
 

the matched-control group, and 80% of the high-dose males lived
 

15
 
TIME ON STUDY (WEEKS)
 

Figure 1. Growth Curves for Rats - Proflavine
 

16
 
to termination of the study at 109 weeks. Figure 2 shows the
 

Kaplan and Meier survival curves for male and female rats.
 

In female rats, the matched-controls and the high-dose group
 

exhibited similar death rates, but the low-dose group experienced
 

a higher rate of survival than either of the other two groups.
 

Consequently the statistics on survival show a departure from
 

linear trend (P - 0.018). Seventy percent of the high-dose
 

females were alive at the end of the study.
 

One low-dose male rat was reported lost due to accidental death
 

at 6 weeks; otherwise all deaths were due to natural causes.
 

C· Pathology (Rats)
 

Histopathologic findings are tabulated in Appendix A, tables AlA8.
 

Numerous inflammatory, degenerative, and proliferative lesions
 

commonly observed in aged Fischer rats occurred with approximately equal frequency in test and control rats. These lesions
 

included chronic tracheitis; multifocal alveolar macrophage
 

aggregates in lung parenchyma; alveolar epithelial-cell hyperplasia (table A5); chronic nephritis with scarring, tubular
 

dilatation, and tubular regeneration; hyperplasia of epithelium
 

of the renal pelvis and urinary bladder (table A4); suppurative
 

endometritis and oophoritis; cystic endometrial hyperplasia
 

17
 
TIME ON STUDY (WEEKS)
 

Figure 2. Survival Curves for Rats - Proflavine
 

18
 
(table A6); testicular atrophy; and C-cell hyperplasia of thyroid
 

(table Al).
 

Other nonneoplastic proliferative lesions included hyperplasia of
 

thyroid follicular cells (table Al), adrenal medulla (table Al),
 

parathyroid (table Al), endometrial stroma (table A6), mesothelium of vaginal tunic (table A6), mammary epithelium (table
 

A6), gastric mucosa (table A2), and hepatocytes (table A2).
 

With regard to liver lesions, the term "focal hyperplasia" was
 

used in this study to indicate the presence of one or more foci
 

of hepatocytes with increased cytoplasmic basophilia and a slight
 

increase in the amount of nuclear chromatin; many of these
 

hepatocytes also had a slight increase in nuclear:cytoplasmic
 

ratio when compared with adjacent normal hepatocytes, and infrequently mitotic figures or hepatocytes with double nuclei were
 

observed. These foci of hyperbasophilic hepatocytes were thought
 

to represent areas of hyperplasia and were diagnosed as such in
 

this study. They did not compress adjacent hepatic parenchyma.
 

These lesions are similar morphologically to those described by
 

Squire and Levitt (1975) as "basophilic foci". The one lesion
 

classified as nodular hyperplasia was similar to the hyperplastic
 

foci, but was larger, more discrete, and compressed adjacent
 

hepatic parenchyma, thus was classified as a "nodule" of
 

hyperplasia.
 

19
 
Lesions classifed as "hepatocytomegaly" consisted of foci of
 

enlarged hepatocytes, many of which contained large, vesicular
 

nuclei and numerous fine cytoplasmic vacuoles which gave the
 

cytoplasm a "ground-glass" appearance. Distortion of lobular
 

architecture in these foci was minimal, and trabeculae were
 

continuous with adjacent normal hepatocytes· These lesions
 

correspond morphologically to those described by Squire and
 

Levitt as "eosinophilic foci," "ground-glass foci," or
 

"clear-cell foci."
 

Lesions classified in this study (table A2) as "neoplastic
 

nodules" (Squire and Levitt, 1975) had many similarities to all
 

three of the previously described proliferative hepatocytic
 

lesions. However, the lesions classifed as neoplastic nodules
 

were larger, and contained more distinct abnormality of lobular
 

architecture; liver cords at the periphery of the neoplastic
 

nodules were oriented perpendicular to cords of adjacent normal
 

hepatic parenchyma, and distinct compression of adjacent normal
 

liver was evident. Hepatocellular carcinoma was diagnosed when
 

the lesion contained areas of complete loss of normal lobular
 

architecture. The most frequent abnormality in this type of
 

lesion was the presence of widely dilated sinusoids lined by rows
 

or nests of hepatocytes several cells thick, sometimes with
 

papillary projections of hepatocytes into the sinusoidal space.
 

20
 
Formation of pseudo-acini or solid sheets of hepatocytes was less
 

frequently observed.
 

Endocrine tissues were the most frequent sites of neoplasms in
 

both treated and control rats in this study. Interstitial-cell
 

tumors of the testis were observed in nearly all male rats in all
 

groups (table A6); a high spontaneous incidence of this tumor is
 

characteristic of aged Fischer 344 rats. Pituitary adenomas were
 

also found in a high incidence in all groups, especially females
 

(table Al). Other endocrine neoplams seen with approximately
 

equal frequency in test and control rats included follicular-cell
 

and C-cell tumors of thyroid, islet-cell tumors of pancreas and
 

pheochromocytomas of adrenal (table Al). In some proliferative
 

endocrine lesions, differentiation between benign and malignant
 

neoplasms was difficult. Thyroid C-cell lesions were classified
 

as adenomas when the proliferating C-cells were present in
 

nodular masses which widely separated thyroid follicles and
 

distorted follicular architecture. In many of the larger
 

adenomas, the C-cells were present in interlacing bundles of
 

elongated, spindling cells, rather than the polyhedral to
 

spherical shape characeristic of normal C-cells. When invasion
 

of thyroid capsule, adjacent tissues, or vessels was present, or
 

when metastasis was detected, the lesion was classified as C-cell
 

carcinoma. Pulmonary metastasis occurred in four of six C-cell
 

21
 
carcinomas, and invasion of trachea and adjacent soft tissue was
 

observed in one of these four tumors.
 

Follicular-cell neoplasms occurred less frequently than C-cell
 

neoplasms (table Al). The follicular-cell adenoma appeared
 

microscopically as a well-circumscribed mass composed of enlarged
 

follicles lined by hyperbasophilic follicular cells which were
 

increased in number per unit area by papillary infolding of
 

simple cuboidal or columnar epithelium into the follicular lumen
 

and stratification of follicular cells surrounding the lumen.
 

Distinct compression of adjacent normal thyroid parenchyma, with
 

some evidence of fibrous encapsulation, was present.
 

Follicular-cell lesions were classified as carcinoma based upon
 

the presence of anaplasia and histologic arrangement in
 

disorderly nests and/or sheets. Areas with papillary patterns
 

were also present. Fibrous stroma often intermingled with, but
 

did not encapsulate, the follicular-cell carcinomas.
 

Pheochromocytomas of adrenal medulla and islet-cell tumors of the
 

pancreas both occurred more frequently in male rats (table Al).
 

The diagnosis of pheochromocytoma was made when the adrenal
 

medullary lesion was present as a discrete mass which compressed
 

adjacent normal adrenal parenchyma. These neoplasms were
 

composed of sheets, nests, and/or cords of polyhedral to
 

spherical cells with abundant, slightly basophilic, cytoplasm and
 

22
 
large nuclei with abundant chromatin. Islet-cell adenomas
 

appeared as discrete, encapsulated nodules of islet cells which
 

compressed adjacent normal pancreas; diagnosis of islet-cell
 

carcinoma was based on invasion of the capsule surrounding the
 

neoplasm, the size of the neoplasm, a high mitotic index, and
 

atypia of the neoplastic cells.
 

Malignant neoplasms of the intestinal tract occurred in five male
 

rats from the high-dose group. Three of these were diagnosed as
 

leiomyosarcomas, all in small intestine. Two ot these three
 

lesions were noted grossly as masses approximately one inch in
 

diameter in the wall of the ileum. The third lesion was
 

described grossly as a mass in mesenteric lymph node, but at
 

microscopic examination the neoplasm was observed in the wall of
 

the small intestine, mesentery, and pancreas. Microscopically,
 

all three of these neoplasms were composed of a pleomorphic
 

population of cells, with a predominance of spindling cells with
 

elongated, blunt-ended nuclei, occurring in interlacing bundles,
 

and lesser numbers of plump, oval to round cells occurring in
 

sheets and nests. Bone formation within the neoplasm was evident
 

in one rat. Staining with Masson's trichrome stain demonstrated
 

moderate amounts of finely divided collagen in the neoplasm in
 

one rat; the same technique demonstrated only scanty amounts of
 

collagen in the other two neoplasms. Diagnosis of leiomyosarcoma
 

was based on the location of the neoplasm in the intestinal wall,
 

23
 
size and shape of the predominant neoplastic cell type, the
 

predominance of blunt-ended nuclei in neoplastic cells, and the
 

relatively small amount of collagen demonstrated with Masson's
 

trichrome stain· Differential diagnoses in these lesions would
 

include fibrosarcoma, other spindle-cell sarcomas, and malignant
 

histiocytic tumors.
 

The histologic characteristics of another tumor observed in the
 

area of the colon were partially masked by autolysis; no good
 

landmarks were present histologically to identify location of the
 

neoplasm, but fatty tissue was present adjacent to the mass,
 

which was described grossly as "involving the colon 5 cm from the
 

cecum". This neoplasm also was made up of a pleomorphic population of cells, varying from spindle-shaped to round. Many
 

mitotic figures were present, and numerous foci of necrosis and
 

acute inflammation were evident. This neoplasm was diagnosed as
 

a sarcoma, but not further classified due to autolysis, lack of
 

evident differentiation of cells, and inability to determine the
 

exact anatomic location of the neoplasm.
 

The other neoplasm occurring in the intestinal tract in this
 

group was a 4-cm diameter mass in the duodenum, which on cut
 

section had a cystic, fluid-filled center. On histologic
 

examination, this mass was found to be composed of numerous
 

cystic spaces partially filled with mucus, lined by a thickened,
 

24
 
papillary epithelium with a loose stroma containing foci of
 

inflammatory cells and numerous nests of proliferating epithelial
 

cells. Numerous mitotic figures and atypical cells were present
 

within the epithelium lining the cysts and in the stroma. This
 

neoplasm was classified as an adenocarcinoma of the small
 

intestine.
 

As noted previously, focal hyperplasia of gastric mucosa was
 

observed in two rats (table A2); both of these were males from
 

the high-dose group. The relatively low spontaneous incidence of
 

gastrointestinal tract neoplasms reported in laboratory rats
 

(Rowlatt, 1967) makes the observation of these lesions only in
 

test animals more noteworthy.
 

Malignant lymphomas occurred rather frequently in both control
 

and test groups (table A3). Most of these neoplasms were
 

composed of relatively undifferentiated lymphoreticular cells,
 

and many were generalized, i.e., involved numerous organs and
 

tissues throughout the body. In those lymphomas that were not
 

generalized, the organ most frequently affected was the spleen.
 

Liver, lymph nodes, and thymus were also frequently affected.
 

Evidence of leukemia (masses of neoplastic lymphoreticular cells
 

in vessel lumens) was seen in some cases of generalized lymphoma.
 

Two cases of granulocytic neoplasia were observed (table A3).
 

Neoplasms of urinary tract epithelium, although occurring infre25
 
quently, were observed in both control and test groups and in
 

both sexes in approximately equal frequency (table A4).
 

Carcinomas of transitional epithelium occurred in both renal
 

pelvis and urinary bladder, and were characterized by formation
 

of solid nest and sheets of transitional cells interspersed with
 

small cystic spaces filled with necrotic debris. The carcinoma
 

in the renal pelvis in a high-dose female not only proliferated
 

outward into the lumen, but invaded deeply into the renal
 

parenchyma. The tubular adenoma observed in a low-dose male was
 

a very small encapsulated mass composed of large, well differentiated tubular cells.
 

Pulmonary neoplasms were observed only in males, with approximately equal frequency in test and control groups (table A5).
 

Differentiation between adenoma and carcinoma was based on the
 

degree of anaplasia, mitotic index, size of the neoplasm, and
 

presence of apparent invasion of adjacent pulmonary parenchyma in
 

carcinoma, as opposed to mere compression of adjacent parenchyma
 

and thus a more discrete lesion seen in adenoma. One poorly
 

differentiated adenocarcinoma of the submucosal glands of the
 

larynx was observed in a control female rat.
 

The most frequently occurring reproductive tract neoplasm, other
 

than the previously mentioned interstitial-cell tumor of the
 

testis, was the endometrial stromal polyp of the uterus (table
 

26
 
A6). This lesion was present as a discrete mass protruding into
 

the lumen of the uterus, lined by endometrium, and sometimes
 

associated with suppurative endometritis and/or cystic endometrial hyperplasia. The stroma was usually proliferating in a
 

rather loosely woven pattern, with numerous small vessels interspersed among stromal cells. Endometrial stromal sarcoma, a less
 

frequent lesion, was similar to stromal polyp except larger and
 

more highly cellular, with more numerous mitoses, anaplasia, and
 

evidence of invasion of adjacent tissues. One leiomyoma of
 

uterine musculature was observed in a high-dose female rat.
 

Adenocarcinoma of the endometrium occurred in four rats: three in
 

controls and one in the low-dose group (table A6). These
 

neoplasms proliferated into the lumen of the uterus, and were
 

composed of poorly formed small acini or nests of endometrial
 

cells invading stroma. Numerous mitotic figures were present.
 

The most common neoplasm of the mammary gland was the fibroadenoma; these lesions occurred much more frequently in females,
 

were often multiple, and were seen in both test and control
 

groups (table A6). Mammary carcinomas were also more common in
 

females (table Aβ). No metastasis of any mammary carcinoma was
 

observed·
 

Various other types of malignant and benign neoplasms were
 

observed in low incidence in sections of skin and subcutis (table
 

27
 
A7), and in other organs and tissues throughout the body (table
 

A8). No apparent difference in incidence of these neoplasms
 

between test and control groups was present.
 

There were instances in this study» as noted above and in the
 

summary tables, where neoplastic or hyperplastic lesions occurred
 

only in test animals, or with increased frequency when compared
 

to control groups. In the judgement of the pathologist, the
 

nature, incidence, and severity of the lesions observed provide
 

no clear evidence of carcinogenic effect.
 

For a summary of the incidence of nontumor pathology in rats
 

exposed to proflavine, see Appendix C.
 

D. Statistical Analyses of Results (Rats)
 

Table A9 contains the statistical analyses of the incidence of
 

those tumors which appeared in over 5% of any dosed group. In
 

male rats, the only tumor with a statistically significant ( β
 

0.034) positive dose-related trend was leiomyosarcoma, which was
 

seen in 3/45 (7%) of the high-dose group and in none of the other
 

male or female groups. Two other high-dose males had tumors of
 

the intestinal tract. When the Fisher exact test is applied to
 

the total incidence 5/45, it shows statistical significance at
 

the 0.026 probability level. No other Fisher exact tests between
 

the high or low-dose groups and the controls were significant
 

28
 
( > 0·05) for positive dose-related tumors, nor is there any
 

apparent significance in the analyses of the time of observation
 

of any particular neoplasm. Although experience of the observation of tumors from this laboratory is insufficient to compare
 

with the observations reported in the control group matched to
 

the dosed group, historic results on this strain of rats from all
 

laboratories in the Bioassay Program show that the male matched
 

controls on this study showed a greater incidence of pituitary
 

tumors (14/49 [29%] compared with 86/846 [10%] in the historic
 

results) and in hematopoietic neoplasms (17/50 [34%] compared
 

with 55/846 [6.5%] in the historic results). As a result of
 

these disparities, both these tumor sites show a negative dose
 

relationship in the proportions of tumors observed.
 

No positive dose-related trends were present in the tumor
 

proportions reported in the female rats, and in these rats a
 

significantly higher proportion (15/50, 30%) of hematopoietic
 

tumors were reported in the matched control compared with the
 

historic control data from all laboratories in the Bioassay
 

Program (45/840, 5.4%).
 

29
 

IV. RESULTS - MICE
 

A. Body Weights and Clinical Signs (Mice)
 

Gains in body weight in male and female tested mice were not
 

different from those of the control groups at any dose concentration during either the first or second year of the test (see
 

figure 3).
 

During the first year of the study, the appearance and behavior
 

of the treated and control mice were generally comparable. Focal
 

alopecia, focal dermatitis, and small palpable masses in the perineal area were observed in increasing numbers of male mice after
 

7 months on test. These lesions were associated with male
 

animals fighting. The first palpable mass for a control male
 

mouse was recorded on day 294 and for a control female mouse on
 

day 629. The first palpable mass in a treated mouse was recorded
 

for a high-dose male on day 546 and for a high-dose female on day
 

574. Palpable masses were recorded on day 519 for both male and
 

female low-dose mice.
 

B. Survival (Mice)
 

All three groups of male mice had similar survival rates, and 68%
 

of the high-dose males lived to the termination of the study at
 

103 weeks. Figure 4 shows the Kaplan and Meier survival curves
 

for male and female mice. In the female mice, survival curves
 

31
 
TIME ON STUDY (WEEKS)
 

Figure 3. Growth Curves for Mice - Proflavine
 

32
 
TIME OF STUDY (WEEKS)
 

Figure 4. Survival Curves for Mice - Proflavine
 

33
 
were comparable up to 90 weeks, but after that time the matched-


control group had a higher survival rate than the dosed groups.
 

The poorest survival was experienced by the low-dose group, in
 

which 54% of the animals survived to the termination of the test.
 

In neither sex were the statistics for positive dose association
 

significant (P > 0.05). There were no animals reported missing
 

or accidentally killed, and no statistically significant relationship of tumors to early deaths was apparent.
 

C. Pathology (Mice)
 

Histopathologic findings are tabulated in Appendix B, tables BlB8. Several chronic inflammatory, degenerative, and proliferative lesions which often occur spontaneously in aged laboratory
 

mice were observed with approximately equal frequency and
 

severity in test and control animals. These lesions included
 

cystic ovaries, suppurative oophoritis and endometritis, cystic
 

endometrial hyperplasia, and chronic nephritis.
 

The incidence of proliferative endocrine lesions is summarized in
 

table Bl. The thyroid carcinoma observed in a control male mouse
 

was a poorly differentiated neoplasm with some microscopic
 

features of a C-cell tumor (sheets and nests of pleomorphic
 

cells); however, the most differentiated portion of the neoplasm
 

contained areas of cuboidal epithelial cells forming follicles,
 

34
 
thus the neoplasm was considered to be of follicular-cell origin.
 

Follicular-cell adenomas and hyperplastic foci of follicular
 

cells were similar histologically in that they were both composed
 

of several follicles lined by large, hyperbasophilic follicular
 

cells which were increased in number per unit area by papillary
 

infolding of simple cuboidal or columnar epithelium into the follicular lumen and stratification of follicular cells surrounding
 

the lumen. Differentiation of follicular-cell adenoma from hyperplasia was based largely on presence of distinct compression
 

of adjacent normal thyroid parenchyma with some evidence of
 

encapsulation in adenoma; other criteria were the size of the
 

lesion, the degree of difference in cellular morphology and
 

follicular architecture between the mass and adjacent normal
 

thyroid, and the presence of a single lesion in adenoma as
 

opposed to multiple foci in hyperplasia.
 

A total of six primary neoplasms occurred in sections of adrenal
 

glands from all groups. Four of these were pheochromocytomas,
 

one was a cortical carcinoma, and one lesion was diagnosed as a
 

carcinoma, but not further classified due to extensive autolysis.
 

The latter neoplasm, found in low-dose female, was described
 

grossly as a large mass in the area of the left adrenal. It had
 

no normal tissue attached to it when examined microscopically,
 

and the histologic pattern was partially masked by autolysis. A
 

mass of similar tissue was present in the lung section. Despite
 

35
 
the autolytic changes and the uncertainty as to location, the
 

most likely primary site of this tumor was considered to be the
 

adrenal gland; the histologic features were those of carcinoma;
 

therefore it was classified as carcinoma, N.O.S., adrenal gland,
 

with metastasis to lung.
 

Table B2 summarizes the incidence of pertinent lesions of the
 

digestive system, including the liver. Numerous hepatocellular
 

carcinomas were observed, the morphologic pattern varying from
 

small, discrete, compressing nodules of enlarged hepatocytes with
 

moderate deviation from normal hepatic architecture to very large
 

masses involving entire lobes of liver, composed of anaplastic
 

hepatocytes forming pseudoacini, solid sheets of cells, or cords
 

separated by angiectatic sinusoids, and in some cases containing
 

foci of necrosis. Pulmonary metastasis of hepatocellular carcinoma occurred only in male mice: 5/20 in controls, 1/28 in low-


dose animals, and 4/30 in the high-dose group.
 

Numerous hemangiosarcomas were also observed in liver parenchyma
 

(table B2), as well as in other sites (table B5).
 

Hemangiosarcoma and hepatocellular carcinoma occurred concurrently in four low-dose male mice, one control male mouse, and two
 

high-dose female mice. Foci of necrosis were also seen in some
 

liver sections containing hemangiosarcoma (two control males, one
 

control female, and three low-dose males).
 

36
 
Diagnosis of hepatic hemangiosarcoma was based on the presence of
 

atypical endothelial cells lining sinusoids or appearing in solid
 

masses. Most hemangiosarcomas were composed of (1) areas of
 

angiectatic sinusoids lined by plump endothelial cells with
 

large, hyperchromatic nuclei, (2) densely cellular areas composed
 

of endothelial cells similar to those described in (1) with only
 

a few small vascular spaces, and (3) areas with a morphology
 

intermediate between (1) and (2).
 

Hepatic lesions classified as nodular hyperplasia consisted of
 

discrete nodules of proliferating hepatocytes which compressed
 

adjacent liver parenchyma, but which did not have sufficient
 

variation in cellular morphology or lobular architecture to
 

warrant diagnosis of neoplasia. The liver section from one
 

low-dose female contained a hyperplastic nodule as well as a
 

hepatocellular carcinoma. The diagnosis of hepatocytomegaly
 

indicates the presence of one or more foci of enlarged hepatocytes containing large amounts of finely vacuolated cytoplasm.
 

Compression of adjacent parenchyma by these foci was minimal or
 

absent.
 

Although no neoplasms were observed in sections of stomach,
 

several hyperplastic lesions of the glandular or squamous mucosa
 

were seen, all occurring in test groups (table B2). These were
 

small, focal lesions. Hyperkeratosis of the squamous gastric
 

37
 
mucosa was noted in three mice, two of which were controls. One
 

papillary adenoma of the duodenum was observed in a high-dose
 

female mouse. Microscopically, this was a focal, polypoid mass
 

protruding into the lumen of the bowel, near the pylorus.
 

Numerous hematopoietic neoplasms occurred in all groups; the vast
 

majority were malignant lymphomas (table B3). These were classified as "generalized malignant lymphoma" when they involved
 

numerous organs and tissues, and as "malignant lymphoma" when
 

only a few organs were involved. The organs most frequently
 

involved with malignant lymphoma were the mesenteric lymph nodes,
 

liver, Peyer's patches, spleen, and thymus. Several morphologic
 

types were observed, including the mixed type, which contained a
 

mixture of reticulum cells, lymphoblasts, and a lesser number of
 

other leukocytes; and the undifferentiated type, in which the
 

vast majority of neoplastic cells were large, undifferentiated
 

reticulum cells. The mixed type and undifferentiated type of
 

lymphomas correspond morphologically to Dunn's reticulum cell
 

sarcoma, types Β and A, respectively (Dunn, 1954). In some
 

cases, subclassification of lymphoma was not practical due to
 

extensive autolysis; these are classified as malignant lymphoma,
 

N.O.S. Two generalized granulocytic sarcomas were observed. Two
 

mast-cell sarcomas were also observed, one of which involved
 

numerous organs and one of which involved only spleen and liver.
 

Both lesions diagnosed as mast-cell sarcoma were composed of
 

38
 
large oval cells with round central nuclei, abundant cytoplasm,
 

and a distinct cytoplasmic membrane. Although stains for
 

metachromasia were negative (Dr· J· L. Emerson, personal
 

communication), mast-cell sarcoma remains the most plausible
 

diagnosis, based on cellular morphology and anatomic location.
 

Differential diagnoses include lymphoma and granulocytic sarcoma.
 

Table B4 summarizes the incidence of proliferative urinary tract
 

lesions. Two carcinomas of renal tubular epithelial cells were
 

observed, in a low-dose female mouse and in a high-dose female
 

mouse. Both these neoplasms occurred in renal cortex, as
 

papillary fronds and nests of large epithelial cells with large
 

hyperchromatic nuclei, numerous mitotic figures and foci of
 

necrosis. Neither of the neoplasms was encapsulated. A low
 

incidence of epithelial hyperplasia of urinary bladder was
 

observed, and epithelial hyperplasia of urethra was noted in two
 

control male mice.
 

The incidence and morphology of hepatic hemangiosarcomas have
 

already been discussed; other primary cardiovascular neoplasms
 

are summarized as to location and incidence in table B5.
 

Hemangiosarcoma of the spleen was observed concurrently with
 

hepatic hemangiosarcoma in six cases. The morphology of the
 

lesion in the spleen was somewhat similar to that described for
 

hepatic hemangiosarcoma, except for a decreased incidence of
 

39
 
angiectatic areas in the splenic neoplasms. The hemangiosarcomas
 

observed in the subcutis and the bone marrow both were predominantly cavernous; plump endothelial cells with hyperchromatic
 

nuclei lined the cavernous spaces. Hemangiomas, which occurred
 

in subcutis and myocardium were also cavernous in architecture.
 

The incidence of genital and mammary neoplasms is summarized in
 

table B6. Several types of primary ovarian neoplasms were
 

observed, all in females from the test groups. Four of these
 

were papillary adenomas, which appeared microseropically as
 

nodules composed of well-differentiated epithelial cells in rows,
 

small nests, and papillae, located within or bulging from the
 

surface of the ovary. One papillary carcinoma was observed.
 

This neoplasm had some similarities to the papillary adenomas,
 

but was composed of epithelial cells in solid sheets as well as
 

papillae, with numerous mitoses. This tumor appeared to infiltrate the adjacent bursa at one point. Four granulosa-cell
 

tumors occurred; these tumors had a prominent stromal component,
 

but also contained more rounded cells and rosette-like structures
 

reminiscent of Call-Exner bodies. A large, infiltrating spindle-


cell neoplasm was observed in the wall of the uterus of a low-


dose female mouse. This lesion was diagnosed as leiomyosarcoma
 

based upon the densely cellular, whorling pattern and the blunt-


ended nuclei seen in many neoplastic cells. Differential
 

40
 
diagnoses would include fibrosarcoma, other spindle cell
 

neoplasms, and endometrial stromal sarcoma.
 

Only two mammary neoplasms were observed, both adenocarcinomas;
 

one in a low-dose female and one in a high-dose female. The
 

latter neoplasm invaded adjacent skeletal muscle.
 

Primary pulmonary neoplasms were observed in all test and control
 

groups (table B7). Twenty-two of these were alveolar/bronchiolar
 

carcinomas, and 19 were alveolar/bronchiolar adenomas. Differentiation between adenoma and carcinoma was based on the degree of
 

anaplasia, mitotic index, size of the neoplasm, and microscopic
 

evidence of invasion of adjacent pulmonary parenchyma (carcinoma), as opposed to mere compression of adjacent parenchyma, and
 

thus a more discrete lesion (adenoma). Incidence of hyperplasia
 

of pulmonary epithelium is also summarized in table B7. This
 

lesion usually consisted of one to several small foci of alveolar
 

epithelial-cell hyperplasia.
 

Miscellaneous neoplasms encountered in low incidence are summarized in table B8. Lacrimal gland adenomas were often noted
 

grossly, and consisted of papillary proliferation of lacrimal-


gland epithelium. The location of the bone involved with osteochondrosarcoma is not known. The osteoma was located in a
 

vertebra, and was a small lesion composed of well-formed bone in
 

41
 
a dense trabecular arrangement, with numerous well-differentiated
 

osteoblasts lining the trabeculae.
 

There were instances in this study, as noted above and in the
 

summary tables, where neoplastic or hyperplastic lesions occurred
 

only in test animals, or with increased frequency when compared
 

to control groups. Particularly striking is the high incidence
 

of hepatocellular carcinoma, especially in test female mice, when
 

compared with controls of the same sex. These differences undoubtedly will have significance statistically. The incidence of
 

nodular hyperplasia of the liver in the test females in the
 

absence of this lesion in female controls is supportive evidence
 

of an effect of the compound on the liver of the female mouse,
 

since this lesion is considered by many to represent a precursor
 

to neoplasia of hepatocytes. Several factors are present, however, which cast doubt upon the biologic significance of these
 

differences in incidence. Especially significant is the unusually high incidence of primary hepatic neoplasms observed in the
 

male control group. It is difficult to justify declaring this
 

compound a carcinogen in female mice based on an incidence of
 

hepatocellular carcinoma of 41-44% in exposed animals, when the
 

incidence of the same tumor in the controls of the opposite sex
 

is 41%. Another factor is the occurrence of hemangiosarcomas in
 

rather high incidence in the controls as well as the test mice;
 

these neoplasms rarely occur spontaneously in mice.
 

42
 
Data were obtained on another group of B6C3F1 control mice from
 

another bioassay study (2~amino-5-nitrothiazole) which was
 

carried out in the same room (Dr. J. L. Emerson, personal communication) ; these mice were procured from the same source as
 

those used in the proflavine study, although at a different time.
 

The incidences of hepatocellular carcinoma and hepatic
 

hemangiosarcomas in these mice were as follows:
 

Control Males Control Females
 

Hepatocellular Carcinoma 5/50 1/50
 

(10%) (2%)
 

Hemangiosarcoma, Liver 0/50 0/50
 

The unusually high incidence of these two types of neoplasms in
 

the controls as well as test mice in the proflavine study leads
 

one to consider several possible explanations: (1) some stimulus
 

other than exposure to the test compound induced these lesions in
 

both the test and control mice in the proflavine study; (2) this
 

particular group of mice had an unusually high incidence of
 

spontaneously occurring hepatocellular carcinoma and hepatic
 

hemangiosarcoma; (3) the proflavine control mice were
 

inadvertently exposed to the test compound, which did indeed
 

induce these lesions. The latter explanation seems rather
 

unlikely, since stringent procedures were in effect to prevent
 

43
 
this specific event from happening (Dr. J. L. Emerson, personal
 

communication). In any event, unless some explanation of the
 

inconsistency of the data is forthcoming, the high incidence of
 

these neoplasms in the control mice would seem to preclude consideration of the data as conclusive evidence of a carcinogenic
 

effect of proflavine on the liver of the mouse.
 

For a summary of the incidence of nontumor pathology in mice
 

exposed to proflavine, see Appendix D.
 

D. Statistical Analyses of Results (Mice)
 

Table B9 contains the statistical analyses of those tumors which
 

were observed in more than 5% of any dosed group. The male
 

matched controls exhibited significantly higher tumor proportions
 

than those seen in the 1,132 control mice whose results have been
 

recorded in the Bioassay Program in tumors of the lung (19% vs.
 

9.2%), hematopoietic system (22% vs. 2%), liver (41% vs. 15.6%),
 

and endocrine system (10% vs. 4%). In female mice, only the
 

incidence of hematopoietic tumors differed statistically from the
 

results on the entire Bioassay Program (46% vs. 6.8%). In both
 

sexes, at all tumor sites except the liver, the proportion of
 

tumors seen in the dosed groups were not statistically different
 

(P > 0·05) than the control groups.
 

In an experiment with 2-amino-5-nitrothiazole which was made at
 

44
 
the same time in the same laboratory, the incidences of hematopoietic tumors and liver tumors in control mice were not
 

significantly different from the combined historic controls of
 

1,132 B6C3F1 mice across all laboratories in the Bioassay
 

Program. Therefore, the incidence in the controls for proflavine
 

of the tumors listed above is higher than that expected from the
 

experience available.
 

The proportions of hepatocellular carcinoma in both dosed groups
 

of female mice have probability levels of less than 0.001 by the
 

Fisher exact test for positive dose-related effects, and the
 

Cochran-Armitage test for positive dose-related trend is significant (P < 0.001). In male mice the high-dose group differs
 

from the matched controls (P • 0.044) and the Cochran-Armitage
 

test for trend has probability level Ρ = 0.057. Figure 5 shows
 

that in both sexes of mice the observation of these hepatic
 

tumors occurred mainly at the end of the study, and prior to that
 

time there is no statistically significant difference between the
 

dosed groups and their controls. In conclusion, the statistical
 

inference is made that in female mice, there is a strong association of carcinoma of the liver with the dosage of proflavine
 

given. In male mice, there is a slight suggestion of such a
 

relationship shown in the high-dose group, but the data on these
 
TIME ON STUDY (WEEKS) 

IJigure 5· Kaplan and Meier Curves of the Proportion of Mice Surviving Without
 

 Observed Hepatocellular Adenoma or Carcinoma (Proflavine Test)
 

46
 
groups are inconclusive due to the high proportion of carcinoma
 

of the liver seen in the matched control.
 

47
 

V. DISCUSSION
 

The doses of proflavine used in this study were slightly toxic,
 

since comparisons of average group weights (figures 1 and 3,
 

above) show that animals on the high doses (600 ppm for rats and
 

400 ppm for mice) consistently gained less weight than their
 

respective controls, while average weights of low-dose animals
 

were indistinguishable from those of the controls· Thus, the
 

doses which were used were high enough to provide an acceptable
 

bioassay of carcinogenicity. The survival rates of the treated
 

rats and mice did not differ from those of the controls except
 

for lower survival among the female mice·
 

Proflavine is absorbed from the gastrointestinal tract, since (1)
 

yellow discoloration of the urine was noted in the subchronic
 

toxicity studies using doses of 1,000 ppm or higher and (2) in
 

vivo staining of nuclei of mammalian cells has been reported
 

following parenteral or oral administration (de Bruyn et al·,
 

1951).
 

Five malignant neoplasms of the intestinal tract consisting of
 

three leiomyosarcomas of the small intestine, a sarcoma near the
 

colon area, and an adenocarcinoma of the small intestine were
 

observed only in five of the high-dose male rats. The relatively
 

low spontaneous incidence of gastrointestinal tract neoplasms
 

49
 
usually found in laboratory rats, in contrast to these data,
 

suggests but does not provide clear evidence of a proflavine
 

induced carcinogenic effect in these animals. This is supported
 

by the statistical evaluation of the combined intestinal tumor·
 

Although the Ρ value given in table A9 for these tumors is 0.026,
 

correction for simultaneous use of controls raises the effective
 

 value above 0.05.
 

The results of the present study are suggestive of, but do not
 

provide strong evidence for the carcinogenicity of proflavine in
 

mice. Fisher exact tests for individual doses showed significant
 

increases in proportions of tumor-bearing animals for
 

hepatocellular carcinoma in female mice administered proflavine
 

at concentrations of 200 ppm (P < 0.001) or 400 ppm (P < 0.001).
 

This effect was of borderline significance in male mice given 400
 

ppm (P * 0.044). Armitage and Cochran tests for dose-related
 

trend showed levels of significance of Ρ < 0.001 for female mice,
 

and Ρ = 0.057 for male mice. However, the control mice in this
 

bioassay had an unusually high incidence of tumors. For example,
 

the male control mice had a 41% incidence of hepatocellular
 

carcinoma and an unusually high incidence of hemangiosarcoma
 

(12%), while female control mice showed an abnormally high
 

incidence of lymphoma (46%). The unusually high incidence of
 

these tumors may be due to the fact that the mice were housed in
 

50
 
4 

the same room as those treated with a positive-control compound
 

N-2-fluorenylacetamide.
 

Previous studies have demonstrated that proflavine has disruptive
 

influences on cellular processes, such as chromatid breakage in
 

leukocytes, fibroblasts, and HeLa cells (Ostertag and Kersten,
 

1965), inhibition of cellular uptake of amino acids (Birkmayer
 

and Balda, 1971), and aggregation of nucleoli with decrease in
 

their size (Recher et al., 1971). However, in vivo staining of
 

cell nuclei is possible without affecting the vitality of the
 

cells, as evidenced by lack of toxicity of doses of proflavine
 

used for the staining and by normal regeneration of liver in
 

animals treated with proflavine and subjected to partial
 

hepatectomy (de Bruyn et al., 1951). Work of Salaman and
 

Glendenning (1951) demonstrated the promoting action of repeated
 

intradermal injections of proflavine on tumor development at or
 

near sites of topical DMBA application in mice. Repeated
 

applications of proflavine alone to the skin, or subcutaneous
 

implantation of the compound, showed neither promoting nor
 

carcinogenic action.
 

Although the present bioassay may suggest that proflavine is
 

carcinogenic in female mice, serious questions have been
 

identified regarding the validity of the bioassay. These
 

consisted of an unusually high incidence of hepatocellular
 

51
 
carcinoma in the control male mice and an unusually high
 

incidence of lymphomas in all female mouse groups including the
 

controls. Furthermore, the positive control compound N-2­

fluorenylacetamide, was tested in the same room as proflavine and
 

have contributed to the findings.
 

52
 
VI. BIBLIOGRAPHY
 

Amstey, Μ· S. 1973. Current concepts of herpesvirus infection
 

in the woman. Am. J. Obstet. Gynecol. 117:5, 717-725.
 

Armitage, P. 1971. Statistical Methods jji Medical Research.
 

John Wiley and Sons, New York. p. 135.
 

Berenblum, I., ed. 1969. Carcinogenicity Testing. UICC
 

Technical Report Series, Vol. 2. International Union
 

Against Cancer, Geneva.
 

Birkmayer, G. D. and Balda, B. R., 1971. Evidence for proflavine
 

sensitive proteins in malignant hamster melanoma. Hoppe
 

Seylers Z. Physiol. Chem. 352:780-790.
 

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

61-65.
 

Cox, D. R. 1972. Regression models and life tables. J^ Roy.
 

Statist. Soc. £ 3Λ:

187-220.
 

deBruyn, P. P. H., Robertson, R. C. and Fair, R. S., 1951. In
 

vivo affinity of diaminoacridines for nuclei. Anat. Rec.
 

108:279-295.
 

Dunn, T. Normal and Pathologic Anatomy of Reticular Tissue in
 

Laboratory Mice. J. Nat'l. Cancer Inst. 14:1281-1433, 1954.
 

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

ratio in the combination of 2 χ 2 tables with fixed
 

marginals. Biometrika 57:471-475, 1970.
 

Giarman, N. J. 1958. Chemotherapy of bacterial infections I:
 

Antiseptics and Germicides. In: Drill, V. A. (ed.)
 

Pharmacology in Medicine. McGraw-Hill, New York.
 

Goodman, L. S.and Gilman, A. 1968. The Pharmacological Basis of
 

Therapeutics, 3rd ed. MacMillan Co., New York. p. 1045.
 

Kaplan, E. L. and Meier, P. 1958. Nonparametric estimation from
 

incomplete observations. Jj^ Amer. Statist. Assn.
 

53:457-481.
 

53
 
Linhart, Μ. S., Cooper, J. Α., Martin, R· L., Page, N. P., and
 

Peters, J· A. 1974. Carcinogenesis bioassay data system.
 

J^ Comp. Biomed. Res. 2:

230-248.
 

Miller, R. G., Jr. 1966. Simultaneous Statistical Inference.
 

McGraw-Hill, New York.
 

Mitchell, G. A. G.and Buttle, G. A. H. 1942. Proflavine powder
 

in wound therapy. Lancet ii, 416.
 

Ostertag, W. and Kersten, W. 1965. The action of proflavine and
 

actinomycin D in causing chromatid breakage in human cells.
 

Exp. Cell Res. _3£· 296-301.
 

Recher, L., Parry, N. T·, Briggs, L. G., and Whitescarves, J.
 

1971. Difference in effects of proflavine and actinomycin D
 

on mammalian cell nucleoli. Cancer Res. 31:1915-1922.
 

Rowlaft, V. 1967. Neoplasms of the alimentary canal in rats and
 

mice, Pathology of Rats and Mice. Cotchin, E. and Roe, F.
 

J. C , ed. Oxford, Blackwell Scientific Publications, pp.
 

57-84.
 

Salaman, M. H. and Glendenning, 0. M. 1957. Tumor promotion in
 

mouse skin by sclerosing agents. Brit. J. Cancer
 

JU: 434-444.
 

Squire, R. A. and Levitt, M. 1975. Report of a workshop on
 

classification of specific hepatocellular lesions in rats,
 

Cancer Research, 35, 3214-3223, November.
 

Tarone, R. E. 1975. Tests for trend in life table analysis.
 

Biometrika j>2;.679-682.
 

Weisburger, E. K., memorandum of personal communication,
 

September 1976.
 

54
 
APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS AND PROLIFERATIVE
 

LESIONS IN RATS FED PROFLAVINE IN THE DIET
 

55
 

TABLE Al
 

PROLIFERATIVE ENDOCRINE LESIONS
 

MALE RATS FEMALE RATS 

Control Low Dose High Dose Control Low Dose 

THYROID 

Follicular-Cell 1/41 0/50 0/50 1/48 0/50 

Carcinoma (2%) (2%) 

Follicular-Cell 0/41 0/50 0/50 1/48 0/50 

Adenoma (2%) 

C-cell Carcinoma 1/41 2/50 1/50 0/48 2/50 

(2%) (4%) (2%) (4%) 

C-cell Adenoma 1/41 6/50 5/50 7/48 4/50 

(2%) (12%) (10%) (15%) (8%) 

Follicular-Cell 1/41 1/50 1/50 0/48 0/50 

Hyperplasia (2%) (2%) (2%) 

C-cell Hyperplasia 23/41 25/50 35/50 27/48 39/50 

(56%) (50%) (70%) (56%) (78%) 

PARATHYROID 

Hyperplasia 5/25 0/40 1/46 2/30 3/45 

(20%) (2%) (6%) (7%) 

PITUITARY 

Chromophobe Adenoma 14/49 6/49 7/48 26/50 28/46 

(29%) (12%) (15%) (52%) (61%) 

PANCREAS 

Islet-Cell Carcinoma 0/47 0/50 1/48 0/50 0/49 

(2%) 

Islet-Cell Adenoma 3/48 5/49 4/48 2/50 3/49 

(6%) (10%) (8%) (4%) (6%) 

ADRENAL 

Pheochromocytoma 4/50 1/50 4/50 1/50 0/49 

(8%) (2%) (8%) (2%) 

Hyperplasia 0/50 7/50 12/50 0/50 2/49 

(14%) (24%) (4%) 

Hyperplasia, Cortex 2/50 0/50 0/50 0/50 0/49 

(4%) 

High Dose
 

1/48
 

(2%)
 

0/48
 

0/48
 

6/48
 

(13%)
 

2/48
 

(4%)
 

26/48
 

(54%)
 

0/41
 

23/49
 

(47%)
 

1/48
 

(2%)
 

0/48
 

0/50
 

0/50
 

0/50
 

57
 
TABLE A2
 

PROLIFERATIVE LESIONS
 

DIGESTIVE SYSTEM
 

Control 

MALE RATS 

Low Dose High Dose 

FEMALE RATS 

Control Low Dose High Dose 

LIVER 

Hepatocellular 

Carcinoma 

0/49 1/50 

(2%) 

1/49 

(2%) 

0/50 2/50 

(4%) 

1/50 

(2%) 

Neoplastic Nodule 0/49 0/50 2/49 

(4%) 

0/50 0/50 0/50 

Nodular Hyperplasia 0/49 1/50 

(2%) 

0/49 0/50 0/50 0/50 

Focal Hyperplasia 0/49 4/50 

(8%) 

5/49 

(10%) 

0/50 25/50 

(50%) 

22/50 

(44%) 

Hepatocytomegaly 5/49 

(10%) 

11/50 

(22%) 

20/49 

(41%) 

2/50 

(4%) 

1/50 

(2%) 

1/50 

•(235) 

STOMACH 

Focal Mucosal 

Hyperplasia 

0/50 0/49 2/50 

(4%) 

0/49 0/50 0/49 

SMALL INTESTINE 

Leiomyosarcoma 0/41 0/48 3/48 

(6%) 

0/45 0/48 0/47 

Adenocarcinoma 0/42 0/48 1/48 

(2%) 

0/46 0/49 0/47 

LARGE INTESTINE 

Sarcoma, N.O.S. 0/44 0/48 1/49 

(2%) 

0/47 0/49 0/49 

58
 
TABLE A3 

HEMATOPOIETIC NEOPLASMS 

Control

MALE RATS

 Low Dose High Dose Control

 FEMALE RATS 

 Low Dose High Dose 

Generalized Malignant 10/47 6/50 1/49	 10/50 6/50 4/50

(20%) (12%) (8%) Lymphoma*	 (21%) (12%) (2%)
 

Malignant Lymphoma* 7/47 4/50 4/49	 5/50 1/50 2/50 

(10%) (2%) (4%) (15%) (8%) (8%)
 

Granulocytic Sarcoma/ 0/50 1/50 0/50 0/50 0/50

 1/50 

(2%) Leukemia	 (2%)
 

^Tabulation of incidence of this neoplasm is divided into those animals in which


the neoplasm was generalized, i.e., involved numerous organs and tissues, and those


animals in which it was seen in only a few organs. Since the spleen is the organ


most often involved with this lesion in the Fischer rat, the number of spleens


examined microscopically was used as the denominator.
 

59
 
TABLE A4
 

PROLIFERATIVE LESIONS
 

URINARY TRACT
 

KIDNEY
 

Transitional-Cell
 

Carcinoma, Renal
 

Pelvis
 

Tubular Adenoma
 

Epithelial Hyper­ plasia
 

Renal Pelvis
 

BLADDER
 

Transitional-Cell
 

Carcinoma
 

Epithelial Hyper­ plasia
 

Control
 

1/50
 

(2%)
 

0/50
 

6/50
 

(12%)
 

0/47
 

4/47
 

(9%)
 

MALE RATS
 

Low Dose
 

0/49
 

1/49
 

(2%)
 

1/49
 

(2%)
 

0/44
 

0/44
 

High Dose
 

0/50
 

0/50
 

1/50
 

(2%)
 

1/45
 

(2%)
 

2/45
 

(4%)
 

Control
 

1/50
 

(2%)
 

0/50
 

0/50
 

1/42
 

(2%)
 

3/42
 

(7%)
 

FEMALE RATS
 

Low Dose
 

0/50
 

0/50
 

0/50
 

1/45
 

(2%)
 

0/45
 

High Dose
 

1/49
 

0/49
 

1/49
 

(2%)
 

0/45
 

0/45
 

60
 
TABLE A5
 

PROLIFERATIVE LESIONS
 

RESPIRATORY TRACT
 

LUNG
 

Alveolar/Bronchi olar
 

Carcinoma
 

Alveolar/Bronchiolar
 

Adenoma
 

Alveolar Epithlial­ cell Hyperplasia
 

LARYNX
 

Adenocarcinoma,
 Submucosal Glands
 

Control
 

1/49
 

(2%)
 

0/49
 

2/49
 

(4%)
 

0/43
 

MALE RATS
 

Low Dose
 

1/50
 

(2%)
 

0/50
 

3/50
 

(6%)
 

0/49
 

High Dose
 

1/50
 

(2%)
 

1/50
 

(2%)
 

6/50
 

(12%)
 

0/50
 

Control
 

0/50
 

0/50
 

3/50
 

(6%)
 

1/48
 

(2%)
 

FEMALE RATS
 

Low Dose
 

0/50
 

0/50
 

0/50
 

0/48
 

High Dose
 

0/50
 

0/50
 

3/50
 

(6%)
 

0/43
 

61
 
TABLE A6
 

PROLIFERATIVE LESIONS
 

REPRODUCTIVE SYSTEM AND MAMMARY GLAND
 

Control 

MALE RATS 

Low Dose High Dose Control Low Dose High Dose 

FEMALEE RAT RATS S

TESTIS 

Interstitial-Cell 

Tumor 

46/50 

(92%) 

46/49 

(94%) 

49/50 

(98%) ... 

Mesothelioma 0/50 0/49 0/50 — — — 

Mesothelial Hyper­ 0/50 

plasia, Vaginal Tunic 

2/49 

(4%) 

1/50 

(2%) 

... ... ... 

OVARY 

Granulosa-Cell Tumor -­- ... ... 0/49 1/48 

(2%) 

0/50 

UTERUS 

Adenocarcinoma ... ... ... 3/49 

(6%) 

1/48 

(2%) 

0/50 

Endometrial Stromal 

Sarcoma 

... ... ... 1/49 

(2%) 

1/48 

(2%) 

0/50 

Endometrial Stromal 

Polyp 

... ... ... 9/49 

(18%) 

4/48 

(8%) 

3/50 

(6%) 

Leiomyoma — 0/49 0/48 1/50 

(2%) 

Cystic Endometrial 

Hyperplasia 

... ... ... 6/49 

(12%) 

8/48 

(17%) 

4/50 

(8%) 

Endometrial Stromal 

Hyperplasia 

... ... ... 0/49 1/48 

(2%) 

1/50 

(2%) 

MAMMARY GLAND 

Adenocarcinoma 1/33 

(3%) 

0/20 0/20 2/46 

(4%) 

1/44 

(2%) 

1/45 

(2%) 

Fibroadenoma 2/33 

(6%) 

0/20 0/20 7/46 

(15%) 

12/44 

(27%) 

4/45 

(9%) 

Lobular Hyperplasia 0/33 0/20 0/20 1/46 

(2%) 

3/44 

(7%) 

0/45 

62
 
TABLE A7
 

PROLIFERATIVE LESIONS
 

SKIN AND SUBCUTIS
 

SKIN
 

Squamous-Cell
 

Carcinoma
 

Basal-Cell Carcinoma
 

Keratoacanthoma
 

Trichoepithelioma
 

Dermal Inclusion Cyst
 

SUBCUTIS
 

Fibrosarcoma
 

Fibroma
 

Lipoma
 

Control
 

2/50
 

(4%)
 

0/5 0/0
 

0/50
 

0/50
 

0/5 0/50
 0

0/50
 

1/50
 

(2%)
 

0/50
 

MALE RATS
 

Low Dose
 

0/50
 

0/50
 

1/50
 

(2%)
 

1/50
 

(2%)
 

0/50
 

0/50
 

1/50
 

(2%)
 

0/50
 

High Dose
 

0/50
 

1/50
 

(2%)
 

0/50
 

0/50
 

2/50
 

(4%)
 

1/50
 

(2%)
 

1/50
 

(2%)
 

1/50
 

2%
 

Control
 

0/50
 

0/50
 

0/50
 

0/50
 

0/50
 

0/50
 

0/50
 

0/50
 

FEMALE RATS
 

Low Dose
 

0/50
 

0/50
 

0/50
 

0/50
 

0/50
 

0/50
 

0/50
 

0/50
 

High Dose
 

0/50
 

0/50
 

1/50
 

(2%)
 

0/50
 

0/50
 

0/50
 

0/50
 

0/50
 

63
 
TABLE A8
 

MISCELLANEOUS PROLIFERATIVE LESIONS
 

Control 

MALE RATS 

Low Dose High Dose Control 

FEMALE RATS 

Low Dose High Dose 

Generalized Malignant 

Fibrous Histiocytoma 

0/50 1/50 

(2%) 

0/50 0/50 0/50 0/50 

Carcinoma, Zymbal*s 

Gland 

1/50 

(2%) 

0/50 1/50 

(2%) 

0/50 0/50 0/50 

Carcinoma, Preputial 

Gland 

4/50 

(8%) 

1/50 

(2%) 

2/50 

(4%) 

...... _—— 

Adenoma/Carcinoma, Clitoral Gland 

..... «• •» _ 5/50 

(10%) 

1/50 

(2%) 

0/50 

Schwannoma, Mediastinum 0/50 0/50 1/50 

(2%) 

0/50 0/50 0/50 

Glioma, Brain 0/50 1/50 

(2%) 

2/50 

(4%) 

0/49 0/50 0/50 

Granular-Cell Tumor, Brain 

0/50 1/50 

(2%) 

0/50 0/49 0/50 0/50 

Fibrosarcoma, Salivary Gland 

0/47 0/47 1/50 

(2%) 

0/47 0/49 0/43 

Mesothelioma, Peritoneum 1/50 

(2%) 

1/50 

(2%) 

1/50 

(2%) 

0/50 0/50 0/50 

Mesentery, Sarcoma, N.O.S. 

0/50 0/50 1/50 

(2%) 

0/50 0/50 0/50 

Papilloma, Tongue 0/50 0/50 0/50 1/50 

(2%) 

0/50 0/50 

Liposarcoma, Skeletal Muscle 

0/50 0/50 1/50 

(2%) 

0/50 0/50 0/50 

Osteogenic Sarcoma, 

Bone 

0/50 1/50 

(2%) 

0/50 0/50 0/50 0/50 

Heart, Metastatic 0/46 

Adenocarcinoma, Primary 

Unknown 

0/50 1/50 

(2%) 

0/50 0/49 0/49 

Hemangiosarcoma, Spleen 

0/47 0/50 0/49 0/50 1/50 

(2%) 

0/50 

64
 
Table A9. Analyses of the Incidence of Primary Tumors at Specific Sites in Rats, Proflavine3 

MALE FEMALE 

Untreated Low High Untreated Low High 

Topography: Morphology Controls Dose Dose Controls Dose Dose 

Integumentary System: All Tumors 3/50(6) 4/50(8) 4/50(8) 0/50(0) 0/50(0) 1/50(2) 

 Values0 N.S. N.S. 

First Tumor Incidence (weeks) 109 85 110 — — 94 

Reproductive System: All Tumors 46/50(92) 46/50(92) 49/50(98) 22/50(44) 19/50(38) 7/50(14) 

 Values0 N.S. N.S. 

First Tumor Incidence (weeks) 88 85 92 88 104 110 

Thyroid: C-cell Adenoma or 

Carcinoma 2/41(5) 8/50(16) 6/50(12) 7/48(15) 6/50(12) 6/48(13) 

 Values0 N.S. N.S. 

First Tumors Incidence (weeks) 109 100 98 82 110 83 

Pituitary: Chomophobe Adenoma 14/49(29) 6/49(12) 7/48(15) 26/50(52) 28/46(61) 23/49(47) 

 Values0 = 0.076(Neg) N.S. 

First Tumor Incidence (weeks) 94 79 110 77 88 83 

Adrenal: Pheochromocytoma 4/50(8) 1/50(2) 4/50(8) 1/50(2) 0/49(0) 0/50(0) 

 Values0 N.S. N.S. 

First Tumor Incidence (weeks) 109 109 98 98 

Pancreatic Islets: Adenoma or 

Carcinoma 3/47(6) 5/50(10) 5/48(10) 2/50(4) 3/49(6) 1/48(2) 

 Values0 N.S. N.S. 

First Tumor Incidence (weeks) 108 103 110 109 110 — 
Table A9. Analyses of the Incidence of Primary Tumors at Specific Sites in Rats, Proflavine
 

continued
 

MALE FEMALE
 

Untreated Low High Untreated Low High
 

Topography: Morphology Controls Dose Dose Controls Dose Dose
 

Hematopoietic System: All Turn orsb

 17/50(34) 10/50(20) 5/50(10) 15/50(30) 7/50(14) 7/50(14)
 

 Values0 = 0.003(Neg) = 0.029(Neg)
 

First Tumor Incidence (weeks) 78 74 94 97 95 64
 

Intestine;
 

Tumorsb

>e 

0/46(0) 0/41(0) 5/45(11) 0/44(0) 0/44(0) 0/41(0)
 

 Valuesc = 0.006 » 0.026d N.S.
 

<J\
 First Tumor Incidence (weeks) 99
 

Liver: Neoplastic Nodule or
 

Hepatocellular Carcinoma 0/49(0) 1/50(2) 3/49(6) 0/50(0) 2/50(4) 1/50(2)
 

 Values0  = 0.063 N.S.
 

First Tumor Incidence (weeks) 109 110 110 110
 

fDosed groups received time-weighted average dose of 300 and 600 ppm in feed.
 

Number of tumor-bearing animals/number of animals examined at site (percent).
 

°Beneath the incidence of each of the controls is the probability level for the Armitage test for dose-related trend in proportions
 

when it is below 0.10 and when the response of both dose groups is not zero; otherwise N.S. - not significant.
 

Departure from linear trend is noted when it is below 0.05 for any comparison.
 

Beneath the dose group incidence is the probability level for the Fisher exact (conditional) test for increased proportion in that dose
 

-group compared with the untreated-control group when it is below 0.05.
 

Correction for simultaneous use of controls raises the effective Ρ value above 0.05.
 

^ive intestinal tumors combined: one sarcoma N.O.S. in the colon, three leimyosarcomas in the small intestine and one adenocarcinoma in the
 

duodenum.
 
APPENDIX R
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS AND PROLIFERATIVE
 

LESIONS IN MICE FED PROFLAVINE IN THE DIET
 

67
 

TABLE Bl
 

PROLIFERATIVE ENDOCRINE LESIONS
 

MALE MICE FEMALE MICE 

Control Low Dose High Dose Control Low Dose High Dose 

THYROID 

Foil i oil ar-Cell 1/39 0/46 0/46 0/38 0/45 0/46 

Carcinoma (3%) 

Follicular-Cell 2/39 1/46 0/46 1/38 2/45 1/46 

Adenoma (5%) (2%) (3%) (4%) (2%) 

Foilicular-Cell 2/39 2/46 2/46 5/38 10/45 4/46 

Hyperplasia (5%) (4%) (4%) (13%) (22%) (9%) 

PARATHYROID 

Hyperplasia 0/15 0/27 1/31 1/12 0/32 0/31 

(3%) (8%) 

Adenoma 0/15 0/27 1/31 0/12 0/32 0/31 

(3%) 

PITUITARY 

Chromophobe Adenoma 0/36 0/13 0/15 3/34 2/24 2/8 

(9%) (8%) (25%) 

ADRENAL 

Pheochromocytoma 2/45 1/46 0/50 0/45 1/49 0/48 

(4%) (2%) (2%) 

Cortical Carcinoma 0/45 0/46 0/50 0/45 0/49 1/48 

(2%) 

Carcinoma, N.O.S. 0/45 0/46 0/50 0/45 1/49 

(2%) 

0/48 

PANCREAS 

Islet-Cell Hyperplasia 3/48 9/44 2/44 0/44 0/46 0/46 

6% (20%) (5%) 

69 
TABLE B2
 

PROLIFERATIVE LESIONS
 

DIGESTIVE SYSTEM
 

MALE MICE 

(Control Low Dose High Dose Control 

LIVER 

Hepatocellular 20/49 28/49 30/50 4/50 

Carcinoma (41%) (57%) (60%) (8%) 

Hemangiosarcoma 6/49 6/49 3/50 1/50 

(12%) (12%) (6%) (2%) 

Total Primary Hepatic 26/49 34/49 33/50 5/50 

Neoplasms (53% (69%) (66%) (10%) 

Nodular Hepatocyte 3/49 4/49 4/50 0/50 

Hyperplasia (6%) (8%) (8%) 

Hepatocytomegaly 5/49 1/49 1/50 1/50 

(10%) (2%) (2%) (2%) 

Angiectasis 1/49 10/49 8/50 2/50 

(2%) (20%) (16%) (4%) 

Focal Necrosis 2/49 9/49 6/50 5/50 

(4%) (18%) (12%) (10%) 

STOMACH 

Hyperplasia, Glandular 0/47 2/46 0/48 0/45 

mucosa (4%) 

Hyperplasia, Squamous 0/47 1/46 1/48 0/45 

mucosa (2%) (2%) 

Hyperkeratosis 1/47 0/46 1/48 1/45 

(2%) (2%) (2%) 

SMALL INTESTINE 

Papillary Adenoma 0/46 0/41 0/45 0/44 

FEMALE MICE
 

Low Dose
 

20/49
 

(41%)
 

0/49
 

20/49
 

(41%)
 

8/49
 

(16%)
 

0/49
 

4/49
 

(8%)
 

10/49
 

(20%)
 

1/49
 

(2%)
 

0/49
 

0/49
 

0/44
 

High Dose
 

22/50
 

(44%)
 

2/50
 

(4%)
 

24/50
 

(48%)
 

9/50
 

(18%)
 

0/50
 

5/50
 

(10%)
 

3/50
 

(6%)
 

0/45
 

1/45
 

(4%)
 

0/45
 

1/41
 

(2%)
 

70
 
TABLE B3
 

HEMATOPOIETIC NEOPLASMS
 

MALE MICE	 FEMALE MICE
 

Control Low Dose High Dose Control Low Dose High Dose
 

Generalized Malignant 2/50 9/50 3/50 15/50 8/50 13/50
 

Lymphoma* (4%) (18%) (6%) (30%) (16%) (26%)
 

Malignant Lymphoma*	 9/50 2/50 10/50 8/50 8/50 7/50
 

(18%) (4%) (20%) (16%) (16%) (14%)
 

Generalized Granulo- 1/50 0/50 0/50 1/50 0/50 0/50
 

cytic Sarcoma (2%) (2%)
 

Mast-Cell Sarcoma	 1/50 0/50 0/50 1/50 0/50 0/50
 

(2%) (2%)
 

TABLE B4
 

PROLIFERATIVE LESIONS
 

URINARY TRACT
 

MALE MICE	 FEMALE MICE
 

Control Low Dose High Dose Control Low Dose High Dose
 

KIDNEY
 

Tubular-Cell 0/50 0/49 0/47 0/49 1/50 1/49
 

Carcinoma (2%) (2%)
 

BLADDER
 

Epithelial Hyperplasia 4/42 3/43 0/39 2/38 2/40 1/40
 

(10%) (7%) (5%) (5%) (2.5%)
 

URETHRA**
 

Epithelial Hyperplasia 2/50 0/50 0/50 0/49 0/50 0/50
 

(4%)
 

•Tabulation of incidence of this neoplasm is divided into those animals in which
 

the neoplasm was generalized, i.e., involved numerous organs, and those animals
 

in which it was observed in only a few organs. The number of animals necropsied
 

was used as the denominator.
 

**Samples of urethra are not routinely examined histologically; therefore the
 

number of mice from the group necropsied was used as the denominator.
 

71
 
TABLE B5
 

CARDIOVASCULAR NEOPLASMS
 

MALE MICE	 FEMALE MICE
 

Control Low Dose High Dose Control Low Dose High Dose
 

LIVER
 

Hemangiosarcoma* 6/49 6/49 3/50 1/50 0/49 2/50
 

(12%) (12%) (6%) (2%) (4%)
 

SPLEEN
 

Hemangiosarcoma 2/46 1/45 5/45 1/50 1/49 3/47
 

(4%) (2%) (11%) (2%) (2%) (6%)
 

SUBCUTIS
 

Hemangiosarcoma 1/50 0/50 0/50 0/49 0/50 0/50
 

(2%)
 

Hemangioma 0/50 0/50 0/50 0/49	 1/50 2/50
 

(2%) (4%)
 

HEART
 

Sarcoma, N.O.S. 0/48 1/49 0/49 0/49 0/49 0/49
 

(2%)
 

Hemangioma	 1/48 0/49 0/49 0/49 0/49 0/49
 

\C/o)
 

BONE MARROW
 

Hemangiosarcoma 0/48 1/49 0/50 0/48 0/49 0/49
 

(2%)
 

•Incidence of hepatic hemangiosarcoma is also tabulated in table B2.
 

72
 
TABLE B6
 

GENITAL AND MAMMARY NEOPLASMS
 

MALE MICE FEMALE MICE 

Control Low Dose High Dose Control Low Dose High Dose 

OVARY 

Papillary Carcinoma — -— — 0/43 1/42 

(2%) 

0/43 

Granulosa-Cell Tumor — 0/43 2/42 2/43 

(4%) (5%) 

Papillary Adenoma — ...... ... 0/43 2/42 2/43 

(5%) (4%) 

Total Primary — ... ... 0/43 5/42 4/43 

Ovarian Tumors (12%) (9%) 

UTERUS 

Leiomyosarcoma — — ... 0/46 1/46 

(2%) 

0/46 

MAMMARY GLAND 

Adenocarcinoma 0/50 0/50 0/50 0/49 1/50 

(2%) 

1/50 

(2%) 

TABLE B7 

PROLIFERATIVE LESIONS 

RESPIRATORY SYSTEM 

MALE MICE FEMALE MICE 

Control Low Dose High Dose Control Low Dose High Dose 

LUNG 

Alveolar/Bronchiolar 4/48 6/50 6/50 0/49 2/50 4/49 

Carcinoma (8%) (12%) (12%) (4%) (8%) 

Alveolar/Bronchiolar 5/48 5/50 2/50 4/49 1/50 2/49 

Adenoma (10%) (10%) (4%) (8%) (2%) (4%) 

Sarcoma, N.O.S. 0/48 1/50 0/50 0/49 0/50 0/49 

(2%) 

Alveolar/Bronchiolar 2/48 6/50 5/50 0/49 1/50 0/49 

Hyperplasia (4%) (12%) (10%) (2%) 

73
 
TABLE Ε58
 

MISCELLANEOUS NEOPLASMS
 

FAT
 

Sarcoma, N.O.S.
 

LACRIMAL GLAND
 

Adenoma
 

BONE
 

Osteochondrosarcoma
 

Osteoma
 

SKELETAL MUSCLE
 

Neurofi brosarcoma
 

SUBCUTIS
 

Sarcoma, N.O.S.
 

Fibrosarcoma
 

Neurofi brosarcoma
 

Control
 

0/50
 

1/50
 

(2%)
 

1/50
 

(2%)
 

0/50
 

0/50
 

0/50
 

0/50
 

0/50
 

MALE MICE
 

Low Dose
 

0/50
 

1/50
 

(2%)
 

0/50
 

0/50
 

0/50
 

0/50
 

1/50
 

(2%)
 

0/50
 

High Dose
 

0/50
 

0/50
 

0/50
 

0/50
 

1/50
 

(2%)
 

1/50
 

(2%)
 

0/50
 

1/50
 

(2%)
 

Control
 

0/49
 

1/49
 

(2%)
 

0/49
 

0/49
 

0/49
 

0/49
 

0/49
 

0/49
 

FEMALE MICE
 

Low Dose
 

1/50
 

(2%)
 

1/50
 

(2%)
 

0/50
 

1/50
 

(2%)
 

0/50
 

0/50
 

0/50
 

0/50
 

High Dose
 

0/50
 

1/50
 

(2%)
 

0/50
 

0/50
 

0/50
 

0/50
 

0/50
 

0/50
 

74
 
Table B9. Analyses of the Incidence of Primary Tumors at Specific Sites in Mice, Proflavine 

Topography: Morphology
 

Lung: Alveolar/Bronchiolar Adenoma
 

or Carcinoma
 

 Values0
 

First Tumor Incidence (weeks)
 

Hematopoietic System: Lymphomasb
 

 Values0
 

First Tumor Incidence (weeks)
 

Liver: Hepatocellular Adenoma
 

or Carcinoma
 

 Values0
 

First Tumor Incidence (weeks)
 

Any Site: Hemangiosarcoma
 

 Values0
 

First Tumor Incidence Jweeks)
 

b
 

Endocrine System: All Tumors
 

 Values0
 

Departure from Linear Trend
 

First Tumor Incidence (weeks)
 

Reproductive System: All Tumors
 

 Values0
 

First Tumor Incidence (weeks)
 

MALE FEMALE 

Untreated Low High Untreated Low High 

Controls Dose Dose Controls Dose Dose 

9/48(19) 11/49(22) 8/49(16) 4/49(8) 3/50(6) 6/49(12) 

N.S. N.S. 

75 46 86 77 104 104 

11/50(22) 11/50(22) 13/50(26) 23/50(46) 16/50(32) 21/50(42) 

N.S. N.S. 

68 44 55 51 67 67 

20/49(41) 28/49(57) 30/50(60) 4/50(8) 20/49(41) 22/50(44) 

 ­ 0.057 = 0.044 < 0.001 < 0.001 < 0.001 

78 64 47 103 73 70 

9/50(18) 8/50(16) 8/50(16) 2/50(4) 1/50(2) 5/50(10) 

N.S. N.S. 

75 95 98 103 88 104 

5/49(10) 3/49(6) 1/50(2) 4/50(8) 6/50(12) 5/49(10) 

 ­ 0.089(Neg) N.S. 

 = 0.020 N.S. 

78 104 104 103 91 96 

0/50(0) 0/50(0) 0/50(0) 0/49(0) 7/48(14) 5/47(10) 

N.S. ­ 0.057 

__ 103 88 

^Untreated controls, dosed groups received time-weighted average dose of 200 and 400 ppm in feed.
 

Number of tumor-bearing animals/number of animals examined at site (percent).
 

Beneath the incidence of each of the controls is the probability level for the Armitage test for dose-related trend in proportions
 

when it is below 0.10, otherwise N.S. - not significant. Departure from linear trend is noted when it is below 0.05 for any comparison.
 

Beneath the dose group incidence is the probability level for the Fisher exact (conditional) test for increased proportion in that dose
 

group compared with the untreated control group when it is below 0.05.
 

APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONTUMOR PATHOLOGY
 

IN RATS FED
 

PROFLAVINE IN THE DIET
 

77
 

TABLE CI
 

SUMMARY OF THE INCIDENCE OF NONTUMOR PATHOLOGY
 

IN MALE RATS TREATED WITH PROFLAVINE
 

HIGH DOSE
 

50
 

50(100%)
 

50
 

50 (100%)
 

* (β%)
 

3
 

2
 

1
 

1
 

1
 

24 (48%)
 

8
 

3
 

5
 

18
 

4
 

9
 

6
 

1 (2%)
 

ANIMALS INITIALLY IB STUDY
 

ANI9ALS NECROPSIES
 

AWBALS ElAHINED» HISTOPAIBOLOGICALLY
 

ANIMALS WITR NOB-TUMOR PATHOLOGY
 

IBTEGOHENTARY SYSTEM*
 

SKIN
 

DERMAL INCLUSION CYST
 

HYPERKERATOSIS
 

StlBCOT TISSUE
 

CYST
 

ABSCESS
 

NECROSIS PAT
 

RESPIRATORY SYSΤKB
 

TRACHEA
 

INFLAMMATION SUPPURATIVE
 

INFLAMMATION CHROI1C
 

LONG/BRONCHUS
 

BRONCHIECTASIS
 

LONG/BRONCHIOLE
 

INFLARHATIOB SOPPuRATlVt
 

LONG
 

CONGESTION
 

&DEHA
 

HEMORRHAGE
 

ABSCESS
 

ALVEOLAR BACROP0AGES
 

HYPERPLASIA ALVEOLAR-CELL
 

HEMATOPOIETIC SYSTEM
 

BLOOD
 

RETICOLOCYTOSIS
 

CONTROL
 

50
 

50 (100%)
 

50
 

50 (100%)
 

34 (68%)
 

17
 

3
 

14
 

3
 

3
 

26
 

7
 

1
 

14
 

1
 

7
 

2
 

8 (16%)
 

3
 

LOW DOSE
 

50
 

50(100%)
 

50
 

50 (100%)
 

2 |4%)
 

2
 

1
 

1
 

22 (44%)
 

12
 

2
 

10
 

1
 

1
 

2
 

2
 

11
 

8
 

3
 

6 (12%)
 

2
 

1
 

•SYSTEB PERCENTAGES ARE BASED ON THE RUBBER OF ANIMALS HECROPSIED
 

79
 
TABLE CI MALE RATS: NONTUMOR PATHOLOGY (CONT.)
 

LYMPHOCYTOSIS
 

AHISOCYTOSIS
 

SPHEROCYTOSIS
 

LEPTOCYTOS1S
 

SPLEEN
 

HEHORRHAGE
 

HEMATOMA
 

FIBROSIS
 

FIBROSIS FOCAL
 

PHAGOCYTIC CELL
 

HYPERPLASIA RETIC0LUH-CELL
 

HEHATOPOIESIS
 

LYMPH WODE
 

HEBORRHAGE
 

BRONCHIAL LYMPH KODB
 

HEBORRHAGE
 

THYMUS
 

HEB0RRHA6E
 

CIRCULATORY SYSTEH*
 

HEART
 

HBH0RRHA6E
 

PERIARTERITIS
 

CALCIFIC!TIOH
 

KYOCARDIOfl
 

IHFLABHATIOW FOCAL
 

FIBROSIS
 

FIBROSIS FOCAL
 

DIGESTIVE SYSTER
 

SALIVARY GLAN2>
 

INFLAMMATION CHRONIC FOCAL
 

FIBROSIS FOCAL
 

LIVER
 

HEMORRHAGE
 

GRANULOMA
 

«SYSTEB PERCENTAGES ABE BASED OB THE

CONTROL LOW DOSE
 

1 1
 

2
 

1
 

1
 

3 α
 1
 

1
 

1
 

1
 

1
 

1 2
 

1
 

1
 

1
 

1
 

1
 

1
 

16 (32%) 10 |20%)
 

1 1
 

1
 

1
 

15 9
 

in 9
 

1
 

36 (72%) 35 (70%)
 

1 1
 

1
 

1
 

8 18
 

1
 

 HDPIBER OF HIHALS HECBOPSIED 

80
 

HIGH DOSE
 

1
 

1
 

17 (34%)
 

1
 

1
 

17
 

1
 

17
 

44 (88%)
 

26
 

1
 
TABLE CI MALE RATS: NONTUMOR PATHOLOGY (CONT.)
 

CONTROL LOU DOSE HIGH DOSE
 

NECROSIS FOCAL 1
 

&EXJBORPHOSIS F%TTY 2 4 3
 

DEPOSI11ON OF CRYSTALS 1
 

HSPATOCYTOHFGALT 5 T1 20
 

HYPERPLASIA KODITLAR 1
 

HYPERPLASIA FOCAL 4 5
 

PEHATOPOILSIS 1
 

bILE DOCT 21 17 20
 

FIBROSIS 2
 

HYPERPLASIA 21 17 20
 

PANCREAS 11 1 3
 

FIBROSIS ϋ 2
 

FIBROSIS FOCAL
 

FIBROSIS DIFFUSE 1
 

PERIARTERITIS 3 1 1
 

IANCREATIC ACINUS 8 10 14
 

ATROPHY 5 10 14
 

ATROPHY FOCAL 3
 

STOBACH 1 1 2
 

JNFLABBIHON SUPPURATIVE 1
 

INFLABHATION AC0TE 1
 

EPOSIVE 1KFLABHAT1ON 1 1
 

GASTRIC MUCOSA 1 1 2
 

INFLAMMATION ACUTE 1
 

CALCIFICATION 1
 

HYPERPLASIA FOCAL 1
 

HYPERKERATOSIS 1
 

SMALL INTESTINE 1
 

ULCER FOCAL 1
 

LARGE INTESTINE 8 5 7
 

NEMATODIASIS 8 s
 7


COLON 1
 

INFLAMMATION SUPPURATIVE 1
 

UNINARY SYSTEM* 44 (88%) 47 (94%) 44 (88%)
 

KIDNEY 44 47 43
 

INFLABBATION CHRONIC 44 47 43
 

•SYSIEB PERCENTAGES ARE BASED ON THE NUMBER OF AN1BALS NECROPSIED
 

81
 
TABLE CI MALE RATS: NONTUMOR PATHOLOGY (CONT.)
 

CONTROL LOW DOSE HIGH DOSE
 

RIDNEY/CORSEX 2
 

PIGMENTATION 2
 

KIDNEY/TUBOLF 1 1
 

PIGMENTATION 1
 

REGENERATION 1
 

KIDNEY/PELVIS 6 6 7
 

INFLAHHATION SUPPURATIVE 1
 

NECROSIS 1
 

HYPERPLASIA EPITHELIAL 6 6 7
 

URIWARY BLADDER k 2
 

INFLAMMATION SUPPURATIVE 1
 

HYPERPLASIA EPITHELIAL 4 2
 

U.&LADDER/HUSCULABIS 1
 

HEHORRHAGE 1
 

URETHRA 1
 

CALCULUS 1
 

HEMORRHAGE 1
 

ENDOCRINE SYSTEM* 28 (56%) 29 (5β%) 39 (78%)
 

ADRENAL 1
 

HFHORRHAGE 1
 

ADRENAL CORTEX 3
 

HEMORRHAGE 1
 

HYPERPLASIA FOCAL 2
 

ADRENAL BEDULLA 7 12
 

HYPERPLASIA 1 5
 

HYPERPLASIA FOCAL β 7
 

THYROID 25 26 35
 

ULTIBOBRAHCHIAL CYST 1
 

CYSTIC FOLLICLES 1
 

HYPERPLASIA C-CBLL 23 25 35
 

HYPERPLASIA FOLLICULAR-CELL 1 t 1
 

PARATHYROID 5 1
 

HYPERPLASIA 5 1
 

•SYSTEM PERCENTAGES ARE BASED ON THE NUBBER OF ANIHALS HFCROPSIED
 

82
 
TABLE C1 MALE RATS: NONTUMOR PATHOLOGY (CONT.) 

CORTROL LOW DOSE HIGH DOSE
 

PARCBEATIC ISLETS Τ
 HYPERPLASIA 1
 

RSPkODOCTIYE SYSiXli* 37 (74%) 29 (58%) 46 (92%)
 

ΠAHΗART GLA1D 1
 

GALACTOCtLE 1
 

PREPOfIAL GLAR3 3
 

METAPLASIA SOOAHODS 3
 

PROSIATΕ 6 7 14
 

IRPLAHH&TIOR SUPPURATIVE 3 7 14
 

IWPLABHATIOll CE*O*XC 3
 

ISSTIS 34 27 42
 

ATROPHY 3% 27 42
 

T01ICA VAGINALIS 1
 

HYPERPLASIA BESOTHELIAL 1
 

•ERYOUS sYsrra 2 <*%> 2 (*%> 3 (6%)
 

BRAIN 2 1 2
 

HYDROCEPHALUS 1
 

HEM0RRHAGE 2 1
 

ABSCESS 1
 

CERCEBELLUM 1 1
 

CYTOPLASIIIC YACOOLIZATIO1 t
 1
 

SURA L CORD 1
 

HEBOMHAGi 1
 

SPECIAL S2RSE ORGAIS 2 (4%) « (8%) 1 (2%)
 

EYE 1
 

HEIIORRBAGE 1
 

EYE POSTERIOR CflABBE 1
 

H&flORRflAGE 1
 

EYE/CORRSA 1 3
 

I1FLA9HAT1OI SUPPURATIVE 1
 

•SISTIH PEBCBRAGBS ARE bftSSS OH TBS ROBBER OF IBIBALS MBCROPSIID
 

83
 
TABLE C1 MALE RATS: NONTUMOR PATHOLOGY (CONT.)

 

Ill FIGURATION CRROKIC
 

IHPLAHMATIO* CHRONIC FOCAL
 

EYE/CILIART BODY
 

1NFLABMATION SOPPOfilTIVE
 

EYF/lftIS
 

IWFLAHHA1ION SUPPURATIVE
 

EAR CAHAL
 

INFLABHAT1O» SUPPURATIVE
 

BUSCULOSKEL2TAL SYSTBB*
 

SKELETAL HUSCLB
 

A1ROPHY
 

BODY CAVITIES
 

PERITOBLU!!
 

INFLABBATIOli CKROH1C
 

ALL OTHER SYSTEMS
 

LESEM1ERY
 

1ECROSIS FAT
 

SPECIAL H0FP30L0GY SUBHARY
 

NONE
 

COMTROL
 

1
 

1
 

1 (2%)
 

1
 

1
 

84
 

LOW DOSE
 

2
 

1
 

1
 

1
 

1
 

1
 

2 (4%)
 

2
 

2
 

HIGH DOSE
 

1
 

1
 

1 (2%)
 

1
 

1
 

1 (2%)
 

1
 

1
 
TABLE C2

 

SUMMARY OF THE INCIDENCE OF NONTUMOR PATHOLOGY 

IN FEMALE RATS TREATED WITH PROFLAVINE 

CO1TROL LOW DOSE HIGH DOSS
 

AV1HALS INITIALLY IB STUDY 50 50 50
 

A11HALS NBCROPSIED 50 (100%) 50(100%) 50(100%)
 

AHIHALS EXAMINED HISTOPATHOLQGICALLY 50 50 50
 

&*I*ALS WITH HO»-*UHOR PATHOLOGY 49 (98%) 50 (100%) 46 (92%)
 

IYTEGOHEHTARY SYSTEM* 1 (2%) 2 (4%)
 

SKI* 1 1
 

LACERATED »OOWD 1
 

IltFLAHBATIOS SUPPURATIVE 1
 

suBcai ussufi 1
 

£DKRA 1
 

RESPIRATORY SISTEB 32 (64%) 26 (52%) 19 (38%)
 

TEACHEA 20 15 6
 

1NFLAHMATTOH SDPPORATIVL 5 5
 

I9FLARHATION CPROHIC 15 10 6
 

LDNG/bROSCHUS 1
 

BHOSCHIfiCTASIS 1
 

LO MG/BROMCHIOLE 4 1
 

FOBB1GII BODY 1
 

IHFLABHATIO» SUPPURATIVE 3 1
 

LONG 22 15 15
 

CONGESTION 7
 

EDEBA 1
 

dEHORRHAGE 4
 

IHFLAHBATIO8 1
 

ABSCESS 2
 

ALVEOLAR MACROPHAGES 12 15 11
 

HYPERPLASIA ALVEOLAR-CSLL 3 3
 

HEUATOPOISTIC SiSTIJI 6 (12%) 1 12%) 3 (6%)
 

FLOOD 1
 

LEUKOCYTOSIS 1
 

•SYSTEH PERCENTAGES ARE BASED ON THE NUBBFR OF ANIMALS NECROPSIED
 

85
 
TABLE C2 FEMALE RATS: NONTUMOR PATHOLOGY (CONT.)

 

CONTROL LOW DOSE HIGH DOSE
 

SPLSES 4 1 3
 

HEMOSIDEROSIS 2
 

HEH1TOPOIESIS 2 1 3
 

SFLK1IC RED PULP 1
 

ATROPHY 1
 

CIRCULATORY SYSTEB* 16 (32%) 14 (28%) 7 (14%)
 

K£ARI 1
 

PERIARTERITIS 1
 

HtAfll/ATRIUh 1
 

THROBBOSIS 1
 

RYOCARDIOH 16 12 7
 

FIBROSIS 16 12 7
 

LORTA 1
 

PERIARTERITIS 1
 

PULRONARY ARTERY 1
 

THROff&OS ORGANIZED 1
 

HEPATIC VEIN 1
 

THROHbOSIS 1
 

DIGfcSTIfE SYS1EW 26 (52%) 34 (68%) 35 (70%)
 

TOUGOE 1
 

ACANTHOSIS 1
 

PAROTID GLA«a 1
 

ATROPBT 1
 

LIVER 9 26 26
 

HEMORRHAGE 1
 

NE^OSIS DIPPOSE 1
 

HETAHORPHOSIS FATTY 7 2 2
 

HEPATOCYTOHIGAL1 2 1 1
 

HYPERPLASIA FOCAL 23
 

ANGIEdASIS 1 1
 

L1VER/C2HTRILOEDLAR 1
 

NECROSIS DIFFUSE 1
 

•SYSTEB PERCENTAGES ARE BASED ON THE lUHbER OF ANIMALS 8ECROPSIED
 

86
 
TABLE C2 FEMALE RATS:


BILE BU d
 

IBFLABΗATIΟΙ
 

F1B&OS1S
 

E1P£*PLASIA
 

PA9CRKAS
 

FIBROSIS
 

PAKCkL&TIC ACliOS
 

ATRGPB1
 

ATROPBT FOCAL
 

GASTRIC HOCOSA
 

ISFLABBATIOH ACOΤI FOCAL
 

S3 ALL IMESTIIE
 

ULC£R FOCAL
 

LARGE 11TESI113
 

HEHATODIAS1S
 

DRIIART SISTEB*
 

KI1»*EI
 

PTBfiOftBPBROSIS
 

HOLtlPLE CISTS
 

F£BORRHAGS
 

1SFLAHBAT1O9 CHHO11C
 

K1DM£T/CORTEX
 

CIST
 

PIGffBVTATIOl
 

ftlDlEI/PILTIS
 

IIFLAflHATlOll SOPPORATIVE
 

ETPESPLASIA EPITHELIAL
 

OIIllAhT BLADDER
 

EUERA
 

9IP£3PLASIA EPITHELIAL
 

EHDOCfclHE SISTEK
 

HT01TARI
 

HLBOfcfiHAGL
 

•SYS1EB PEfcCEBTAGES ARE hkSTD OR THE

 NONTUMOR PATHOLOGY (CONT.)
 

COHTROL
 

13
 

t
 

13
 

2
 

2
 

9
 

7
 

2
 

1
 

t
 

3
 

3
 

38 (76%)
 

34
 

1
 

33
 

7
 

7
 

i
 

1
 

3
 

3
 

30 (6C%)
 

3
 

3
 

LOW DOSE HIGH DOSE
 

6 12
 

1
 

6 12
 

11 8
 

11 8
 

1
 

1
 

5
 

5 5
 

34 (68%) 18 (36%)
 

32 17
 

1
 

1
 

32 16
 

1
 

1
 

1
 

1
 

1
 

1
 

40 (80%) 28 (56%)
 

1
 

1
 

 ROBBER OF ARIBALS WECROPSIED 

87
 
TABLE C2 FEMALE RATS: NONTUMOR PATHOLOGY (CONT.) 

ALiiNAL
 

RJECR0S1S FOCAL
 

RuTAHORPHOSlS FATTY
 

ADKKSAL CORTLX
 

CYTG»EGALY
 

JLdFBHAL ffEDOLLA
 

BYPSRPLASTA FOCAL
 

iEYROID
 

HYPERPLASIA C-CILL
 

HYPERPLASIA FOLLICULAR-CELL
 

PARATHYROID
 

HYPERPLASIA
 

PAHCR2ATIC ISLETS
 

PYPERPLASIA
 

R£iEODUC1ITE SYSTKH* 

ΆΑϋΒ&Ιί GLAND
 

CALACTOCtLL
 

CYSTIC IrUCTS
 

IBFLABSATION SUPPURATIVE
 

HYPERPLASIA SODOLAR
 

HYPERPLASIA FOCAL
 

CLITOKJLL GLAUD
 

!!£YAPLAS3A SQUAMOUS
 

UTERUS
 

HYDEOflETiiA
 

HFBORRHAGE
 

ABSCESS
 

BTEROS/BBPOHCTRIU?!
 

IHFLABRATIO» SOPPOii£TIVE
 

HYPERPLASIA
 

fcYPERPLASIA CYSTIC
 

HYPERPLASIA SIPOHAL
 

OtAfY/OfIDOCT
 

IBFLJUTHATIOir SUPPURATIVE
 

•SYSItB PFRCEStfAGES ARE BASED ON THE


CONTROL LOW ROSE 

2
 

1
 

t
 

2 

2 

2 6 39
 

2 6 39
 

2 t 

2 1 

1 

1 

29 (58%) 25 (50%) 

4 12 

11 

3 

1 

3 

1
 

1
 

5 1 

4 

1 

1 

18 15 

16 13 

2 

6 6 

1 

3
 

3
 

 NOUBER OP AHIHALS RECROPSIED
 

88
 

HIGH DOSE 

2 

2 

27 

26 

2 

14 (28%) 

7 

7 

1 

9 

7 

4 

1 
TABLE C2 FEMALE RATS: NONTUMOR PATHOLOGY (CONT.) 

CONTROL
 

11
 

ε
 

6
 

1
 

1
 

1 (2%)
 

1
 

1
 

2 (4%)
 

1
 

1
 

1
 

1
 

OVARY
 

CYST
 

INFLAMMATION SUPPURATIVE
 

NECROSIS FAT
 

«ESOtARlOT
 

NECROSIS FAT
 

NERVOUS SYSTEM*
 

BitAIH
 

HEMORRHAGE
 

CEREBRAL CORTEX
 

HEMORRHAGE
 

GLIOSIS
 

SPECIAL SENSE ORGANS
 

EYE/CORNIA
 

INFLAMMATION CHRONIC
 

EYELID
 

ABSCESS
 

MUSCULOSKELETAL SYSTEM
 

BODY CAViTiES
 

PERITONEUM
 

INFLAMMATION FOCAL
 

EPICARDIUM
 

INFLAMMATION
 

ALL OTHER SYSTEMS
 

NONE
 

SPECIAL MORPEOLOGY SUMMARY
 

LOW DOSE
 

8
 

6
 

3
 

1 (2%)
 

1
 

1
 

1
 

1 (2%)
 

1
 

1
 

•SYSTEM PERCENTAGES ARE BASED OH THE NUMBER OF ANIMALS
 NECROPSIED
 

89
 

HIGH DOSE
 

1
 

1
 

1 (2%)
 

1
 

1 

APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONTUMOR PATHOLOGY
 

IN MICE FED
 

PROFLAVINE IN THE DIET
 

91
 

TABLE D1
 

SUMMARY OF THE INCIDENCE OF NONTUMOR PATHOLOGY
 

IN MALE MICE TREATED WITH PROFLAVINE
 

COWTROL LOW DOSE HIGH DOSE
 

A5IHALS IHiriALLY I* STUDY 50 50 50
 

A*3HALS 1ECROPSIED 50 (100S) SO(100%) 50(100%)
 

ANIMALS BXABINfcD HISTOPATEOLOGICALLY 50 50 50
 

A9IHALS WITH fOR-TUHOR PATHOLOGY 45 (90%) 44 (88%) 44 (88%)
 

INTEGUMENTARY SVS1S.M* 1 (2%) 2 (4%) <• (8%)
 

SKIN 1 2 2
 

IfFLAHHATIOH FOCAL 1
 

ULCLR FOCAL 1
 

INFLAMMATION ACUTE t
 

ACARIASIS 2
 

SUBCOT TISSUE 2
 

CIST 1
 

GRANULOMA 1
 

RESPIRATORY SYSIEB 10 (20%) 6 (T2%) 10 (20%)
 

LUNG/bRONCHUS 1
 

HYPERPLASIA FOCAL 1
 

LUHG/BROllCHIOLt 1
 

IKFLAMATIOK SUPPURATIVE 1
 

LUWG 9 6 9
 

COMGLSTIOli 1
 

HEMORRHAGE 3
 

BSO9CHOPHEURONIA 1
 

ABSCESS 1
 

IMfLAH SUPPURATIVE GAAliULARATOUS 1
 

ALVLOLAU BACROPBAGES 3 t 3
 

HYPERPLASIA ALVEOLAR-CELL 1 6 5
 

LUKG/ALVBOL1 1
 

IMFLAKHATIOH SUPPURATIVE 1
 

HEMATOPOIETIC S1STEB 12 (24%) 6 (12%) 5 (10%)
 

BLOOD 3
 

LEUKOCYTOSIS 3
 

•SYST£B PERCENTAGES ARE BASED ON THE HUBB5R OF AN1BALS ΝECRΟPSIED
 

93
 
TABLE D1 MALE MICE: NONTUMOR PATHOLOGY (CONT.) 

HIGH DOSE
 

•SYSTEH PERCENTAGES ARE EASED ON THE K1HBEB OF AHIHALS NECROPSIED
 

94
 
TABLE D1 MALE MICE: NONTUMOR PATHOLOGY (CONT.)
 

CONTROL LOW DOSE HIGH DOSE
 

ANGIECTASIS 1 10 8
 

GALLBLADDER 1
 

HYPERPLASIA EPITHELIAL 1
 

BILE DOCT 3 1 2
 

INFLAMMATION* 1
 

HYPERPLASIA 2 1 2
 

PANCREAS 1
 

CYSTIC DUCTS 1
 

PANCREATIC ACINUS 1 3
 

ATROPHY 1 3
 

STOMACH 2 4 3
 

ULCER FOCAL 1
 

INFLAMMATION SUPPURATIVE 1
 

ABSCESS 1
 

HYPERPLASIA EPITHELIAL 3 1
 

HYPERKERATOSIS 1 1
 

GASTRIC HUCOSA 1 1
 

CYST 1
 

MULTIPLE CYSTS 1
 

CALCIFICATION FOCAL 1
 

SMALL INTESTINE 1 2
 

CTST 1
 

HYPERPLASIA EPITHELIAL 1
 

LYRPHOID HYPERPLASIA 1
 

PEYERS PATCH 1
 

HYPERPLASIA RETICULUM-CELL 1
 

LARGE INTESTINE 1 4 1
 

NEMATODIASIS 1 4 1
 

RECTUS 1
 

INFLAMMATION CHRONIC FOCAL 1
 

ANUS 1
 

HYPERPLASIA PSEUDOEPITHELIOMΑTΟU 1
 

URINARY SISTES* 37 (74%) 17 (34%) 24 (48%)
 

KIDNEY 35 14 24
 

MULTIPLE CYSTS 1
 

*SYSTEB PtRCHHTAGES ABE BASED OK THE KOHBEB OF AWIBtLS BECBOPSIED
 

95
 
TABLE D1 MALE MICE: NONTUMOR PATHOLOGY (CONT.)
 

COHTROL LOW DOSE HIGH DOSE
 

IBPLABBATIOB CKROKIC 11 14 24
 

HFLABIIATIOB CPKOB1C FOCAL 1
 

PELIfASCDLAR CUFFING 31
 

KIMEY/CORTEX 2
 

PIGREKTATIOX 1
 

JYTOPLASBTC YACDGLIZAIIOM 1
 

KlDHEI/GLOBEfiOLOS 1
 

ABYLOIDOSIS 1
 

XlDBEY/PELfIS 1
 

DILA1ATIO* 1
 

IftFLABBATIO» SUPPURATIVE 1
 

HYPOPLASIA EPITBELIAL 1
 

3R1BAFY BLADDFi 5 3
 

l)iFLABBATIG.t SDPPORATIVK 1 1
 

BTP£&FLAS1A EPITHELIAL 4 3
 

ΌΒΕΤΒΗΑ
 CALCULUS 4
 

HYPEKPLASIA EPITBEL1AL 2
 

LUDOCRIME SI5TEB* 5 (10%) 11 (22%) 9 (18%)
 

ADR£HAL COHIIX 2
 

CTTOBEGALT 1
 

0TPSKPLASIA FOCAL 1
 

A!#S£ffAL SEDOLLA 1
 

HYPERPLASIA FOCAL 1
 

TBYBOIB 2 3
 

CYSriC FOLLICLES 1
 

FIBROSIS 1
 

HYPERPLASIA FOLLICULAR-CELL 1 2 2
 

PARATHYROID 1
 

BYPFRPLASIA 1
 

PAMCR^AFIC ISLETS 3 9 2
 

HYPERPLASIA 3 9 2
 

fcEPKODBCTIVE SYSTSH 3 (6%) 4 (8%) 5 (10%)
 

FtlilS 1
 

CALCDLOS 1
 

•SYSME PERCZUTAGES ARE EASED OH THE BOBBER OF ABI8ALS BECROPSIED
 

96
 
TABLE 01 MALE MICE: NONTUMOR PATHOLOGY (CONT.) 

HIGH DOSE 

2 

1 

1 

1 

1 

1 

1 

1 

1 

26 (52%) 

26 

26 

2 (<*%) 

2 

1 

1 

1 (2%) 

1 

PREPUTIAL GLAND 

CYST 

CTSTIC DUCTS 

ABSCESS 

INFLAMMATION CHRONIC 

METAPLASIA SQUAMOUS 

FBOSTATB 

CYST 

INFLAMMATOIN SUPPURATIVE 

SEMINAL VESICLE 

DILATITION 

EPIDIDYMIS 

NECROS1S FAT 

NERVOUS SYSTEM* 

COBPOBA AMYLACEA 

SPECIAL SENSE OFGAMS 

EYE/LACRIMAL GLAND 

HYPERPLASIA 

HYPERPLASIA EP1THELIAL 

KOSCOLoSKELSIAL STSTSR 

PMHISIA L £ON£ 

0STEOSCL3BOSIS 

BO&Y CAflTIZS 

A1X OIBSii SISTtftS 

ADIPOSE TISSUE 

1SFLARHA2XOI 

COBTKOL LOW DOSS 

1 4 

2 

1 2 

1 

1 

1 

1 

2 |4%) 14 (28%) 

2 14 

2 m 

1 (2%) 

t 

1 

2 (*%) 2 (4X) 

1 1 

1 

•S1STE» PERCEHTAGES ARE BASED OS THE ROBBER OF AHIHALS WECROPSIED
 

97
 
TABLE D1 MALE MICE: NONTUMOR PATHOLOGY (CONT.) 

COWTROL LOW DOSE HIGH DOSE
 

INFLAHMATIOH FOCAL 1
 

INFLAMMATION CHRONIC
 

BECROSIS FIT 1
 

CALCIFICATION FOCAL 1
 

K£5£HT£fiY 1 1
 

CYST 1
 

«ECROS1S FA Τ
 

SPECIAL HORPHOLOGY SUMMARY
 

AOTO/i£CROPSY PBRF/HISTO PFRF
 

98
 
TABLE D2
 

SUMMARY OF THE INCIDENCE OF NONTUMOR PATHOLOGY
 

IN FEMALE MICE TREATED WITH PROFLAVINE
 

COHTROL (OBTR) LOW DOSE HIGH DOSE
 

2IXHALS INITIALLY IB STUDY 50 50 50
 

ABIEALS BZCROPSI&D 49 (100%) 50(100%) 50(100%)
 

AKIBALS RXTR NOH-TOflOK PATHOLOGY 41 (&4%) 41 (82%) 35 (70%)
 

AttI£>LS EXARLB2I* HISTOPATftGLOGICALLY 46 50 50
 

1ITEGU3EHTAHY SYSX2B* 1 (2%)
 

SOBCOT TISSUE 1
 

ABSCESS 1
 

R2SPIRATOFY SYSTEM 5 (10%) 2 (4%)
 

LUWG 5 2
 

RERORREAGS 1
 

IXFLARRATIOS GKAUD1.OHAT0QS 1
 

ISFLAHSATIOU FOCAL GfilUQLOBATOUS 1
 

ALVEOLAR H1CBOPBAGES 3
 

EYPfHPLASIA ALYBOLAK-CELL 1
 

B£BATOPOIETXC SYSTKH 11 (22%) 6 (12%) 2 (4%)
 

b LOO i> 3
 

LEUKOCYTOSIS 3
 

ΒΟΙ UA&ROU 1
 

HYPERPLASIA 1
 

SΡLEES 6 1 2
 

HYPERPLASIA FETICULOΗ-CELL 1
 

LYHfHOlD HYPERPLASIA 3 1 1
 

HEMATOPOIESIS 2 1
 

LYHPH «ODE 1
 

VECROSIS 1
 

BRO1CHIAL LYNPB NODE 1
 

LYSPEOID HYPERPLASIA 1
 

PESEIITEllXC LYRPHHOBE 2 s
 

HiftORRHAGS 1 3
 

•SYSTEH PEEC2H1AGES ARE BISLB OH THB 1URBER OF AWIHALS KECROPSIED
 

99
 
TABLE D2 FEMALE MICE: NONTUMOR PATHOLOGY (CONT.)

 

INPLAHMATIO» SUPPURATIVE
 

ASGIECTAS1S
 

HYPERPLASIA LETICULUR-CELL
 

LYftPHGID FYPERPLAS1A
 

XBYBUS
 

LYBPHOID PYPERPLAS1A
 

CIRCULATORY SYS2E1I*
 

HEART
 

LYBPriOCYTIC INFLAB INFILTRATE
 

UYOCARDIUH
 

FIBROSIS FOCAL
 

CALCIFICATION FOCAL
 

ENDOCARDIUB
 

ISFLABBATION ACUTE FOCAL
 

DIGESTIVE SYSTEH
 

SALIVARY GLAKI
 

ATROPHY
 

LIVER
 

SEBORRHAGE
 

IfcFLAKBATION SUPPURATIVE
 

GRANULOMA
 

M2CR0SIS
 

NECROSIS FOCAL
 

BETAHORPHOS1S FATTY
 

HEPATOCYTOBEGALY
 

HYPERPLASIA »0!>ULAH
 

ASG1ECTASIS
 

LYBPHOID HYPERPLASIA
 

GALLBLADDER
 

DILATATION
 

fclLE DUCT
 

CYST
 

HYPERPLASIA
 

PANCREAS
 

CYSTIC DOCTS
 

COITROL LOW DOSE HIGH DOSS
 

1
 

2
 

1
 

1
 

1
 

1
 

1 (2%) 3 (6%) 1 (2%)
 

t
 

1
 

2 1
 

1
 

2
 

1
 

1
 

17 (35%) 27 (54%) 18 (36%)
 

1
 

1
 

10 22 16
 

1
 

1
 

1
 

1
 

6 to
 3


1 2 1
 

1
 

8 9
 

3 <» 6
 

1
 

1
 

1
 

1 1
 

1
 

1
 

3 1 1
 

1
 

*SYSTSH P2P.CENIAGBS ARB EASED OB THE HDHBEH OP IHIHALS HBCBOPSIED
 

100
 
TABLE D2 FEMALE MICE: NONTUMOR PATHOLOGY (CONT.)
 

COVTROL LOW DOSE HIGH DOSE
 

IFBOHBOS1S 1 1
 

1KFLAHHATI09 SUPPURATIVE 1
 

FIBROSIS 1
 

PERIFISU&LAK CUFFIBG 1
 

H5CROSIS FOCAL 1
 

K&CF.OSIS SZI 1
 

PAKCfcEATIC ACINUS 2 1
 

ATROPHY 2 1
 

STOMACH 2 2 2
 

CYST 1
 

ABSCESS 1 1
 

PEEIAIiTKJtITIS 1
 

FYfrERPLASI* EPITHELIAL 1
 

HYPERPLASIA FOCAL 1
 

FTPFHKERA2OSIS 1
 

GASTRIC B0C05* 1
 

HIPERPLAMA FOCJIL 1
 

SHALL HTESTlNr 1 1
 

COKG2STI0K 1
 

INFARCT 1
 

LARGE IHTFSTIHS 1
 

B£eATODXASIS 1
 

OBIVART STSiil!!* 17 (35%) 10 (20%) 11 (22%)
 

K1DKFY 17 8 10
 

I1FLA8HATIO1 CB9OVIC 4 7 10
 

PEFI¥ASCULAP CUFFIIG 13
 

SfXJvOSIS FAT 1
 

C1LCIFICATI01 FOCAL 1
 

ORXWART BLADDER 2 3 τ
 1VFLAHRA7IOB CFROB1C 1
 

HYPERPLASIA EPXTRBL1AL 2 2 1
 

LHKICRIBS SY5TEB b (16%) 11 (22%) 7 (14%)
 

ADR?1AL 1
 

A9GXECXASIS 1
 

•STSTEB P£fiC£»T»GES IBS EiSEL· OH THE BOBBER OF tHlBALS WECROPSIED
 

101
 
TABLE D2 FEMALE MICE: NONTUMOR PATHOLOGY (CONT.)
 

ADHEHAL CORΪEX
 

CYTO3EGALT
 

HYPERPLASIA FOCAL
 

ADREBAL ΒΕDOLLA
 

HYPERPLASIA FOCAL
 

THYROID
 

HYPERPLASIA FOLLICULAB-CELL
 

PARATHYROID
 

HYPERPLASIA
 

REPRODUCTIVE SYSTOfl*
 

VAGIBA
 

IB7LAHBATIOB SUPPURATIVE
 

HYPERPLASIA PSEUDO2PIIBELIOBATOU
 

UTERUS
 

BYDROHETRA
 

CYST
 

ABSCESS
 

U12RUS/SBIMHISTRIU8
 

IHFLABBATIO1 SUPPURATIVE
 

HYPERPLASIA CYSTIC
 

OVARY
 

cirsr
 

HEBOREBAGE
 

ABSCESS
 

BECROSIS FAT
 

1SFVOUS SYS1E9
 

bRAIB
 

CORPORA ABYLACEA
 

SPIRAL COR&
 

&RGEBERATIOB
 

SPECIAL SEBSE ORGABS
 

IRFLARHATIOB FOCAL
 

*SYST£]1 PEBCEBTAGES AEE BASED OB THE
 

COBTROL
 

1
 

1
 

6
 

6
 

1
 

1
 

30 (61%)
 

2
 

1
 

1
 

6
 

6
 

21
 

3
 

20
 

11
 

9
 

1
 

1
 

1 (2%)
 

1
 

1
 

1 (2%)
 

1
 

1
 

BUBBER OF
 

LOV DOSE
 

1
 

1
 

1
 

1
 

10
 

10
 

11 (22%>
 

3
 

2
 

1
 

4
 

1
 

3
 

6
 

6
 

1
 

5 (10%)
 

5
 

5
 

ABIBALS BECROPSIED
 

HIGH DOSE
 

2
 

2
 

4
 

4
 

8 (16%)
 

2
 

2
 

6
 

6
 

2
 

8 (16%)
 

8
 

8
 

102
 
TABLE D2 FEMALE MICE: NONTUMOR PATHOLOGY (CONT.)
 

GRANULATION TISSUE
 

EYE/LACRIHAL GLAND
 

HYPERPLASIA EPITHELIAL
 

MUSCULOSKELETAL SYSTEM*
 

SKELETAL MUSCLE
 

PERIVASCULAR CUFFING
 

BODY CAVITIES
 

NONΕ
 

ALL OTHER SYSTEMS
 

MULTIPLE ORGANS
 

LYMPHOID HYPERPLASIA
 

MESENTERY
 

CYST
 

NECROSIS FAT
 

SPECIAL MORPHOLOGY SUMMARY
 

NO LESION REPORTED
 

NO NECROPSY PERFORMED
 

AUTO/NECROPSY PERF/HISTO PERF
 

AUTOLYSIS/NECROPSY PERF/NO HISTO
 

CONTROL
 

1
 

1
 

1
 

1 (2%)
 

1
 

1
 

5 (10%)
 

3
 

3
 

2
 

2
 

1
 

1
 

103 

LOW DOSE HIGH DOSE
 

4 (8*) 1 (2%) 

Τ 1 

3 1 

1 

1 1 

U. S. GOVERNMENT PRINTING OFFICE : 1977 241-161/3039 


DHEW Publication No. (NIH) 77-805