National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No.7 

January 1977 

BIOASSAY OF 

PHENFORMIN 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 114-86-3 

NCI-CG-TR-7 

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


Public Health Service 


National Institutes of Health 


BIOASSAY OF
 


PHENFORMIN
 


FOR POSSIBLE CARCINOGENICITY
 


Carcinogen Bioassay and Program Resources Branch
 


Carcinogenesis Program
 


Division of Cancer Cause and Prevention
 


National Cancer Institute
 


Bethesda, Maryland
 


DHEW Publication No. (NIH) 77-807
 


BIOASSAY OF
 


PHENFORMIN
 


FOR POSSIBLE CARCINOGENICITY
 


Carcinogenesis Program
 


Division of Cancer Cause and Prevention
 


National Cancer Institute
 


CONTRIBUTORS: This report presents the results of the carcinogen
 


bioassay of phenformin conducted by the Carcinogen Bioassay and
 


Program Resources Branch, Carcinogenesis Program, Division of
 


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


Bethesda, Maryland. The bioassay was conducted at Southern
 


Research Institute, Birmingham, Alabama, initially under direct
 


contract to the NCI and currently under a subcontract to Tracer
 


Jitco, Inc., prime contractor for the NCI carcinogen bioassay
 


program.
 


The experimental design and doses were determined by Drs. D. P.
 


Griswoldl and J. D. Prejean^; Dr. Griswold was the principal
 


investigator. The NCI Project Officers were Drs. E. K.
 


Weisburger^ and J. H. Weisburger^.^
 


I. Brown^ and J. Belzer* were responsible for the care of
 


laboratory animals. Histopathologic examinations of treated and
 


control animals were performed by Drs. S. D. Kosanke^ J. C.
 


Peckhaml and Dr. R. A. Renne. 4,5 The diagnoses in this report
 


represent the interpretation of Dr. Renne. Pathologists at NCI
 


and at Tracer Jitco have reviewed selected slides and concur with
 


the overall pathologic evaluation of the study.
 


Compilation of individual animal survival and summary tables was
 


performed by C. A. Dominick^, and the pathology tables were
 


compiled at Southern Research Institute. The statistical
 


analyses were performed by Dr. J. R. Joiner", using methods
 


selected for the Bioassay Program by Dr. J. J. Gart^. Chemicals
 


used in this bioassay were analyzed under the direction of Dr. E.
 


iii
 

Murrill^, and the analytical results were reviewed by Dr. S. S.
 


Olin6.



This report was prepared at Tracer Jitco° under the direction of
 


Dr. J. F. Robens with the assistance of. Dr. G. L. Miller and L.
 


A. Waitz. Dr. E. W. Gunberg was the technical editor, assisted
 


by Y. E. Presley. The final report was reviewed by members of
 


the participating organizations^'^*°.
 


^•Southern Research Institute, 2000 Ninth Avenue South,
 


Birmingham, Alabama.
 


^Carcinogenesis Program, Division of Cancer Cause and Prevention,
 


National Cancer Institute, National Institutes of Health,
 


Bethesda, Maryland.
 


with The Naylor Dana Institute for Disease Prevention,
 


American Health Foundation, Hammond House Road, Valhalla,
 


New York.
 


^Experimental Pathology Laboratories, 17 Pine Street, Herndon,
 


Virginia.
 


with Battelle Pacific Northwest Laboratories, Battelle
 


Boulevard, Richland, Washington.
 


^Tracer Jitco, Inc., 1776 East Jefferson Street, Rockville
 


Maryland.
 


^Field Studies and Statistics, Division of Cancer Cause and
 


Prevention, National Cancer Institute, National Institutes of
 


Health, Bethesda, Maryland.
 


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


Missouri.
 


iv
 

SUMMARY
 


A bioassay of the carcinogenicity of phenformin hydrochloride was
 


conducted using Fischer 344 rats and B6C3F1 mice. The compound
 


was administered in the diet for 78 weeks to groups of 35 animals
 


of each species and sex, using concentrations of 15,000 and
 


30,000 ppm for rats and concentrations of 1,200 and 2,500 ppm for
 


mice. Treatment was followed by a period of observation of 26
 


weeks. Control groups consisted of 15 untreated animals of each
 


species and sex.
 


Average weights attained by treated groups of rats and mice were
 


consistently lower than those of control groups in all tests
 


except that for male rats, in which case the weights shown by
 


treated and control animals were indistinguishable. Survival was
 


apparently unaffected in both species by treatment with
 


phenformin, but was poor in mice due to intercurrent disease.
 


Tumors appearing in treated rats and mice were similar in type
 


and number to those in controls, and no pathologic or statistical
 


evidence of induction of tumors in these species by phenformin
 


was found.
 


V 

TABLE OF CONTENTS
 


Page
 


I. Introduction 1



II. Materials 3



A. Chemicals 3



B. Dietary Preparation 3



C. Animals 4



D. Animal Maintenance 4



E. Subchronic Studies 6



F. Design of the Chronic Study 7



G. Clinical and Pathologic Examinations 7



H. Data Recording and Statistical Analyses 10
 


III. Results - Rats 13
 

A. Body Weights and Clinical Signs (Rats) 13
 


B. Survival (Rats) 13
 

C. Pathology (Rats) 16
 

D. Statistical Analyses of Results (Rats) 21
 


IV. Results - Mice 27
 

A. Body Weights and Clinical Signs (Mice) 27
 


B. Survival (Mice) 27
 

C. Pathology (Mice) 29
 

D. Statistical Analyses of Results (Mice) 35
 


V. Discussion 43
 

VI. Bibliography 47
 

APPENDIXES
 


Appendix A Summary of the Incidence of Neoplasms
 


and Other Proliferative Lesions in Rats
 


Fed Phenformin in the Diet 49
 


Table Al Proliferative Lesions of the Endocrine
 


System 51
 

Table A2 Proliferative Lesions of the Reproductive
 


System and Mammary Gland 53
 


vii
 

Page 

Table A3 Proliferative Lesions of the 

Respiratory Tract 54 

Table A4 Proliferative Lesions of the 

Urinary Tract 55 

Table A5 Miscellaneous Proliferative Lesions 56 

Table A6 Proliferative Lesions of the Liver 58 

Appendix B Summary of the Incidence of Neoplasms 

and Other Proliferative Lesions in 

Mice Fed Phenformin in the Diet 59 

Table Bl Proliferative Lesions of the 

Digestive System 61 

Table B2 Neoplasms of the Hematopoietic System 62 

Table B3 Primary Pulmonary Neoplasms 62 

Table B4 Neoplasms of the Vasculature 63 

Table B5 Proliferative Lesions of the 

Reproductive System 65 

Table B6 Proliferative Lesions of the Endocrine 

System 66 

Table B7 Proliferative Lesions of the 

Urinary Bladder 67 

Table B8 Subcutaneous Neoplasms 68 

Appendix C Summary of the Incidence of 

Nonneoplastic Lesions in Rats Fed 

Phenformin in the Diet 69 

Table Cl Summary of the Incidence of 

Nonneoplastic Lesions in Male Rats 

Fed Phenformin in the Diet 71 

Table C2 Summary of the Incidence of 

Nonneoplastic Lesions in Female 

Rats Fed Phenformin in the Diet 76 

viii
 
Page 

Appendix D Summary of the Incidence of 

Nonneoplastic Lesions in Mice 

Fed Phenformin in the Diet 81 

Table Dl Summary of the Incidence of 

Nonneoplastic Lesions in Male Mice 

Fed Phenformin in the Diet 83 

Table D2 Summary of the Incidence of 

Nonneoplastic Lesions in Female Mice 

Fed Phenformin in the Diet 87 

TABLES 

Table 1 Chronic Study of Phenformin 8 

Table 2 Analyses of the Incidence of Primary 

Tumors at Specific Sites in Male 

Rats Fed Phenformin in the Diet 22 

Table 3 Analyses of the Incidence of Primary 

Tumors at Specific Sites in Female 

Rats Fed Phenformin in the Diet 24 

Table 4 Analyses of the Incidence of Primary 

Tumors at Specific Sites in Male 

Mice Fed Phenformin in the Diet 36 

Table 5 Analyses of the Incidence of Primary 

Tumors at Specific Sites in Female 

Mice Fed Phenformin in the Diet 39 

FIGURES 

Figure 1 Growth Curves for Rats Fed Phenformin 

in the Diet 14 

Figure 2 Survival Curves for Rats Fed Phenformin 

in the Diet 15 

Figure 3 Growth Curves for Mice Fed Phenformin 

in the Diet 28 

Figure 4 Survival Curves for Mice Fed Phenformin 

in the Diet 30 

ix
 

I. INTRODUCTION
 


Phenformin is a synthetic oral hypoglycemic agent used to control
 


maturity-onset diabetes. Pharmacologically, phenformin acts to
 


enhance anaerobic glycolysis, decrease gluconeogenesis, and
 


inhibit intestinal absorption of glucose (Goodman and Oilman,
 


1975). This compound was selected for carcinogen!city testing
 


since, in the treatment of diabetes, it is administered
 


chronically.
 


1

II. MATERIALS AND METHODS
 


A. Chemicals
 


Phenformin hydrochloride (DBr*/) is 1-phenethylbiguanide
 


hydrochloride, manufactured by CIBA-GEIGY, Ardsley, N.Y. The
 


stated purity of lot #22332 was 99.7% (spectrophotometric
 


analysis). One trace impurity identified as biguanide (0.34%)
 


and two other trace impurities were detected by thin-layer
 


chromatography. Purity by perchloric acid titration was 100.9 ±



0.3%. The melting point of this sample was 177-178°C, which was
 


comparable to the literature value of 175-178°C. The infrared
 


and nuclear magnetic resonance spectra conformed to standard
 


spectra for this compound. The phenformin used in this bioassay
 


was stored in plastic containers at 5°C.
 


B. Dietary Preparation
 


Dosed feed mixtures were prepared by combining the total dose of
 


the compound with a small amount of feed and then mixing this
 


premix with the remaining quantity of animal feed for 10 minutes
 


in a twin-shell blender. Mixtures were prepared every two weeks
 


and stored at room temperature. Analyses to determine the
 


concentration or stability of the compound in the feed or the
 


homogeneity of the mix were not performed.
 


3
C. Animals
 


Fischer 344 rats were received from Charles River Breeding
 


Laboratories, Wilmington, Mass., at 40 days of age. B6C3F1 mice
 


were also obtained from Charles River, at 41 days of age.
 


Following a two-week quarantine, those animals with a clinical
 


appearance of health and adequate body weight were assigned to
 


test groups and matched-control groups. Animals were
 


individually identified within cages by ear marking.
 


D. Animal Maintenance
 


Rats and mice were housed five per cage in solid-bottom stainless
 


steel cages (Hahn Roofing and Sheet Metal Co., Birmingham, Ala.).
 


Rat cages were lined with Iso-Drr^ hardwood chips (Carworth,
 


Edison, N.J.), and covered with disposable filter bonnets; mouse
 


cages were lined with Sterolit clay bedding (Englehard Mineral
 


and Chemical Co., New York, N.Y.). Dosed feed mixtures and diets
 


for the untreated controls were prepared with Wayne© Lab Blox
 


Meal (Allied Mills, Chicago, 111.). City tap water was used.
 


Cages, water bottles, feeders, and racks were sanitized weekly.
 


Bedding was replaced weekly. Animal rooms were illuminated for
 


nine hours per day by fluorescent lighting and natural light.
 


Room air underwent 15 changes per hour, and was maintained at
 


20-24° C and 38-42% relative humidity.
 


4
Rats and mice treated with phenformin were housed in rooms where,
 


at one time or another, other chemicals were being tested. These
 


chemicals were as follows:
 


RAT S
 


anthranilic acid
 


pyrazinecarboxamide
 


L-tryptophan
 


1-buty1-3-(p-tolylsulfonyl)urea; tolbutamide
 


4,4'-sulfonyldianiline
 


4,4'-thiodianiline
 


2,6 diamino-3-(phenylazo)pyridine hydrochloride
 


2-ethyl-4-pyridinecarbothioamide; ethionamide
 


5-(4-chlorophenyl)-6-ethyl-2,4-pyrimidinediamine; pyrimethamine
 


4-chloro-N-((propylamino)carbonyl)benzenesulfonamide;
 


chloropropamide
 


*N-9H-fluoren-2-ylacetamide; 2-AAF
 


4—acetyl—N-((cyclohexylamino)carbonyl)benzenesulfonamide;
 


acetohexamide
 


N-(p-toluenesulfonyl)-N'-hexamethyleniminourea; tolazamide
 


MIC E
 


anthranilic acid 

pyrazinecarboxamide
 


L-tryptophan
 


3,3'-iminodi-l-propanol dimethanesulfonate (ester) hydrochloride
 


5-azacytidine
 


(+)-4,4'-(l-methyl-l,2-ethanediyl)bis-2,6-piperazinedione;
 


ICRF-159
 


beta-2'-deoxy-6-thioguanosine
 


1,4-butanediol dimethanesulfonate; busulfan
 


acronine
 


emetine dihydrochloride tetrahydrate
 


N,3-bis(2-chloroethyl)tetrahydro-2H-l,3,2-oxazaphosphorin-2­


amine-2-oxide; isophosphamide
 


tris(l-aziridinyl)phosphine sulfide; thioTEPA
 


1-buty1-3-(p-tolylsulfonyl)urea; tolbutamide
 


4,4'-sulfonyldianiline
 


4,4'-thiodianiline
 


cholesterol (p-(bis(2-chloroethyl)amino)phenyl)acetate;
 


phenesterin
 


estradiol bis( (p-(bis(2-chloroethyl)amino)phenyl)acetate);
 


estradiol mustard
 


5
2,6 diamino-3-(phenylazo)pyridine hydrochloride
 


2-ethyl-4-pyridinecarbothioamide; ethionaraide
 


N-(2-chloroethyl)-N-(l-methyl-2-phenoxyethyl)benzylamine
 


hydrochloride; phenoxybenzamine hydrochloride
 


5-(4-chlorophenyl)-6-ethyl-2,4-pyrimidinediamine; pyrimethamine
 


4-chloro-N-((propylamino)carbonyl)benzenesulfonamide;
 


chloropropamide
 


*N-9H-fluoren-2-ylacetamide; 2-AAF
 


N-(l-methylethyl)-4-((2-methylhydrazino)methyl)benzamide
 


monohydrochloride; procarbazine hydrochloride
 


4-aceyl-N-((cyclohexylamino)carbonyl)benzenesulfonamide;
 


acetohexamide
 


4'-(9-acridinylamino)methansulfon-m-aniside monohydrochloride;
 


MAAM
 


N-(p-toluenesulfonyl)-N'-hexamethyleniminourea; tolazamide
 


2,4,6-tris(dimethylamino)-s-triazine
 


adriamycin
 


*positive control
 


E. Subchronic Studies
 


Feeding studies were conducted to estimate the maximum tolerated
 


dose (MTD) for administration in the chronic study. The low dose
 


given in the chronic study is 1/2 of the high dose. In the
 


subchronic studies, phenformin was administered in the diet to
 


female Fischer 344 rats and B6C3F1 mice for 45 days at five dose
 


concentrations. Five animals were tested at each dose.
 


Following the treatment there was a 45-day rest period, after
 


which all animals were killed and necropsied.
 


For rats doses of 300, 800, 1,500, 3,000, and 6,000 ppm were
 


used. After 84 days the gain in body weight of the treated rats
 


was 90% of that of the controls at 300 ppm; 89% at 800 ppm; 81%
 


6
at 1,500 ppm; 82% at 3,000 ppm; and 69% at 6,000 ppra. Two rats
 


died at 6,000 ppm, but no animals died at lower doses. Based on
 


these results, doses of 400 ppm and 800 ppm were selected for
 


rats in the chronic study.
 


For mice doses of 500, 1,200, 2,500, 5,000, and 10,000 ppm were
 


used. After 84 days the gain in body weight of the treated mice
 


was similar to that of the controls at 500 ppm, 66% of that of
 


the controls at 1,200 ppm, 66% at 2,500 ppm, and 58% at 5,000
 


ppm. All mice treated at 10,000 ppm died, but no mortality
 


occurred in the animals treated at lower doses. No gross
 


abnormalities were noted at necropsy. Based on these results,
 


doses of 1,200 ppm and 2,500 ppm were selected for mice in the
 


chronic study.
 


F. Design of the Chronic Study
 


The design of the chronic study, including both test and matched­


control groups, is shown in table 1. The matched controls were
 


housed in the same rooms as the test animals.
 


G. Clinical and Pathologic Examinations
 


All animals were observed for signs of toxicity twice daily and
 


were palpated for masses at each weighing. Animals appearing
 


moribund at the time of clinical examination were killed and
 


necropsied. In the chronic study, the following tissues and
 


7


Table 1. Chronic Study of Phenformin
 


Group
 


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
 


No. of
 


Animals
 


15
 


35
 


35
 


15
 


35
 


35
 


15
 


35
 


35
 


15
 


35
 


35
 


Dose
 


(ppm)
 


0



400
 


800
 


0



400
 


800
 


0



1,200
 


2,500
 


0



1,200
 


2,500
 


Time on Study
 


Treated Untreated
 


(weeks) (weeks )



78
 


78
 


78
 


78
 


78
 


78
 


78
 


78
 


78
 


78
 


78
 


78
 


26
 


26
 


26
 


26
 


26
 


26
 


26
 


26
 


26
 


26
 


26
 


26
 


8
organs were taken from killed animals and, where possible, from
 


animals found dead: brain, pituitary, lymph nodes (cervical and
 


mesenteric), thyroid, parathyroid, salivary glands, lung, heart,
 


diaphragm, stomach (pylorus and fundus), duodenum, jejunum or
 


ileum, large intestine, pancreas, adrenal gland, kidney 

(longitudinal and transverse), liver, skin, entire gonads, 

urinary bladder, prostate or uterus, and femur with marrow. 

Tissues were preserved in 10% buffered formalin, embedded in
 


paraffin, sectioned, routinely stained with hematoxylin and
 


eosin, and microscopically examined. An occasional section was
 


subjected to special staining techniques for more definitive
 


diagnosis.
 


Only positive histopathologic findings are tabulated. These are
 


based primarily on hematoxylin and eosin-stained paraffin-process
 


sections. Where possible all organs, tissues, and gross lesions
 


were evaluated for every animal as specified in the current
 


pathology protocol. A few tissues are missing from some animals,
 


particularly from early deaths, which were necropsied according
 


to earlier bioassay protocols. Also, occasional animals were
 


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


of autolysis as to preclude histopathologic interpretation and
 


may not have been preserved for such purposes. Thus, the number
 


of animals whose tissues were examined microscopically for any
 


particular organ, tissue, or lesion varies and does not
 


9


necessarily represent the number of animals that were placed on
 


experiment in each group.
 


H. Data Recording and Statistical Analyses
 


Pertinent data on this experiment have been recorded in an
 


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 gener­


ated 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
 


10
 

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
 


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
 


11
 

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 pt(l-pc)/pc(l-pt)



where pt 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).



12
 

III. RESULTS - RATS
 


A. Body Weights and Clinical Signs (Rats)
 


The average body weights of both high- and low-dose female rats
 


were consistently lower than the controls during the treatment
 


period, while body weights of the males were essentially
 


unaffected by the compound (figure 1).
 


Due to signs of respiratory disease in several animals,
 


oxytetracycline was administered in drinking water at 0.6 mg/ml
 


from week 38 to 42 and at 0.3 mg/ml from week 42 to 44. Eye
 


irritation was also reported in several treated animals. Masses
 


were found by palpation in animals from all groups, beginning at
 


week 77.
 


B. Survival (Rats)
 


There was no dose-related mortality apparent in the male rats,
 


since the control and high-dose animals experienced comparable
 


death rates and both groups had lower survival than the low-dose
 


group (figure 2); however, 65% of the high-dose group survived to
 


the end of the test. In the female rats, there were no
 


significant differences between the mortality of the different
 


groups, and 83% of the high-dose group survived to the end of the
 


13
 

Figure 1. Growth Curves for Rats Fed Phenformin in the Diet
 


14 
Figure 2. Survival Curves for Rats Fed Phenformin in the Diet
 


15 
study. The survival in all groups appears adequate to provide
 


meaningful statistical analysis.
 


C. Pathology (Rats)
 


Histopathologic findings are tabulated in Appendix A, tables
 


A1-A6, covering neoplasms and other proliferative lesions, and in
 


Appendix C, tables C1-C2, covering nontumor pathology.
 


Numerous inflammatory, degenerative, and proliferative lesions
 


commonly observed in aged Fischer rats occurred with approxi­


mately equal frequency in drug-treated and control animals.
 


These included chronic tracheitis and bronchiolitis; multifocal
 


alveolar macrophage aggregates in the lung parenchyma; chronic
 


nephritis with scarring, tubular dilatation and tubular regener­


ation; suppurative oophoritis, endometritis and cystic
 


endometrlal hyperplasia (table A2); testicular atrophy; biliary
 


hyperplasia and focal hepatocytomegaly of the liver (table A6);
 


and C—cell hyperplasia of the thyroid (table Al).
 


Other nonneoplastic proliferative lesions included
 


follicular-cell hyperplasia of the thyroid, adrenal medullary
 


hyperplasia, and islet-cell hyperplasia of the pancreas (table
 


Al); endometrial stromal hyperplasia of the uterus (table A2);
 


alveolar epithelial hyperplasia of the lung (table A3);
 


epithelial hyperplasia of the renal pelvis, ureter, bladder,
 


16
 

and/or urethra (table A4); and focal hepatocyte hyperplasia
 


(table A6).
 


Because of the current controversy regarding the nomenclature of
 


proliferative hepatocyte lesions in rodents, the definition of
 


the term "focal hepatocyte hyperplasia" used above requires
 


clarification. The term was used in this study to indicate the
 


presence in a liver section of one or more foci of hepatocytes
 


with basophilic cytoplasm and a slight increase in the amount of
 


nuclear chromatin. Many of these hepatocytes also had a slightly
 


increased nuclear:cytoplasmic ratio when compared with adjacent
 


normal hepatocytes, and infrequently mitotic figures or hepato­


cytes with double nuclei were observed. These foci of hyper­


basophilic hepatocytes were thought to represent areas of
 


hyperplasia and were coded as such in this study. They did not
 


compress adjacent hepatic parenchyma and thus were not considered
 


to represent "nodules" of hyperplasia. These lesions are similar
 


morphologically to those described by Squire and Levitt (1975) as
 


"basophilic foci".
 


The most frequently occurring neoplasm was the interstitial-cell
 


tumor of the testis, which was observed in a very high percentage
 


of sections of testes from both control and test rats (table A2).
 


These tumors were very often bilateral, often occupied a large
 


portion of the testis, and were usually accompanied by atrophy of
 


17
 

seminiferous epithelium. Pituitary adenomas were also very
 


common, especially in females (table Al). Neoplasms of thyroid
 


C-cells also occurred more frequently in females than males,
 


especially C-cell adenomas (table Al). As in many endocrine neo­


plasms, differentiation between benign and malignant C-cell
 


neoplasms was often difficult. C-cell lesions were classified as
 


adenomas when the proliferating C-cells were present in nodular
 


masses which widely separated thyroid follicles and distorted
 


normal follicular architecture. In the larger, more discrete,
 


nodular lesions, the proliferating C—cells sometimes were present
 


as interlacing bundles of enlongated, spindling cells rather than
 


the polyhedral to spherical shape characteristic of normal
 


C-cells. When invasion of the thyroid capsule, adjacent tissues,
 


or vessels was present, or when metastasis was detected, the
 


lesion was classified as C-cell carcinoma. Metastases to the
 


lung and the adrenal gland occurred in the C-cell carcinoma
 


present in a high-dose female rat.
 


Follicular-cell neoplasms occurred much less frequently than
 


C-cell neoplasms (table Al). Follicular-cell adenomas appeared
 


microscopically as well circumscribed masses composed of enlarged
 


follicles lined by hyperbasophilic follicular cells which were
 


increased in number per unit area, either by papillary infolding
 


of simple cuboidal or columnar epithelium into the follicular
 


lumen, or stratification of follicular cells surrounding the
 


18
 

lumen. Distinct compression of adjacent normal thyroid
 


parenchyma, usually 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 papil­


lary patterns were also often present. Fibrous stroma often
 


intermingled with, but did not encapsulate, the tumors. Evidence
 


of invasion of the thyroid capsule was present in both of the
 


lesions that were diagnosed as follicular-cell carcinoma.
 


Pheochromocytomas of adrenal medulla occurred in both test and
 


control rats (table Al). Differentiation between pheochromo­


cytoma and the previously mentioned adrenal medullary hyperplasia
 


was difficult in some cases. The diagnosis of pheochromocytoma
 


was made when the adrenal medullary lesion was present as a



discrete hypercellular mass that compressed adjacent normal
 


adrenal parenchyma.
 


One carcinoma and two adenomas of pancreatic islet cells were
 


present, all occurring in high-dose male rats (table Al). The
 


diagnosis of islet-cell carcinoma was based on the invasion of
 


the capsule surrounding the neoplasm, the size of the neoplasm,
 


and the anaplastic appearance of the neoplastic cells.
 


Two endometrial adenocarcinomas, two endometrial stromal
 


sarcomas, and 10 endometrial stromal polyps were observed in
 


19
 

sections of uterus (table A2). The diagnosis of adenocarcinoma
 


was based on cellular anaplasia and/or invasion of stroma and
 


muscularis. Differentiation between endometrial stromal sarcoma
 


and endometrial stromal polyp was based on cellular anaplasia,
 


the mitotic index, the size of the lesion, and the invasion of
 


adjacent tissues. Differentiation between stromal polyp and
 


stromal hyperplasia was based on the degree of cellularity and
 


the amount of discrete polypoid protrusion into the lumen of the
 


uterus.
 


Fibroadenoma of the mammary gland was quite common in the female
 


rats (table A2). Some of these neoplasms were multiple. All
 


revealed a very distinct fibrous component that contained mature
 


collagen, interspersed with a benign epithelial component. One
 


mammary carcinoma was observed in a control female rat.
 


Malignant lymphomas occurred quite frequently in all groups of
 


rats (table A5). Most of these neoplasms were composed of rather
 


undifferentiated lymphoblastic cells; in some cases, autolysis
 


prevented further classification of the neoplasm. Most involved
 


multiple organs, and the organs most frequently involved were
 


spleen, liver, thymus, and lymph nodes. In some cases, histo­


logic evidence of leukemia was present.
 


A low incidence of various other neoplasms was present, including
 


20
 

the following: alveolar/bronchiolar carcinoma of the lung (table
 


A3); nephroblastoma, hamartoma, and tubular adenoma of the kidney
 


(table A4); Schwannoma, hemangioma, and fibroma of the subcutis
 


(table A5); keratoacanthoma of the skin (table A5); carcinoma of
 


the preputial gland (table A5); mesothelioma of the peritoneum
 


(table A5); and adenocarcinoma of the small intestine (table A5).
 


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 the control groups. In the judgment of the pathologist, the
 


nature, incidence, and severity of the lesions observed provide
 


no clear evidence of carcinogenic effect from exposure to
 


phenformin.
 


D. Statistical Analyses of Results (Rats)
 


Tables 2 and 3 contain the statistical analyses of the
 


proportions of tumors at specific sites, which occurred in more
 


than 5% of a dosed group of rats. There were no statistically
 


significant positive dose-related trends. The proportions of
 


tumors in the matched controls were compared with the overall
 


proportions observed to date for controls in the bioassay program
 


at the laboratory in which the phenformin was tested
 


(lab-historic controls). The proportion of thyroid tumors in the
 


female matched controls (6/13, 46%) was significantly higher than
 


21
 

TABLE 2. Analyses of the Incidence of Primary Tumors at Specific Sites in Male Rats Fed
 


Phenformin in the Dieta



Topography: Morphology
 


Hematopoietic System: Lymphoma^
 


P Values0



95% Confidence Interval (matched)d



Weeks to First Observed Tumor
 


Reproductive System: All Tumors'3



P Values0



95% Confidence Interval (matched)d



Weeks to First Observed Tumor
 


Thyroid: C-cell Adenoma
 


or Carcinoma*5



P Values0



95% Confidence Interval (matched)d



Weeks to First Observed Tumor
 


Matched
 


Control
 


4/15(0.27)



N.S.
 


85
 


14/15(0.93)



N.S.
 


85
 


1/15(0.07)



N.S.
 


103
 


Low
 


Dose
 


4/35(0.11)



(-0.39,0.10)



90
 


35/35(1.00)



(-0.03,1.00)



84
 


3/34(0.09)



(-0.18,0.12)



105
 


High
 


Dose
 


7/34(0.21)



(-0.36,0.18)



75
 


31/34(0.89)



(-0.12,0.18)



63
 


2/32(0.06)



(-0.16,0.09)



93
 

TABLE 2. Analyses of the Incidence of Primary Tumors at Specific Sites in Male Rats Fed
 


Phenformin in the Diet3 


(continued) 


Matched Low High 


Topography: Morphology Control Dose Dose 


Pituitary: Chromophobe Adenoma^* 0/14(0.00) 6/33(0.18) 5/29(0. 17) 

P Values0 N.S. 

95% Confidence Interval (matched)0* (-0.08,1.00) (-0.09, 1.00) 

Weeks to First Observed Tumor 105 99 — 

Adrenal: Pheochromocytoma0 1/15(0.07) 3/34(0.09) 1/33(0. 03)
 


P Values0 N.S.
 


95% Confidence Interval (matched)d (-0.18,0.12) (-0.13, 0.06)
 


Weeks to First Observed Tumor 106 105 105
 


a

Dosed groups received doses of 400 and 800 ppm in feed. 

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

c

Beneath the incidence of each of the control is the probability level for the Cochran-Armitage 

test for dose-related trend in proportions when it is below 0.10, otherwise, N.S. ­

not significant. 

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

Beneath the dose group is the probability level for the Fisher exact (conditional) test 

for comparison of that dose group with the matched-control group when it is below 0.05. 

(N) A negative trend results from lower incidence in dose group(s) than in matched controls. 

"95% confidence interval of the difference in proportions of dose group and matched-control group. 
TABLE 3. Analyses of the Incidence of Primary Tumors at Specific Sites in Female Rats Fed
 


Phenformin in the Diet3



Topography: Morphology
 


Hematopoietic System: Lymphoma^5



P Values0



95% Confidence Interval (matched)^
 


Weeks to First Observed Tumor
 


Reproductive System: All Tumors**
 


P Values0



95% Confidence Interval (matched)d



Weeks to First Observed Tumor
 


Thyroid: C-cell Adenoma
 


or Carcinoma**
 


P Values0



95% Confidence Interval (matched)**
 


Weeks to First Observed Tumor
 


Matched
 


Control
 


5/15(0.33)



N.S.
 


75
 


7/15(0.47)



P = 0.027(N)
 


75
 


5/13(0.38)



N.S.
 


102
 


Low
 


Dose
 


5/34(0.15)



(-0.45,0.09)



79
 


9/34(0.26)



(-0.51,0.12)



51
 


7/33(0.21)



(-0.49,0.14)



96
 


High
 


Dose
 


7/35(0.20)



(-0.43,0.15)



71
 


6/35(0.17)



(-0.57,0.02)



101
 


7/31(0.23)



(-0.48,0.16)



105
 

TABLE 3. Analyses of the Incidence of Primary Tumors at Specific Sites in Female Rats Fed
 


Phenformin in the Dieta



(continued)
 


Matched Low High
 


Topography: Morphology Control Dose Dose
 


Pituitary: Chromophobe Adenoma*3 7/12(0.58) 14/30(0.47) 19/26(0.73)



P Values0 N.S.
 


95% Confidence Interval (matched)'1 (-0.45,0.26) (-0.20,0.50)



Weeks to First Observed Tumor 75 85 97
 


Adrenal:
 


Pheochromocytoma" 0/13(0.00) 0/34(0.00) 3/32(0.09)



P Values0 P = 0.063
 


95% Confidence Interval (matched)^ (-0.13,1.00)

 —



Weeks to First Observed Tumor ~.~_ 105
 


a

Dosed groups received doses of 400 and 800 ppm in feed.
 


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


c

Beneath the incidence of each of the control is the probability level for the Cochran-Armitage
 


test for dose-related trend in proportions when it is below 0.10, otherwise, N.S.­


not significant.
 


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


Beneath the dose group is the probability level for the Fisher exact (conditional) test
 


for comparison of that dose group with the matched-control group when it is below 0.05.



(N) A negative trend results from lower incidence in dose group(s) than in matched controls.
 


"95% confidence interval of the difference in proportions of dose group and matched-control group.
 

that in the female lab-historic controls (5/105, 5%), as also was
 


the proportion of pituitary tumors in females (7/12, 58% vs.
 


20/105, 19%); otherwise the incidence of specific tumors in the
 


matched controls was comparable to that of the lab-historic
 


controls.
 


There is also no statistical evidence of the carcinogenicity of
 


phenformin in rats at the individual dose concentrations
 


administered.
 


As an additional statistic, the 95% confidence interval was
 


calculated and entered in the tables. The implication of this
 


interval is that in 95/100 (95%) of a large number of
 


experiments, the true difference between the tumor rate for
 


treated groups of animals and the rate for the control groups
 


would be inside the interval calculated from the experiment. In
 


each of the intervals shown in the tables, zero is included; this
 


indicates the negative aspects of the results. It should also be
 


noted that each of the intervals has a positive endpoint indica­


ting the theoretical possibility of tumor induction by phenformin
 


which could not be detected under the conditions of this test.
 


26
 

IV. RESULTS - MICE
 


A. Body Weights and Clinical Signs (Mice)
 


The average body weights of both male and female mice were
 


markedly lower than controls during the first 60-75 weeks of
 


administration of compound (figure 3). Because of signs of
 


respiratory disease in some mice, oxytetracycline (0.6 mg/ml
 


during week 63 and 0.3 mg/ml during week 64) was added to the
 


drinking water, and animals were exposed to a propylene glycol
 


mist (weeks 63-64). However, between weeks 63 and 89 eight male
 


and seven female control mice died and were diagnosed as having
 


bronchopneumonia. The disease is believed to be associated with
 


the sharp declines in the mean body weights of the females at 63
 


weeks and the males at 76 weeks.
 


B. Survival (Mice)
 


No significant positive dose-related trend was seen in the
 


survival statistics of either the male or female mice. There
 


were three matched-control mice missing at 8 weeks on study, and
 


one high-dose female mouse was accidently killed at 15 weeks. In
 


both sexes, the mortality in the matched controls was excessive
 


after 75 weeks on study when compared with the dosed groups.
 


The lowest overall survival was experienced in the low-dose male
 


27
 

TIME ON STUDY (WEEKS) 

Figure 3. Growth Curves for Mice Fed Phenformin in the Diet
 


28
 

mouse group, in which 50% of the animals were dead by 74 weeks.
 


Early deaths may increase the probability of making an error by
 


the acceptance of the hypothesis of "no dose-related
 


carcinogenicity" when the hypothesis is false; therefore, the
 


test of the male mice may be inconclusive due to the limited
 


survival (figure 4).
 


C. Pathology (Mice)
 


Histopathologic findings are tabulated in Appendix B, tables
 


B1-B8, covering neoplasms and other proliferative lesions, and in
 


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


Several chronic inflammatory, degenerative, and proliferative
 


lesions frequently observed in aged laboratory mice occurred with
 


approximately equal frequency and severity in test and control
 


animals. These lesions included bronchopneumonia, suppurative
 


oophoritis, endometritis, and cystic endometrial hyperplasia
 


(table B5).
 


The incidence of proliferative lesions of the digestive tract,
 


including the liver, is summarized in table Bl. The term
 


"hepatocellular carcinoma" was used for proliferative lesions of
 


the livers in mice which, in the judgment of the pathologist, had
 


the potential or the capacity for progressive growth, invasion,
 


and metastasis and for causing the death of the host. This
 


29
 

Figure 4. Survival Curves for Mice Fed Phenformin in the Diet
 


30 
judgment was based upon the cytologic and histologic features of
 


the neoplasms and the knowledge that lesions with the same
 


morphologic characteristics have exhibited malignant biologic
 


behavior. The hepatocellular carcinomas observed in the various
 


test and control groups comprised the full spectrum of morphology
 


of this entity. Microscopically, the neoplasms varied in appear­


ance from nodules of hepatocytes with moderate deviation from
 


normal hepatic architecture to large masses of anaplastic
 


hepatocytes, with numerous mitotic figures and complete loss of
 


normal hepatic architecture. Various types of hepatocellular
 


carcinomas described in the literature were seen, including those
 


with an orderly cord-like arrangement of neoplastic cells, those
 


with a glandular pattern resembling adenocarcinoma, and those
 


composed of sheets of highly anaplastic cells with little ten­


dency to form a cord- or gland-like arrangement. Pulmonary
 


metastasis of hepatocellular carcinoma occurred in two low-dose
 


male mice.
 


Two proliferative hepatic lesions were classified as nodular
 


hyperplasia, both from male mice in the high-dose group.
 


Although both of these lesions compressed adjacent liver
 


parenchyma, variations in cellular morphology and lobular
 


architecture were not sufficient to warrant a diagnosis of neo­


plasia in either case.
 


31
 

Focal hepatocytomegaly was diagnosed in one male and one female
 


mouse, both from the high-dose group. These lesions were
 


composed of one or more foci of enlarged hepatocytes containing
 


large amounts of finely vacuolated cytoplasm. Compression of
 


adjacent hepatic parenchyma by these foci was minimal or absent.
 


Many hepatocellular carcinomas had foci of these enlarged
 


hepatocytes within the neoplasm.
 


One papilloma of squamous gastric epithelium was observed in a
 


high-dose male mouse.
 


Malignant lymphomas were observed in all groups of mice (table
 


B2). In summarizing the incidence of lymphomas, the data were
 


divided into two categories, localized and generalized. Those
 


neoplasms classified as generalized were observed in numerous
 


organs and tissues; those classified as localized involved only
 


two or three tissues. The organs most frequently involved with
 


malignant lymphoma were the mesenteric lymph nodes, liver,
 


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


of malignant lymphoma were observed, including the mixed
 


(reticulum-cell sarcoma, type B, of Dunn [1954]), the histiocytic
 


(reticulum-cell sarcoma, type A, of Dunn [1954]), and the undif­


ferentiated. In some cases, autolysis prevented subclassifi­


cation of malignant lymphomas; these were coded as malignant
 


lymphoma, N.O.S.
 


32
 

Primary pulmonary neoplasms were observed only in male mice
 


(table B3). Differentiation between adenoma and carcinoma was
 


based on the degree of anaplasia, the mi to tic index, the size of
 


the neoplasm, and the microscopic evidence of invasion of
 


adjacent pulmonary parenchyma (carcinoma), as opposed to the mere
 


compression of adjacent parenchyma and thus a more discrete
 


lesion (adenoma).
 


The incidence of neoplasms of the vasculature is summarized in
 


table B4. All lesion classified as hemangiomas were cavernous in
 


morphology, with little or no abnormal cytology of the
 


endothelial cells. The hemangiosarcomas also had areas of
 


cavernous spaces, but the endothelial cells lining these spaces
 


often had plump, hyperchromatic nuclei, and there was a gradual
 


transition to more densely cellular areas of endothelial-cell
 


proliferation, with cellular atypia and an increased nuclear:cy­


toplasmic ratio.
 


Endometrial hyperplasia was seen frequently in all three groups
 


of female mice (table B5), but only two neoplasms were present, a



stromal polyp and a leiomyoma.
 


A very low incidence of proliferative lesions was present in the
 


endocrine tissues, involving the thyroid, adrenal, and pituitary
 


glands and the interstitial cells of the testis (table 66).
 


33
 

The incidence of proliferative lesions of the urinary bladder is
 


summarized in table B7. In addition to the one cavernous
 


hemangioma (see also table B4 and the preceding paragraph on
 


neoplasms of the vasculature), proliferative lesions of the
 


epithelium of the bladder were observed in five mice. These
 


epithelial lesions had several similarities: (1) they all
 


consisted of entirely intraluminal proliferation of the tran­


sitional cells with no evidence of invasion of the muscularis;
 


(2) the epithelial cells protruding into the bladder lumen were
 


very large, spherical to columnar in shape, and had a large
 


amount of pale, eosinophilic cytoplasm and a large nucleus with a



prominent nucleolus; (3) some cells appeared to have multiple
 


nuclei; (4) the lesions were not focal, rather the entire
 


epithelial surface appeared to be involved. In one high-dose
 


male mouse the entire lumen of the bladder section was filled
 


with proliferating transitional cells; in addition, numerous
 


mitotic figures were evident. All five of these lesions were
 


classified as epithelial hyperplasia with mild dysplasia.
 


Table B8 summarizes the incidence of subcutaneous neoplasms. In
 


addition to the two vascular tumors already discussed, a malig­


nant Schwannoma was present in the subcutis in the pelvic area of
 


a high-dose female mouse. From the gross description at
 


necropsy, this lesion apparently also involved peritoneal organs;
 


although no section of this portion of the lesion was available,
 


34
 

the mass was described grossly as being located "intraperitoneal­


ly, below the bladder." Microscopically, this neoplasm was
 


composed of oval to spindle cells in dense whorls and, to a



lesser extent, in a loose, fibrillar pattern.
 


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


summary tables, where neoplastic or hyperplastic lesions occurred
 


only in test mice or with increased frequency when compared to
 


control groups. In the judgment of the pathologist the nature,
 


incidence, and severity of the lesions observed provide no clear
 


evidence of carcinogenic effect from exposure to phenformin.
 


D. Statistical Analyses of Results (Mice)
 


Tables 4 and 5 contain the analyses of the incidence of tumors at
 


any specific site where tumors were diagnosed in over 5% of any
 


dosed group. There were no additional matched-control groups
 


that had conditions suitably conformable to the matched-control
 


group of phenformin, so no pooled-control group was formed.
 


However, the proportions of particular tumors in matched-control
 


mice were comparable to the proportions seen for the same tumors
 


in all previous control mice used at the laboratory where
 


phenformin was tested (lab historic controls), except that
 


hematopoietic tumors were observed in 33% of the matched controls
 


of both male and female mice, compared to only 1.5% hematopoietic
 


tumors of the male and 5.4% of the female lab historic controls.
 


35
 

TABLE 4. Analyses of the Incidence of Primary Tumors at 

Phenformin in the Diet3 

 Specific Sites in Male Mice Fed 

Topography: Morphology
 


Lung: Alveolar/Bronchiolar
 


Adenoma or Carcinoma*5



P Values0



Departure from Linear Trend
 


95% Confidence Interval (matched)4*



Weeks to First Observed Tumor
 


Hematopoietic System: Lymphoma'3



P Values0



Departure from Linear Trend
 


95% Confidence Interval (matched)d



Weeks to First Observed Tumor
 


Matched
 


Control
 


1/14(0.07)



N.S.
 


N.S.
 


82
 


5/15(0.33)



P = 0.048(N)
 


P = 0.009
 


48
 


Low
 


Dose
 


4/29(0.13)



(-0.19,0.17)



105
 


1/35(0.03)



P = 0.007*(N)
 


(-0.40,0.05)



98
 


High
 


Dose
 


1/33(0.03)



(-0.14,0.06)



104
 


3/35(0.09)



P = 0.043*(N)
 


(-0.46,0.02)



99
 

TABLE 4. Analyses of the Incidence of Primary Tumors at Specific Sites in Male Mice Fed
 


(continued)
 


Topography: Morphology
 


All Sites: Hemangioma or
 


Hemangio sarcoma"
 


P Values0



Departure from Linear Trend
 


95% Confidence Interval (matched)^
 


Weeks to First Observed Tumor
 


Liver: Hepatocellular Carcinoma*3



P Values0



Departure from Linear Trend
 


95% Confidence Interval (matched)^
 


Weeks to First Observed Tumor
 


Phenformin in the Dieta 

Matched Low High 

Control Dose Dose 

1/15(0.07) 1/35(0.03) 2/35(0. 06) 

N.S. 

N.S. 

(-0.13,0.06) (-0.16, 0.08) 

82 105 105 

1/14(0.07) 6/27(0.22) 4/33(0. 12) 

N.S. 

N.S. 

(-0.15,0.26) (-0.20, 0.15) 

87 78 104 

a

Dosed groups received 1,200 and 2,500 ppm in feed.
 


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

TABLE 4. Analyses of the Incidence of Primary Tumors at Specific Sites in Male Mice Fed
 


Phenformin in the Dieta



(continued)
 


c

Beneath the incidence of each of the controls is the probability level for the Cochran-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 the
 


comparison of that dose group with the matched-control group (*) when it is below 0.05.
 


(N) A negative trend results from lower incidence in dosed group(s) than in matched controls.
 


°95% confidence interval of the difference in proportions of dose group and matched-control group.
 

TABLE 5. Analyses of the Incidence of Primary Tumors at Specific Sites in Female Mice Fed

 

Phenformin in the Diet3

Matched Low High 

Topography: Morphology Control Dose Dose 

Hematopoietic System: Lymphoma^ 4/12(0.33) 7/33(0.21) 3/33(0.09) 

P Values0 P = 0.049(N) 

Departure from Linear Trend N.S. 

95% Confidence Interval (matched)** (-0.46,0.17) (-0.48,0.04) 

Weeks to First Observed Tumor 86 86 90 

All Sites: Hemangioma or
 


Hemangiosarcoma^ 1/12(0.08) 3/33(0.09) 2/33(0.06)



P Values0 N.S.
 


Departure from Linear Trend N.S.
 


95% Confidence Interval (matched)d (-0.23,0.12) (-0.21,0.08)



Weeks to First Observed Tumor 89 104
 

TABLE 5. Analyses of the Incidence of Primary Tumors at Specific Sites in Female Mice Fed
 


Phenformin in the Diet3



(continued) 

Matched Low High 

Topography: Morphology Control Dose Dose 

Liver: Hepatocellular Carcinoma^3 1/10(0.10) 3/29(0.10) 2/33(0. 06) 

P Values0 N.S. 

Departure from Linear Trend N.S.
 


95% Confidence Interval (matched)** (-0.28,0.13) (-0.26, 0.09)
 


Weeks to First Observed Tumor 102 105 88
 


a

Dosed groups received 1,200 and 2,500 ppm in feed.
 


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


c

Beneath the incidence of each of the controls is the probability level for the Cochran-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 the
 


comparison of that dose group with the matched-control group (*) when it is below 0.05.
 


(N) A negative trend results from lower incidence in dosed group(s) than in matched controls.
 


^95% confidence interval of the difference in proportions of dose group and matched-control group.
 

The dosed groups did not exhibit as high proportions of
 


hematopoietic tumors as those of the matched-control groups, with
 


the result that the females showed a negative linear trend (P =



0.038) and the males showed a departure from negative linear
 


trend (P = 0.009) with significant differences between each of
 


the dosed groups and the matched control. There is no
 


statistical evidence in the data that phenformin is carcinogenic
 


to mice at the dose levels given.
 


As an additional statistical test, the 95% confidence intervals
 


were calculated and are entered in the tables. The implication
 


of these intervals is that in 95/100 (95%) of a large number of
 


experiments the true difference between the treatment tumor rate
 


and the control tumor rate would be inside the interval
 


calculated from the experiment. In each of the intervals shown
 


in the table, zero is included; this indicates the negative
 


aspects of the results. It should be noted that each of the
 


intervals has a positive endpoint indicating the theoretical
 


possibility of tumor induction by phenformin which could not be
 


detected by this test.
 


41
 


V. DISCUSSION
 


The phenformin used in this study was stored at 5°C, and there is
 


no reason to suspect instability. However, no actual analysis of
 


the dosed feed for concentration of phenformin was performed
 


during the study. The doses of phenformin used in this study
 


were slightly toxic, since comparisons of average group weights
 


(figures 1 and 3) show that, except in male rats, the treated
 


animals on either high or low doses of the compound gained less
 


weight than the corresponding controls. Differences in weight
 


gain between high- and low-dose animals were much less than
 


differences between low-dose animals and controls. Survival was
 


not dose-related for either sex of either species; however,
 


because there were excessive numbers of deaths in mice,
 


particularly males, these animals may not have been at risk long
 


enough to develop any possible latent tumors. The possibility of
 


an effect on tumor incidence of treatment of the mice with
 


oxytetracycline at weeks 63-64 to control respiratory disease is
 


not known; however, no mechanism is immediately apparent by means
 


of which the antibiotic might be expected to exert an adverse
 


effect on the reliability of the findings.
 


Various types of tumors were observed in the treated animals, but
 


the incidences were not significantly different from those for
 


43
 

the same types seen in control animals. Thus, tumors appearing
 


in hematopoietic systems, thyroids, and pituitaries of treated
 


rats also appeared in untreated rats at the same sites and at
 


insignificantly different rates of incidence. Similarly, tumors
 


appearing in the respiratory systems, circulatory systems, and
 


livers of treated mice also appeared in untreated mice at the
 


same sites and at essentially the same incidences.
 


The observed failure of phenformin to induce tumors when
 


administered to rats and mice extends the negative findings of an
 


earlier report (Stoner et al., 1973) in which the compound was
 


administered to mice at a level of 12.5-62.5 mg/kg/day by intra­


peritioneal injections three times per week for 8 weeks. In the
 


present work, dosages for mice calculated from food consumption
 


were 300-625 mg/kg/day administered in the diet five times per
 


week for 78 weeks. In addition, Dil'man et al., (1974) reported
 


that the incidence of DMBA-induced mammary tumors in 16 female
 


rats that were force-fed phenformin (3 benign tumors; 9 highly
 


differentiated tumors; 1 other type) was much less than that of
 


16 controls treated with DMBA but not phenformin (6 adenomas; 35
 


adenocarcinomas; 3 other types).
 


Thus, there is no evidence that under the conditions of this
 


bioassay phenformin was carcinogenic. However, the stability of
 


the test material in feed was not determined. Furthermore, the
 


44
 

tests performed in male mice may be inconclusive, since early
 


deaths increase the probability of failing to discover carcino­


genicity even when it exists. It should be noted that the
 


confidence intervals for all tumor sites in rats and mice which
 


were subjected to statistical analysis include a positive value
 


indicating the theoretical possibility of tumor induction by
 


phenformin which could not be detected under the conditions of
 


this test.
 


45 

VI. BIBLIOGRAPHY
 


Armitage, P. Statistical Methods in Medical Research, John Wiley
 


and Sons, New York, 1971, p. 135.
 


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


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


Geneva, 1969.
 


Cox, D. R. Analysis of Binary Data, Methuen and Co., Ltd.,
 


London, 1970, p. 62.
 


Dil'man, V. M., Bershtein, L. M., Zabezhinsky, M. A., and
 


Alexandrov, V. A. Concerning the effects of phenphormin on
 


induction of breast tumors in rats. Voprosy. Onkol.
 


_20(9):94-98, 1974.
 


Dunn, T. Normal and pathologic anatomy of the reticular tissue
 


in laboratory mice. J. Natl. Cancer Inst. 14:1281-1433,
 


1954.
 


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


ratio in the combination of 2 x 2 tables with fixed
 


marginals. Biometrika ^7:471-475, 1970.
 


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


Therapeutics., Macmillan Publishing Co., Inc., New York,
 


1975.
 


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


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


Comp. BJoined. Res. 7^:230-2^8, 1974.
 


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


McGraw-Hill, New York, 1966, p. 8.
 


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


classification of specific hepatocellular lesions in rats.
 


Cancer Res. ^5j3214, 1975.
 


Stoner, G. D., Shimkin, M. B., Kniazeff, A. J., Weisburger, J.
 


H., Weisburger, E. K., and Gori, G. B. Test for carcinogeni­


city of food additives and chemotherapeutics agents by the
 


pulmonary tumor response in strain A mice. Cancer Res.
 


13:3069-3085, 1973.
 


47
 


APPENDIX A



SUMMARY OF THE INCIDENCE OF NEOPLASMS
 


AND OTHER PROLIFERATIVE LESIONS
 


IN RATS FED PHENFORMIN IN THE DIET
 


49
 


TABLE Al 

PROLIFERATIVE LESIONS OF THE ENDOCRINE SYSTEM 

THYROID
 


Follicular-cell
 


Carcinoma
 


Follicular-cell
 


Adenoma
 


C-cell Carcinoma
 


C-cell Adenoma
 


Follicular-cell
 


Hyperplasia
 


C-cell Hyperplasia
 


ADRENAL
 


Pheochromocytoma
 


Hyperplasia
 


Adrenal Medulla
 


Control
 

0/15
 


0/15
 


1/15
 


(7%)
 


0/15
 


0/15
 


4/15
 


(27%)
 


1/15
 


(7%)
 


2/15
 


(13%)
 


MALE RATS 


 Low Dose
 


0/34
 


0/35
 


0/35
 


3/35
 


(8.6%)
 


1/35
 


(3%)
 


25/35
 


(71%)
 


3/34
 


(8.6%)
 


6/35
 


(17%)
 


High Dose
 


1/32
 


(3%)
 


1/32
 


(3%)
 


1/32
 


(3%)
 


1/32
 


(3%)
 


2/32
 


(6%)
 


14/32
 


(44%)
 


1/33
 


(3%)
 


1/33
 


(3%)
 


High Dose
 


1/31
 


(3%)
 


0/34
 


1/34
 


(3%)
 


6/34
 


(18%)
 


9/34
 


(3%)
 


21/34
 


(62%)
 


3/32
 


(9%)
 


0/35
 


Control
 

0/13
 


1/15
 


(7%)
 


0/15
 


5/15
 


(33%)
 


0/15
 


10/154
 


(67%)
 


0/13
 


2/15
 


(13%)
 


FEMALE RATS
 


 Low Dose
 


0/33
 


0/33
 


2/33
 


(6%)
 


5/33
 


(15%)
 


0/33
 


16/33
 


(48%)
 


0/34
 


2/34
 


(6%)
 

TABLE Al 

PROLIFERATIVE LESIONS OF THE ENDOCRINE SYSTEM 

(continued) 

MALE RATS 

Control Low Dose High Dose 

FEMALE RATS 

Control Low Dose High Dose 

PITUITARY 

Adenoma 0/14 6/33 

(18%) 

7/29 

(24%) 

7/12 

(58%) 

14/30 

(47%) 

19/26 

(73%) 

PANCREAS 

Islet-cell Carcinoma 0/15 0/32 1/32 0/12 0/34 0/30 

Islet-cell Adenoma 0/15 0/33 2/32 

(6%) 

0/14 0/34 0/33 

Islet-cell Hyperplasia 1/15 

(7%) 

0/33 0/32 0/14 1/34 

(3%) 

0/33 
TESTIS
 


Interstitial-cell
 


Tumor
 


UTERUS
 


Adenocarcinoma
 


Endometrial Stroraal
 


Sarcoma
 


Endometrial Stromal
 


Polyp
 


Endometrial
 


Hyperplasia
 


Endometrial Stromal
 


Hyperplasia
 


MAMMARY GLAND
 


Adenocarcinoma
 


Fibroadenoma
 


Fibroma
 


TABLE A2 

PROLIFERATIVE LESIONS OF THE 

REPRODUCTIVE SYSTEM AND MAMMARY GLAND 

MALE RATS FEMALE RATS 

Control Low Dose High Dose Control Low Dose High Dose 

14/15 34/34 31/34 

(93%) (100%) (91%) 

0/11 0/34 2/31 

(6%) 

1/13 0/34 1/33 

(8%) (3%) 

4/13 4/34 2/33 

(31%) (12%) (6%) 

3/13 5/34 5/33 

(23%) (15%) (15%) 

0/13 2/34 1/33 

(6%) (3%) 

0/15 0/32 0/28 1/13 0/30 0/31 

(8%) 

0/15 0/32 1/28 3/15 5/34 3/35 

(4%) (20%) (15%) (9%) 

0/15 0/32 0/28 0/15 0/34 1/35 

(3%) 
TABLE A3 

PROLIFERATIVE LESIONS OF THE 

RESPIRATORY TRACT 

MALE RATS FEMALE RATS 

Control Low Dose High Dose Control Low Dose High Dose 

LUNG 

Alveolar /Bronchiolar 0/15 0/34 0/34 1/13 0/34 1/32 

Carcinoma (8%) (3%) 

Alveolar Epithelial 1/15 3/35 0/33 0/15 0/34 0/35 

Hyperplasia (7%) (9%) 
KIDNEY 


Nephroblastoma 


Hamartoma 


Tubular Adenoma 


Renal Pelvis/Ureter 


Hyperplasia 


BLADDER/Urethra 


Hyperplasia 


TABLE A4
 


PROLIFERATIVE LESIONS OF THE
 


URINARY TRACT
 


MALE RATS 


Control Low Dose High Dose 


0/15 0/34 	 0/34 


1/15 0/35 	 0/34 


(7%) 


0/15 0/35 	 1/34 


(3%) 


3/15 6/35 	 5/34 


(20%) (17%) 	 (15%) 


0/13 	 1/31 1/22 


(3%) (5%) 


FEMALE RATS 


Control Low Dose 


0/12 	 1/34 


(3%) 


0/11 	 0/32 


0/11 	 0/32 


2/11 	 1/32 


(18%) 	 (3%) 


1/10 	 1/32 


(10%) 	 (3%) 


High Dose 


0/32 


0/35 


0/35 


0/35 


1/29 


(3%) 

TABLE A5
 


MISCELLANEOUS PROLIFERATIVE LESIONS
 


MALE RATS FEMALE RATS 

Control Low Dose High Dose Control Low Dose High Dose 

HEMATOPOIETIC SYSTEM 

Malignant Lymphoma 4/15 4/35 7/34 5/15 5/34 7/35 

(27%) (11%) (21%) (33%) (15%) (20%) 

SKIN/SUBCUTIS 

Preputial Gland 0/15 1/35 1/34 

Carcinoma (3%) (3%) 

Schwannoma 1/15 0/35 0/34 0/15 0/34 0/35 

(7%) 

Hemangioma 0/15 0/35 2/34 0/15 0/34 1/35 

(6%) (3%) 

Fibroma 0/15 1/35 1/34 0/15 0/34 0/35 

(3%) (3%) 

Keratoacanthoma 1/15 0/35 0/34 0/15 0/34 0/35 

(7%) 

Carcinoma, N.O.S. 0/15 0/35 0/34 1/15 0/34 0/35 

(7%) 

Poorly Differentiated 0/15 0/35 0/34 0/15 1/34 0/35 

Adenocarcinoma (3%) 
TABLE A5 

MISCELLANEOUS PROLIFERATIVE LESIONS 

(continued) 

MALE RATS FEMALE RATS 


Control Low Dose High Dose Control Low Dose High Dose 


PERITONEUM 


Mesothelioma 0/15 0/35 1/34 0/15 0/34 0/35 


(3%) 


SMALL INTESTINE 


Adenocarcinoma 0/15 0/34 0/31 0/11 1/32 0/32 


(3%) 


HEART 


Sarcoma, N.O.S. 0/15 0/34 1/33 0/13 0/33 0/32 


(3%) 


MEDIASTINUM 


Osteogenic Sarcoma 0/15 0/35 0/34 0/15 1/34 0/35 


(3%) 

TABLE A6 

PROLIFERATIVE LESIONS OF THE LIVER 

MALE RATS 

Control Low Dose High Dose 

FEMALE RATS 

Control Low Dose High Dose 

HEPATOCYTES 

Focal Hyperplasia 8/15 

(53%) 

3/35 

(9%) 

13/34 

(38%) 

8/15 

(53%) 

28/34 

(82%) 

14/35 

(40%) 

Hepatocytomegaly 3/15 

(20%) 

6/35 

(17%) 

3/34 

(9%) 

1/15 

(7%) 

1/34 

(3%) 

2/35 

(6%) 

BILE DUCTS 

Hyperplasia 12/15 

(80%) 

23/35 

(66%) 

11/34 

(32%) 

5/15 

(33%) 

8/34 

(23.5%) 

4/35 

(11%) 
APPENDIX B



SUMMARY OF THE INCIDENCE OF NEOPLASMS
 


AND OTHER PROLIFERATIVE LESIONS
 


IN MICE FED PHENFORMIN IN THE DIET
 


59
 


LIVER
 


He pa to cellular
 


Carcinoma
 


Nodular Hyperplasia
 


Focal
 


Hepatocytomegally
 


Focal Angiectasis
 


STOMACH
 


Papilloma, Squamous
 


Epithelium
 


ANUS
 


Pseudoepitheliomatous
 


Hyperplasia
 


TABLE Bl
 


PROLIFERATIVE LESIONS OF THE
 


DIGESTIVE SYSTEM
 


MALE MICE
 


Control Low Dose High Dose
 


1/14 6/27	 	 4/33
 


(7%) (22%)	 	 (12%)
 


0/14 0/27	 	 2/33
 


(6%)
 


0/14 0/27	 	 1/33
 


(3%)
 


1/14 0/27	 	 1/33
 


(7%)	 	 (3%)
 


0/13 0/24	 	 1/30
 


(3%)
 


0/14 0/29	 	 0/33
 


High Dose
 


2/33
 


(6%)
 


0/33
 


1/33
 


(3%)
 


0/33
 


0/33
 


1/33
 


(3%)
 


Control
 


1/10
 


(10%)
 


0/10
 


0/10
 


0/10
 


0/11
 


0/12
 


FEMALE MICE
 


Low Dose
 


3/29
 


(10%)
 


0/30
 


0/30
 


0/30
 


0/30
 


0/33
 

TABLE B2
 


NEOPLASMS OF THE HEMATOPOIETIC SYSTEM
 


MALE MICE FEMALE MICE
 

Control Low Dose High Dose Control Low Dose High Dose 

Malignant Lymph oma, 2/14 1/29 2/33 3/12 4/33 1/33 

Generalized* (14%) (3%) (6%) (25%) (12%) (3%) 

Malignant Lymphoma, 3/14 0/29 1/33 1/12 3/33 2/33 

Localized* (21%) (3%) (8%) (9%) (6%) 

TABLE B3 

PRIMARY PULMONARY NEOPLASMS 

MALE MICE FEMALE MICE 

Control Low Dose High Dose Control Low Dose High Dose 

LUNG 

Alveolar /Bronchiolar 1/14 1/29 1/33 0/12 0/30 0/32 

Carcinoma (7%) (3%) (3%) 

Alveolar /Bronchiolar 0/14 3/29 0/33 0/12 0/31 0/32 

Adenoma (10%) 

*The tabulation of the incidence of this neoplasm is divided into those animals in which
 


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


neoplasm was observed in only a few organs. The number of animals necropsied is used as
 


the denominator.
 

SPLEEN 


Hemangiosarcoma 


SUBCUTIS 


Hemangiosarcoma 


Cavernous Hemangioma 


HEART 


Hemangiosarcoma 


MESENTERY 


Cavernous Hemangioma 


TABLE B4
 


NEOPLASMS OF THE VASCULATURE
 


MALE MICE 


Control Low Dose High Dose 


1/14 1/27 	 0/30 


(7%) 	 (4%) 


1/14 0/29 0/33 


(7%) 


0/14 0/29 0/33 


0/14 0/28 	 1/33 


(3%) 


0/14 0/29 	 0/33 


FEMALE MICE 


Control Low Dose 


0/11 1/27 


(4%) 


0/12 0/33 


1/12 0/33 


(8%) 


0/11 	 0/31 


0/12 	 1/33 


(3%) 


High Dose 


0/32 


0/33 


0/33 


0/32 


0/33 

(continued)
 


STERNUM
 


Cavernous Hemangioma
 


BLADDER
 


Cavernous Hemangioma
 


PANCREAS
 


Hemangioma
 


Total Malignant
 


Vascular Neoplasms
 


Total Benign
 


Vascular Neoplasms
 


TABLE B4
 


NEOPLASMS	 OF THE VASCULATURE
 


MALE MICE
 


Control Low Dose High Dose
 


0/14	 	 1/29 0/33
 


(3%)
 


0/14 0/24	 	 1/28
 


(4%)
 


0/14	 	 0/24 0/28
 


2/14	 	 1/29 1/33
 


(14%)	 	 (3%) (3%)
 


0/14	 	 1/29 1/33
 


(3%) (3%)
 


High Dose
 


0/33
 


0/30
 


0/30
 


0/33
 


0/33
 


Control
 


0/12
 


0/10
 


0/10
 


0/12
 


1/12
 


(8%)
 


FEMALE MICE
 


Low Dose
 


0/33
 


0/27
 


1/27
 


(4%)
 


1/33
 


(3%)
 


2/33
 


(6%)
 

TABLE B5
 


PROLIFERATIVE LESIONS OF THE
 


REPRODUCTIVE SYSTEM
 


MALE MICE FEMALE MICE
 


Control Low Dose High Dose Control Low Dose High Dose 


Teratoma, Ovary
 0/11 	 1/28 0/32 


(4%) 


Endometrial Stromal
 0/12 0/27 1/33 


Polyp
 (3%) 


Leiomyoma, Uterus
 0/12 	 1/27 0/33 


(4%) 


Endometrial Hyperplasia
 7/12 9/27 12/33 


(58%) (33%) (36%) 

THYROID
 


Follicular-cell
 


Adenoma
 


Follicular-cell
 


Hyperplasia
 


C-cell Hyperplasia
 


ADRENAL
 


Focal Hyperplasia,
 


Medulla
 


PITUITARY
 


Chromophobe Adenoma
 


TESTIS
 


Interstitial-cell
 


Tumor
 


TABLE B6
 


PROLIFERATIVE LESIONS	 OF THE ENDOCRINE SYSTEM
 


MALE MICE FEMALE MICE
 


Control Low Dose High Dose Control Low Dose
 


0/13 0/28 0/32	 	 1/12 0/30
 


(8%)
 


0/13 0/28	 	 2/32 1/12 1/31
 


(6%) (8%) (3%)
 


0/13	 	 2/28 4/32 0/12 0/31
 


(7%) (13%)
 


0/13 0/27 0/33	 	 1/12 0/30
 


(8%)
 


0/6 0/9 0/19	 	 1/9 0/18
 


(11%)
 


1/14 0/28	 	 1/33
 


(7%)	 	 (3%)
 


High Dose
 


2/33
 


1/33
 


(3%)
 


0/33
 


0/31
 


0/21
 

TABLE B7 

PROLIFERATIVE LESIONS OF THE 

URINARY BLADDER 

MALE MICE 

Control Low Dose High Dose 

FEMALE MICE 

Control Low Dose High Dose 

Cavernous Hemangioma* 0/14 0/24 1/28 

(4%) 

0/10 0/27 0/30 

Epithelial Hyperplasia 

and Dysplasia 

1/14 

(7%) 

1/24 

(4%) 

1/28 

(4%) 

0/10 0/27 2/30 

(7%) 

*This lesion was also tabulated in Table B4, Neoplasms of the Vasculature. 
TABLE B8 

SUBCUTANEOUS NEOPLASMS 

MALE MICE FEMALE MICE 

Control Low Dose High Dose Control Low Dose High Dose 

Hemangiosarcoma* 1/14 0/29 0/33 0/12 0/33 0/33 

(7%) 

Malignant Schwannoma 0/14 0/29 0/33 0/12 0/33 1/33 

(3%) 

Cavernous Hemangioma* 0/14 0/29 0/33 1/12 0/33 0/33 

(8%) 

Adenocarcinoma, N.O.S. 0/14 0/29 0/33 0/12 1/33 0/33 

(3%) 

Zymbal's Gland 0/14 0/29 0/33 0/12 1/33 0/33 

Carcinoma (3%) 

*This lesion was also tabulated in Table B4, Neoplasms of the Vasculature. 
APPENDIX C



SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 


LESIONS IN RATS FED PHENFORMIN
 


IN THE DIET
 


69
 


TABLE C1

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS 

IN MALE RATS FED PHENFORMIN IN THE DIET 

*SYSTEM PERCENTAGES ARE BASED ON THE NUMBER OF ifiI&itS._!iSC£2P.§II2 

71 

HIGH DOSE
 


35
 


34(100*)
 


34
 


34 (100*)
 


23 (68X)
 


19
 


8



11
 


4



4



6



1



3



2



2



2



3 (9X)
 


2



1



ANIMALS INITIALLY IN 51001 


ANIMALS NECROPSIED 


ANIMALS EXAMINED HISTOP&THOLOGICALLY 


ANIHALS KITH NON-TUMOR PATHOLOGY 


INTEGUMENTARY SYSTEM
 


NONE
 


RESPIRATORY SYSTEM 


TRACHEA 


INFLAMMATION SUPPURATIVE 


INFLAMMATION CHRONIC 


LUNG/BRONCHOS 


BRONCHIECTASIS 


INFLAMMATION SUPPORATIVE 


METAPLASIA SQUAHOUS 


LUNG/BRONCHIOLE 


INFLAMMATION SUPPURATIVE 


HYPERPLASIA EPITHELIAL 


LUNG 


CONGESTION 


HEMORRHAGE
 


BRONCHOPNEUHONIA 


PIGMENTATION 


CYTOMEGALY 


ALVEOLAR MACROPHAGES 


HYPEHPLASIA ALVEOLAR-CELL 


LUNG/ALVEOLI 


EMPHYSEMA 


INFLAMMATION SUPPURATIVE
 


HEHATOPOIETIC SYSTEM 


SPLEEN
 


LYMPHOID HY.PERPLA§IA
 


CONTROL LOW DOSE
 


15 35
 


15(100%) 35 (100X)
 


15 35
 


15 (100X) 35 (100X)
 


11 (73%) 29 (83X)
 


7 28
 


7 25
 


3



1 1



1



1



1



2 2



2 1



1



7 9



3 3



1



1



1



3 4



1 2



1



1



2 (13X) 1 (3X)
 

TABLE C1 MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

HEMATOPOIESIS
 


SPLENIC CAPSULE
 


FIBEOSIS FOCAL
 


LYMPH NODE
 


DILATATION
 


LYMPHOID HYPERPLASIA
 


MANDIBULAR L. NODE
 


LYHPHOID HYPERPLASIA
 


CIRCULATORY SYSTEM
 


MYOCARDIUM
 


HEMORRHAGE
 


INFLAMMATION SUPPORATIVE
 


FIBROSIS
 


METAPLASIA OSSEOUS
 


ENDOCARDIUM
 


INFLAMMATION SUPPURATIVE
 


DIGESTIVE SYSTEM
 


LIVER
 


NECROSIS FOCAL
 


HEPATOCYTOMEGALY
 


HYPERPLASIA FOCAL
 


ANGIECTASIS
 


BILE DUCT
 


FIBROSIS
 


HYPERPLASIA
 


PANCREAS
 


FIBROSIS
 


FIBROSIS FOCAL
 


FIBROSIS DIFFUSE
 


PERIARTERITIS
 


PANCREATIC ACINUS
 


ATROPHY
 


ATROPHY FOCAL
 


*SYSTEM PERCENTAGES ARE BASED ON 

CONTROL LOW DOSE
 HIGH DOSE
 


1



1



1



1
 1



1



1



1



1



9 (60X) 17 (49X)
 8 (2UX)
 


9 17
 8



1



1



8 17
 7



1



1



1



12 (SOX) 27 (77X)
 24 (71X)
 


9 7
 15
 


1



3 6
 3



8 3
 13
 


1



12 23
 11
 


2 3



12 23
 11
 


1 1
 2



1



1



1



1



2



1



1



 THE NUMBER OF AN!NJIL.S__NECJROPSIED 

72 
TABLE C1 MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

ESOPHAGUS
 


INFLAMMATION SUPPORATIVE
 


STOMACH
 


INFLAMMATION ACUTE
 


GASTRIC MUCOSA
 


ABSCESS
 


EROSIVE INFLAMMATION
 


SMALL INTESTINE
 


INFLAMMATION SUPPURATIVE
 


SMALL INTESTINAL SBR
 


HEMORRHAGE
 


LARGE INTESTINE
 


NEMATODIASIS
 


URINARY SYSTEM
 


KIDNEY
 


INFLAMMATION CHRONIC
 


HYPERPLASIA TUBULAR-CELL
 


KIDNEY/PELVIS
 


HYPERPLASIA EPITHELIAL
 


URINARY BLADDER
 


CONGESTION
 


URETHRA
 


CALCULUS
 


HYPERPLASIA EPITHELIAL
 


ENDOCRINE SYSTEM
 


ADRENAL
 


METAMORPHOSIS FATTY
 


ADRENAL CORTEX
 


HYPERPLASIA FOCAL
 


ADRENAL MEDULLA
 HIPEBEWSJU
 


CONTROL 


1



1



LOU DOSE HIGH DOSE
 


1



1



1



1



1 1



1



1



1



1



1



1



14 (93%) 35 (100*) 30 (88*)
 


14 35 30
 


14 35 30
 


1



3 6 5



3 6 5



1



1



2 1



1



1 1



6 (40*) 26 (74*) 21 (62%)
 


1



1



1



1



2 6 1



. I.... 2 1



*SYSTSM PERCENTAGES ARE BASED ON THE NUMBER OF ANIflAL.S_N.E.CHOPSIED
 


73
 

TABLE C1 MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

HYPERPLASIA FOCAL 

THYROI D 

ULTIMOBRANCHIAL CYST 

HYPERPLASI A C-CELL 

HYPERPLASIA FOLLICULAR-CELL 

PARATHYROID 

HYPERPLASI A 

PANCREATIC ISLETS 

HYPERPLASIA 

REPRODUCTIVE SYSTEM 

MAMMAR Y GLAN D 

GALACTOCELE 

CYST 

PREPUTIAL GLAND 

CYST 

CYSTIC DUCTS 

ABSCESS 

HYPERPLASI A 

PROSTATE 

INFLAMMATIO N SUPPURATIV E 

PERIARTBRITIS 

TESTIS 

ATROPHY 

NERVOU S SYSTEM 

BHAIN/MENINGE S 

FIBPOSIS 

CEREBRU M 

HEMORRHAGE 

BRAIN 

HEMORRHAGE 

CEREBELLUM 

NECROSIS FOCAL 

5Efi£IAL._5£fiaE_fiaSA.llS 

fSYSTEM PERCENTAGES ARE BASED ON THE 

CONTROL i 

1 

U 

3 

3 

1 

1 

12 (SOX) 

4 

3 

1 

11 

11 

2 (13X) 

2 

2 

 NUMBER OF 

74 

LOW DOSE 

4 

25 

25 

1 

1 

1 

32 (91X) 

3 

2 

1 

1 

1 

1 

6 

6 

30 

30 

1 (3X) 

1 

1 

i 

2_liiL 

 A.N.I{LAL.S._N,£C£2P.S.igD 

HIGH DOSE 

16 

1 

14 

2 

U 

4 

30 (88X) 

1 

1 

2 

1 

1 

2 

2 

30 

30 

3 (9») 

1 

1 

1 

1 

1 

1 

l_lliL__. 
TABLE C1 MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW POSE HIGH DOSE 

EYE/CRYSTALLINE LENS 2 

MINERALIZATION 2 

EYELID 1 

INFLAMMATION SOPPURATIVE 1 

EAR CANAL 1 

INFLAMMATION SUPPORATIVE 1 

HYPERKERATOSIS 1 

MUSCULOSKELETAL SYSTEM 1 (7*) 

SKELETAL MUSCLE 1 

HEMORRHAGE 1 

BODY CAVITIES 

NONE 

ALL OTHER SYSTEMS 1 (3X) 

ADIPOSE TISSUE 1 

INFLAMMATION 1 

SPECIAL MORPHOLOGY SUMMARY 

NO NECROPSY PERFORMED 1 

75
 

TABLE C2

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS 

IN FEMALE RATS FED PHENFORMIN IN THE DIET 

ANIMALS INITIALLY IN STUDY
 


ANIMALS NECROPSIED
 


ANIMALS EXAMINED HISTOPATHOLOGICALLY
 


ANIMALS WITH NON-TUMOR PATHOLOGY
 


INTEGUMENTARY SYSTEM
 


SKIN
 


ULCER FOCAL
 


RESPIRATORY SYSTEM
 


TRACHEA
 


INFLAMMATION SUPPURATIVE
 


INFLAMMATION CHRONIC
 


LUNG/BRONCHUS
 


BRONCHIECTASIS
 


LUNG/BRONCHIOLE
 


INFLAMMATION SUPPURATIVE
 


HYPERPLASIA EPITHELIAL
 


LUNG
 


CONGESTION
 


HEMORRHAGE
 


ALVEOLAR MACROPHAGES
 


HEMATOPOIETIC SYSTEM
 


BONE MARROW
 


HYPERPLASIA HEMATOPOIETIC
 


SPLEEN
 


FIBROSIS FOCAL
 


HEMATOPOIESIS
 


CIRCULATORY SYSTEM
 


MYOCARDIUM
 


INFLAMMATION
 


*SYSTEM PERCENTAGE S ARE BASE D O N TH E 

HIGH DOSE
 


35
 


35(100%)
 


35
 


32 (91%)
 


16 (46%)
 


15
 


12
 


3



1



1



1 (3X)
 


1



1



3 (9%)
 


3



CONTROL
 


15
 


15(100%)
 


15
 


1U (93X)
 


12 (80%)
 


10
 


6



4



1



1



4



1



2



1



3 (20%)
 


1



1



3



3



"* (27%)
 


i*
 


LOH DOSE
 


35
 


3« (1008)
 


34
 


33 (97%)
 


1 (3%)
 


1



1



22 (65%)
 


18
 


11
 


7



1



1



3



3



1



8



6



1



2



3 (9%)
 


3



3



6 (18%)
 


6



1



 NUMBE R O F ANIMAL.S_NECROPSIED 

76 
TABLE C2 FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE
 


FIBROSIS 4 5 3



DIGESTIVE SYSTEM 13 (87%) 32 (94S) 19 (54X)
 


LIVER 10 29 16
 


GRANULCMA 1



NECROSIS FOCAL 1 1



METAMORPHOSIS FATTY 2



HEPATOCYTOMEGALY 1 1 2



HYPERPLASIA FOCAL 8 28 14
 


LIVER/CENTRILOBULAR 1



NECROSIS FOCAL 1



BILE DUCT 5 8 5



FIBROSIS 1



HYPERPLASIA 5 8 4



PANCREAS 1 4 3



FIBROSIS 1 4 3



PANCREATIC ACINUS 3 3



ATROPHY 3 3



STOMACH 2



INFLAMMATION 1



ULCER FOCAL 1



URINARY SYSTEM 12 (80%) 29 (85%) 25 (71%)
 


KIDNEY 10 28 25
 


INFLAMMATION CHRONIC 10 28 25
 


KIDNEY/TUBULE 1



PIGMENTATION 1



KIDNEY/PELVIS 1 1



HYPERPLASIA EPITHELIAL 1 1



URETER 1



HYPEHPLASIA EPITHELIAL 1



URINARY BLADDER 1 1 1



HYPERPLASIA EPITHE.LIAL 1 1 1



*SYSTEM PERCENTAGE S ARE BASE D O N TH E NUMBE R O F ANIMALS_NECROPSIE D 

77 
TABLE C2 FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

U. BLADDER/MUSCULARIS 

EDEMA 

ENDOCRINE SYSTEM 

ADRENA L CORTEX 

CYTOMEGALY 

ADRENA L MEDULL A 

HYPERPLASIA FOCAL 

THYROID 

HYPERPLASIA C-CELL

 

HYPERPLASIA FOLLICOLAR-CELL 

PARATHYROI D 

HYPERPLASIA 

PANCREATIC ISLETS 

HYPERPLASIA 

REPRODUCTIVE SYSTEM 

MAMMAR Y GLAN D 

GALACTOCELE 

MAMMAR Y LOBULE 

HYPERPLASIA 

CLITORAL GLAND 

CYSTIC DDCTS 

HYPERPLASI A 

UTER OS/END OMETRIUM

 

INFLAMMATIO N SOPPORATIV E

 

HYPERPLASIA 

HYPERPLASIA CYSTIC 

HYPERPLASIA STROMAL 

METAPLASIA SQUAMOUS 

OVARY 

CYST 

HJFJiABHATION SUPPURATIVS 

*SYSTEM PERCENTAGES ARE BASED ON THE 

CONTROL 

11 (73X) 

2 

2 

10 

10 

1 

1 

8 (53X) 

2 

2 

6 

5 

3 

2 

2 

1 

 NUMBER OF 

LOW DOSE 

1 

1 

17 (SOX) 

1 

1 

2 

2 

16 

16 

1 

1 

1 

1 

20 (59*) 

3 

3 

1 

1 

16 

13 

5 

2 

6 

u 

 ANI&*yL_NECROPSIE:; 

HIGH DOSE 

22 (63%) 

22 

22 

1 

17 (U9%) 

2 

2 

1 

1 

1 

1 

1 

13 

10 

2 

3 

1 

1 

2 

2 

78 
TABLE C2 FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

ABSCESS 1 1 

INFLAMMATION CHRONIC 1 

NERVOUS SYSTEM 1 (3X) 

BRAIN 1 

GLIOSIS 1 

CORPORA AMYUCEA 1 

SPECIAL SENSE ORGANS 1 (3%) 1 (3*) 

EYE 1 

INFLAMMATION GRANULOBATOUS 1 

EYE/CORNEA 1 

INFLAMMATION CHRONIC FOCAL 1 

MUSCULOSKELETAL SYSTEM 

NONE 

BODY CAVITIES 

NONE 

ALL OTHER SYSTEMS 1 (3X) 

ADIPOSE TISSUE 1 

INFLAMMATION G RANULOHATOUS 1 

SPECIAL MORPHOLOGY SUMMARY 

AUTO/NECROPSY PERP/HISTO PBRF 

AUTOLYSIS/NO NECROPSY PERFORMED 1 

1 

79
 


APPENDIX D



SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 


LESIONS IN MICE FED PHENFORMIN
 


IN THE DIET
 


81
 


TABLE 01

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS 

IN MALE MICE FED PHENFORMIN IN THE DIET 

ANIMALS INITIALLY IN STUDY
 


ANIMALS NECROPSIED
 


ANIMALS EXAMINED HISTOPATHOLOGICALLY
 


ANIMALS WITH NON-TUMOR PATHOLOGY
 


INTEGUMENTARY SYSTEM
 


NONE
 


RESPIRATORY SYSTEM
 


TRACHEA
 


INFLAMMATION SUPPURATIVE
 


LUNG
 


BRONCHOPNEUMONIA
 


ALVEOLAR MACROPHAGES
 


LUNG/ALVEOLI
 


EMPHYSEMA
 


HEMATOPOIETIC SYSTEM
 


SPLEEN
 


AMYLOIDOSIS
 


HEMATOPOIESIS
 


LYMPH NODE
 


HYPEHPLASIA RETICULUM-CELL
 


BRONCHIAL LYMPH NODE
 


PHAGOCYTIC CELL
 


LYMPHOID HYPERPLASIA
 


MESENTERIC LYMPHNODE
 


HYPBRPLASIA RETICULUM-CELL
 


CIRCULATORY SYSTEM
 


MYOCARDIUM
 


INFLAMMATION INTERSTITIAL
 


*SYSTEM PERCENTAGES ARE BASED ON THE 

CONTROL LOW DOSE
 


15 35
 


14 (100%) 29 (100X)
 


14 29
 


13 (93%) 26 (90%)
 


13 (93%) 10 (34%)
 


7 5



7 5



12 10
 


11 10
 


4



2 (7%)
 


1



1



1



1



7 (24%)
 


7



1



 NUMBE R OF ANIMAJ.S_NECROPSIED 

83 

HIGH DOSE
 


35
 


33(100%)
 


33
 


33 (100%)
 


27 (82%)
 


14
 


14
 


25
 


21
 


5



1



1



3 (9%)
 


1



1



1



1



1



1



1



1



4 (12%)
 


4


TABLE D1 MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOH DOSE HIGH DOSE 

INFLAMMATIO N SUPPORATIVE 4 1 

PIBROSIS 14 3 

FIBROSIS POCAL 1 

CALCIFICATION 1 

AORTA 1

THROMBOSIS 1

DIGESTIVE SYSTEM 3 (21X) 1 (3X) 7 (21*) 

LIVER 2 1 3 

NECROSIS FOCAL 1 

METAMORPHOSIS PATTY 1 

HEPATOCYTOMEGALY 1 

HYPERPLASIA NODULA R 2 

ANGIECTASIS 1 1 

HEMATOPOIESIS 1 

PANCREATIC ACINUS 2

ATROPHY 2

ESOPHAGUS 1

INFLAMMATIO N SUPPURATIVE 1

LARGE INTESTINE 1 2 

NEMATODIASIS 1 2 

URINAR Y SYSTEM 3 (21*) 10 (3UX) 19 (58X) 

KIDNEY 2 10 18 

INFLAMMATIO N CHRONIC 2 10 18 

AMYLOIDOSIS 1 

URINAR Y BLADDER 1 1 1 

HYPERPLASI A EPITHELIAL 1 1 1 

DYSPLASIA EPITHELIAL 1 1 1 

ENDOCRINE SYSTEM 1 (7*) 3 (10X) 6 (18X) 

ADRENA L CORTEX 1

HYPERPLASI A 1

THYROI D 3 6

INFIA MBATI ON_F OCAL.G RAN Ut2nATO US 1

*SYSTE« PERCENTAGES ARE BASED ON THE NUMBE R OF ANIMALS_NECEC)PSIEr 

84 
TABLE D1 MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE 


HYPERPLASIA C-CELL 2 


HYPERPLASIA F OLLICOLAR-CELL 


REPRODUCTIVE SYSTEM 1 (7X) 


PREPOTIAL GLAND 1 


INFLAMMATION CHRONIC 1 


NERVOUS SYSTEM 1 (7X) 9 (31X) 


BRAIN 1 9 


HEMORRHAGE 1 


CORPORA AMYLACEA 1 8 


SPECIAL SENSE ORGANS 


MIDDLE EAR 


INFLAMMATION SUPPURATIVE 


MUSCULOSKELETAL SYSTEM 


NONE 


BODY CAVITIES 


NONE 


ALL OTHER SYSTEMS 1 (7%) 1 (3*) 


MULTIPLE SITES 1 


LYMPHOID HYPERPLASIA 1 


MULTIPLE ORGANS 1 1 


PERIARTERITIS 1 


HYPERPLASIA RETICULUM-CELL 1 


SPECIAL MORPHOLOGY SUMMARY 


NO LESION REPORTED 1 1 


NO NECROPSY PERFORMED 1 6 


*SYSTEM PERCENTAGE S ARE BASE D ON THE NUMBE R OF ANIMALS_NECRO£SIE D 

HIGH; DOSE
 


U



2



1 (3X)
 


1



1



10 (30X)
 


10
 


10
 


1 (3%)
 


1



1



._ __ 2_
 


85 
TABLE 01 MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOR DOSB HIGH DOSS 

AUTO/HBCROPSY PBRF/HISTO PBRP 1 

86 
TABLE D2

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS 

IN FEMALE MICE FED PHENFORMIN IN THE DIET 

CONTROL
 LOW DOSE HIGH DOSE
 

ANIMALS INITIALLY IN STUDY
 

ANIHALS HISSING
 

ANIMALS NECROPSIED
 

ANIMALS EXAMINED HISTOPATHOLOGIC ALLY
 

ANIMALS WITH NON-TUMOR PATHOLOGY
 

15
 

3


12(100S)
 

12
 

12 (100X)
 

35 

33(100S) 

33 

25 (76X) 

35
 

33(100)1)
 

33
 

30 (91X)
 

INTEGUMENTARY SYSTEM
 1 (3*)
 

SKIN
 

METAPLASIA SQUAMOUS
 

1


1


RESPIRATORY SYSTEM
 10 (83X)
 16 (U8X) 21 (6KX)
 

TRACHEA
 

INFLAMMATION SUPPURATIVE
 

3


3


U 

4 

8


8


LUNG
 

HEMORRHAGE
 

BRONCHOPNEUMONIA
 

BRONCHOPNEUMONIA FOCAL
 

INFLAM SUPPURATIVE GRANULAMATODS
 

ALVEOLAR MACROPHAGES
 

HYPERPLASIA ALVEOLAR-CELL
 

10
 

9


1


15 

6 

1


1 

7 

21
 

1


11
 

<i
 

6


1


HEMATOPOIETIC SYSTEM
 

SPLEEN
 

HEMATOPOIESIS
 

1 (8%)
 

1


1


2 (61)


1


1


LYMPH NODE
 

LYMPHOID HYPERPLASIA
 

1


1


MESENTERIC LYMPHNODE
 

HYPEBPLASIA RETICULUM-CELL
 

1


1


CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE. SYSIEM _ .
 §_118Xi 6.118S)
 

*SYSTEM PERCENTAGES ARE BASED ON THENUMBER OF ANIMAL.S_NECROPSIED 

87 
TABLE D2 FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL L00 DOSE 

LIVER 2 

NECROSIS FOCAL 1 

HEPATOCYTOMEGALY 

HEMATOPOIESIS 1 

PANCREAS 2 

CYSTIC DUCTS 2 

INFLAMMATIO N ACUTE FOCAL 

ABSCESS 

INFLAMMATION CHRONIC 

PANCREATIC ACINU S 2 

ATROPHY 2 

LARGE INTESTINE 3 

NEMATODIASIS 3 

RECTUM 

INFLAMMATIO N CHRONIC 

ANUS 

HYPERPLASIA PSEUDOEPITHELIOMATOU 

URINAR Y SYSTEM 5 («2X) 7 (2U) 

KIDNEY 5 7 

INFLAMMATIO N CHRONIC 5 7 

URINAR Y BLADDER 

HYPERPLASIA EPITHELIAL 

DYSPLASIA EPITHELIAL 

ENDOCRINE SYSTEM 2 (17*) 1 OX) 

ADRENA L MEDULLA 1 

HYPERPLASIA FOCAL 1 

THYROID 1 1 

HYPERPLASIA FOLLICULAR-CELL 1 1 

REPRODUCTIVE SYSTEM 8 (67*) 10 (30«) 

UTERUS/ENDOMETRIUM 7 9 

IN FIAMNA TION.SU PPURAT£V£ . 2 

*SYSTEM PERCENTAGES ARE BASED ON THE NUMBER OF AMIBALS_NECRO£SIED 

88 

HIGH DOSE 

2 

1 

1 

3 

1 

1 

1 

1 

1 

1 

1 

1 

1 

1 

9 (27*) 

8 

8 

2 

2 

2 

1 (3*) 

1 

1 

18 (55*) 

14 

3 
TABLE 02 FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL L OW DOSE HIGH DOSE 

HYPEHPLASI A 

HYPERPLASI A CYSTIC 

HYPERPLASI A STROMA L 

METAPLASI A SQUAMOU S 

1 

6 

1 

9 

1 

12 

OVAR Y 

CYST 

HEMORRHAG E 

INFLAMMATIO N 

ABSCESS 

 SUPPUHATIV E 

3 

1 

2 

i» 

K 

1 

14 

3 

1 

NERVOU S SYSTEM 

BRAI N 

HEMORRHAG E 

CORPORA AMYLACE A 

2 (6X) 

2 

1 

1 

5 (15S) 

5 

5 

SPECIAL SENSE ORGAN S 

NONE 

MUSCULOSKELETAL SYSTEM 

NON E 

BODY CAVITIES 

PERITONEU M 

ABSCESS 

1 (3*) 

1 

1 

A LL OTHER SYSTEMS 

MESENTER Y 

ABSCESS 

1 (3X) 

1 

1 

SPECIAL MORPHOLOG Y SUMMAR Y 

NO LESION REPORTED 

ANIMA L MISSING/NO NECROPS Y PERF 

NO NECROPS Y PERFORME D 

AUTO/NECROPSY PERF/HISTO PERF 

3 

6 

2 

1 

1 

1 

*SYSTEM PERCENTAGES ARE BASED ON THE NUMBER OF ANJMALS_NECROPSIED 

89 
TABLE D2 FEMALE MICE. NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE
 

AUTOLYSIS/NO HECROPSY PERFORMED
 1 

90
 


A U. S. GOVERNMENT PRINTING OFFICE : 1977 241-161/3052 

DHEW Publication No. (NIH) 77-807