THE UNIVERSIT Y OF MICHIGAN COLLEGE OF ENGINEERING Fission Products Laboratory and SCHOOL OF MEDICINE Department of Pathology Terminal Report on GROWTH, REPRODUCTION, MORTALITY, AND PATHOLOGIC CHANGES IN RATS FED GAMMA-IRRADIATED POTATOES L. E. Brownell C. H. Burns.G.. D Abrams UMAI Project 2507 under contract with: DEPARTMENT OF THE ARMY OFFICE OF THE SURGEON GENERAL CONTRACT NO. DA-49-007-MD-581 WASHINGTON, D. C. administered by: THE UNIVERSITY OF MICHIGAN RESEARCH INSTITUTE ANN ARBOR February 1959

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ACKNOWLEDGMENTS The authors wish to express their appreciation for the professional assistance of Kenneth Ao KLeyn, Robert Lo Kruger, and Robert No Berkman, D.V.M., for performing the routine examination and gross autopsies of the animals, and for theconscientious assistance of Arthur A. Smiltens who managed the animal colony. The authors also wish to acknowledge the contribution of Ruth C.o Wanstrom, MoDo. Do Lo Hinerman, M.Do, and Ao James French, M.D., Chairman, of the Department of Pathology, The University of Michigan Medical School, whose original pathological diagnoses led us to the use of this animal colony for a study of hypertensive vascular disease. ii

TABLE OF CONTENTS Page LIST OF ILLUSTRATIONS iv ABSTRACT v OBJECTIVE v I. INTRODUCTION 1 IIo EXPERIMENTAL 1 A. Source and Storage Behavior of Potatoes 1 B. Diet Formulation and Preparation 3 C. The Animal Colony 5 D. Procedures Used6 III. RESULTS 7 A. Growth 7 B. Food Efficiency 7 Co Reproduction 7 Do Blood Cell Counts 12 E. Mortality 12 F. Gross Tissue Weights 16 IV. PATHOLOGIC CHANGES 18 A. Brain 18 B. Cardiovascular System 19 1. Heart 19 2. Blood Vessels 20 C. Respiratory System 20 D. Digestive System 21 1. Salivary Glands 21 2. Gastrointestinal Tract 21 3. Liver 21 4. Pancreas 21 E. Genitourinary System 22 1. Kidneys 22 2. Reproductive Organs 22 3. Mammary Glands 23 F. Endocrine Glands 23 G. Bone Marrow, Spleen, and Lymph Nodes 24 H. Skin 24 I. Ears 24 J. Eyes 25 Ko Neoplasms 25 V. DISCUSSION 25 VI. REFERENCES 29 iii

LIST OF ILLUSTRATIONS Table Page I Percent Decay by Weight Occurring in Potatoes and Included in the Diet from the Fifteenth to the Twenty-Fifth Week of Feeding 2 II Mean Body Weights and Standard Deviations for First-Generation Males and Females up to Thirty-Six Weeks 9 TII Reproductive Performance of First-, Second-, and Third-Generation Animals 11 IV Blood Cell Counts of Representative Animals at Four, Ten, and Eighteen Months of Age 13 V Mortality Data for First- and Second-Generation Males and Females During Periods Specified 16 VI Mean Percentage of Organ to Body Weight of Six Tissues from FirstGeneration Animals Fed the Potato Diets 17 VII Incidences of the Most Common Non-Neoplastic Pathological Processes in the First-Generation Colony 19 VIII Incidences of Neoplasms in the First-Generation Colony 25 IX Number of Second-Generation Animals and Number Dead as of 15 October 1957 According to Whether Neither, One, or Both Parents Had Died by Same Date, by Dietary Group 26 X Major Pathological Findings in the First 42 First-Generation Males Which Died or Were Sacrificed (by About 16 Months) 28 Figure 1 Growth curves for first-, second-, and third-generation male and female rats fed diets containing irradiated potatoes. 8 2 Efficiency of utilization of food for growth by males and females of the first generation fed diets containing irradiated potatoes ("lX" and "2X") relative to those fed the control diet ("1OX")o 10 3 Survival curve of first- and second-generation males fed irradiated potato diets. 14 4 Survival curve of first- and second-generation females fed irradiated potato dietso 15 iv

ABSTRACT For two years a colony of albino rats was fed diets of which one third contained potatoes which had been exposed to ionizing radiation for sprout inhibitiono Growth. food consumption, reproductive performance, hematologic changes, mortality, and pathologic.changes in these rats were compared with the same in animals fed a nonirradiated potato diet. The growth, reproductive performance, and pathologic changes up to 30 weeks of second- and third-generation animals were also compared with corresponding controls. Three results emerged from this studyo (1) There were no consistent effects due to irradiation of potatoes which could be established by these criteriao (2) There was a slightly greater mortality rate among males of the first generation fed the irradiated potato diets which was of questionable statistical significance and may be related to the poor condition of the irradiated potatoes relative to the nonirradiated controls. Second-generation males and females fed the irradiated potato diets also experienced a higher mortality rate but this is attributed to genetic factors. (3) An unusually high incidence of a necrotizing arteritis resembling "peri-arteritis nodosa" occurred in the first- and secondgeneration animals in this experiment~ The combination of a genetic and a dietary factor is implicated in causing this disease, but irradiation of the potatoes is not a factor. Studies are currently in progress on hypertensive vascular disease in descendants of the animals used in the above experiment. OBJECTIVE The objective of this experiment was to test the wholesomeness of irradiated potatoes using albino rats as the experimental animals. v

Io IJNTRODUTTION The use of ionizing radiation to arrest sprout development in tubers and thereby to prolong their storage life has been extensively reported.1-3 Since only about 7 to 15 kilorads are required to achieve this, many of the problems which involve prolonged research and development on the radiation sterilization of foods, which requires doses of 4000 kilorads or more, are avoided. For this reason, the irradiation of potatoes attracted attention early as a means of gaining civilian and military acceptance of other food preserved by radiation. Accordingly, potatoes were one of the first items on the list in the extensive program sponsored by the Office of the Surgeon General of the U. S. Army, on the basis of procedures set up by the Food and Drug Administration,4 for evaluating the wholesomeness of a number of irradiated foods. A long-term feeding study using rats was prescribed, and the following is a report of the experiment, conducted in the Fission Products Laboratory over the past three years. The potato varieties selected for the test were Idaho Russet Burbanks, Maine Russet Burbanks, and Maine Katahdins, and they were used in equal quantities. Two dose ranges of 15.5-20 and 27-40 kilorads of gamma radiation from reactor fuel elements were tested on each variety. The higher level was to provide a test of potatoes given an overdose. The evaluation of wholesomeness was based upon the growth, food consumption, reproductive performance, longevity, and hematologic and pathologic changes in rats fed a diet containing 73% of the above potatoes on a wet-weight basis for 2 years. In addition, growth and reproductive performance of second- and third-generation animals were to be determined. IIo EXPERIMENTAL A. SOURCE AND STORAGE BEHAVIOR OF POTATOES The potatoes used during the first year of the feeding experiment were from the 1955 harvest and were irradiated in December at the National Reactor Test Site, Arco, Idaho. Those used during the second year were from the 1956 harvest. All the potatoes from the first harvest and most of those from the second were stored at Presque Isle, Maine, under the supervision of Professor Matthew Highlands, University of Maine. An additional quantity of second-year lots were stored in Idaho under the supervision of Donald Hunsaker, Philips Petroleum Company, Idaho Falls. Nonirradiated lots of each variety were treated identically, except for the irradiation step, to serve as controls. The controls will be referred to as "OX" potatoes, and the irradiated lots, as "IX" and "2X." 1

The potatoes were stored and shipped either in 50- or 100-lb perforated paper-board drums with waxed bottoms or in bushel baskets with wooden coverso All "OX" potatoes were manually desprouted periodically. Further details on the storage conditions and storage behavior of these potatoes may be obtained through the TU S. Army Quartermaster Food and Container Institute.5 The two varieties grown in Maine were subjected to shipping from that state to Idaho and back again for storage. As required for the feeding experiment, they were then shipped to Ann Arbor, Michigan, At The University of Michigan, they were accommodated through the courtesy of Mro Herbert Wagner at the Food Service's regular potato-storage locker held normally at 40~F, In the laboratory they were held in a household type of refrigerator until made into diet. The feeding part of the experiment began 3 months after the potatoes were irradiated. The skins and eyes as well as the whites were included in the potato diet. A month or more after the experiment was begun, some decay began to appear in the potatoes as received at the University. The decay at first was small in amount and difficult to remove, and any attempt to do so would have introduced more rather than less prejudice into the experiment. The amount of decay increased steadily in the potatoes as received, and starting with the twentyfifth week of feeding, nearly all were culled and discarded. During the last 10 weeks of this 25-week period, the amount of decay occurring in the potatoes and the proportion of decayed material included in the diet were approximated by weighto The results for the 9 lots of potatoes are shown in Table I. With each variety there was a progressive increase in decay with the irradiation dose; the Katahdin variety appeared more susceptible than did the Burbank varieties. This is consistent with the results obtained in storage studies of irradiated potatoes.4 TABLE I PERCENT DECAY BY WEIGHT OCCURRING IN POTATOES AND INCLUDED IN THE DIET FROM THE FIFTEENTH TO THE TWENTY-FIFTH WEEK OF FEEDING Variety.....Irradiation Dose* Variety OX 1X 2X Maine Katahdin 0.7 5.4 8.3 Maine Russet Burbank 1.4 2.6 3 8 Idaho Russet Burbank 1.5 1.9 4.4 Whole diet** 0.9 2.4 3.9 *"OX," no irradiation; "lX," 13.5-20 kilorads; "2X," 27-40 kilorads. The potatoes were fed in equal quantities, and the average for the three varieties is reduced by the fraction (.72) of whole diet consisting of potatoeso 2

Bo DIET FORMULATION AND PREPARATION Most irradiated foods in the Surgeon General's program are tested, on a dryweight basis, at 355 of the total diet solids. This is equivalent to 11.7% for each of the three varieties of potatoes tested in this experiment. The composition of the diet was as follows: Dry Weight, Wet Weight, percent grams per batch Maine Katahdin 1107 1300 Maine Russet Burbank 11.7 1300 Idaho Russet Burbank 11.7 1300 Purified casein 15 355 Lactalbumin 10 235 Alphacel 5 120 Corn starch 14 350 Lard and other oils 15 355 Salt mixture 4 95 Vitamin mixture 1-2 25-45 The variation in solids content from potato to potato made sampling of individual batches of potatoes unreliable, and the presence of decay and other factors made estimates of solids content difficult on the basis of specific gravity. The use of 1300 grams of each variety per batch is based on an average dry weight of 21% of all varieties as determined by oven-dried samples of various batches at this laboratory over a period of several weeks The work of M. E. Highlands showed that the solids content of the Idaho Russets, while in storage in Maine, varied from 18 to 26% over a 13-month period, Maine Russets, from 14 to 19%, and Maine Katahdins, from 14 to 17%. Irradiation dosage appeared to have little effect on the dry weight, however, and the average of all lots did not change (19o9% in December, 1956, and 20.1% in January, 1958). For the first month and a half, the vitamin mixture consisted of the "Vitamin Diet Fortification Mixture" as compounded by the Nutritional Biochemicals Corporation, Cleveland. This contained all the vitamins known to be required, plus Vitamins D and C, and constituted 2% of the diet. After the first month and a half, Vitamins A, D, C, E, and choline were removed from the mixture; the first two were furnished in cod li ver oil (1%) which was fed to provide a source of unsaturated fatty acid. Vitamin E was administered as an oral supplement in the form of alpha-tocopherol acetate and choline was added to the diet at 0.2% level from a 50% solutionn What remained of the original vitamin mixture was added to the diet at a level of 0.85% (20 g/batch) and had the following compositiono 3

grams/kilo Inositol 5.0 Menadione 2.25 p-Aminobenzoic acid 5.0 Niacin 4o5 Riboflavin 1.0 Thiamine HC1 1.0 Calcium pantothenate 3.0 milligrams/kilo Biotin 20 Folic acid 90 Vitamin B-12 1.35 grams/kilo Extender (casein) 974 After the first year of the experiment, the thiamine and biotin levels were increased twofold and the riboflavin level, threefold. These increases were made because of an ocular inflammation in the animal colony which may have been caused by marginal levels of these vitamins. For the first month and a half the salt mixture consisted of "Salt Mixture UoSoP. XIV," prepared by the Nutritional Biochemicals Corporation. Since the potatoes in the diet furnished over 1% potassium (dry-weight basis),7 it was desirable not to furnish additional potassium through the salt mixture o Thereafter, a salt mixture of the following composition was used at a 3.5% level: Percent CaHP04 2H20 48o0 NaCl 22.1 2aCOs 15.0 MgS0477H20 8.7 Fe citrate 6H20 5.5 MnS04 a:O0 0.52 YI.0o8 CuS04.o5H20.07 ZnCl2.025 CcC12.005 After one year of the experiment, calcium carbonate was removed from the salt mixture because of the possibility that it was promoting auto-oxidation of some of the vitamins.8 The calcium was supplied by additional calcium phosphate. Potassium iodide was furnished in the choline chloride solution because it tends to oxidize in salt mixtures containing cupric salts and moistureo Zinc chloride and cobaltous chloride were also removed from the salt mixture because the large proportion of potatoes i.n the diet furnished these trace elements. 910 The sim

plified salt mixture, added to the diet at the 355% level, had the following composition Percent CaHP04 ~2H20 67.2 NaCl 20 2 MgS04o 7H20 7o1 Fe citrate 6H20 5.0 MnS04 H20 0.5 CuC04 5H20 0.06 To prepare the diet, the potatoes were freed of decay (except during the first few months of feeding), but not of the eyes or skin, washed quickly, cut up, and steamed for 10 to 15 minutes at low steam pressure. They were then pushed through a meat grinder having a plate with 3/16-in.-diameter holes, which cut the skins finely without making a paste of the whites. The dry ingredients were added to the cocked and mashed potatoes and, while being mixed, lard, in a melted state, and other liquids were added. When the batch of "dough" reached the completely cohesive stage (further beating makes it adhesive), it was rolled out in a flat stainless-steel pan 3.5 cm deep. The diet was scored on top into 180 cubes so that when the animals were fed on a per-cube basis, as an alternative to weighing out the diet, the portion of nutrients furnished to the animals was known in spite of variations in the thickness of the diet or moisture contento The diet was stored in a refrigerator until use, sometimes covered with polyethylene or cheesecloth to control moisture accumulation on the surface. The diet was never used after the second day. Co THE ANIMAL COLOITY' The parent-generation colony was organized into 5 replicates. Each replicate consisted of 5 litters of Holtzmann strain rats (Madison, Wisconsin), and each litter had 4 males and 4 females. The replicates were started one week apart, the first on 14 March 1956; all rats were 22 days old when started. Each of the 3 potato diets was fed to one male and one female in each of the 25 litterso The groups thus constituted will be referred to as the "OX," "1X," and "2X" groups. The fourth male and fourth female of each litter were fed Purina Laboratory Chow, and these animals will be referred to as the "XL" (extralittermate) group The parent generation of animals was maintained for about 2 years. At 15 weeks of age, 4 of the 5 females fed each diet in each replicate were mated in weekly rotation with 4 males in the same group. The second-generation animals obtained from the second breeding of the parents were fed the parental diet until two breedings had been completed, after which it was no longer necessary to maintain them for the purposes of this experiment. They were then all fed the "OX" diet, and later the same diet with fresh market potatoes, to make certain 5

observations of pathological interest.11 The third-generation animals were also fed the experimental diets until their young had been weaned, at which time they too were continued on the fresh-potato diet. The fourth-generation animals were examined at weaning, but were not fed the irradiated-potato diet thereafter. All animals fed the irradiated potato diets were housed in individual wirebottom cages and provided with water and diet ad libitum. Animals fed Purina Laboratory Chow were housed five to a large cage', and were not mated. The animal quarters were air-conditioned (70~F) with ambient humidity. The usual standards of cleanliness, periodic cage and water-bottle washing, and shavings change were followed. D. PROCEDURES USED All animals were weighed weekly to the nearest gram on a Toledo balance. Food consumption was measured for the first 12 weeks after weaning for first- and second-generation animals. Each animal's daily allotment of fresh food was weighed and samples from the same batch of diet were air-dried to constant weight. Food left in jars was collected daily and combined, after fecal matter was separated, with food spilled which was caught on a piece of mosquito screen; this was dried in air and its weight was subtracted from the total of food, dry basis, fed for the week as calculated from the daily fresh food weights and fraction of solids. Reproduction data consisted of apparent pregnancies, pups born/litter, survival of pups to first and twenty-first days, and average body weights of pups at the seventh and fourteenth days of lactation for the last three of the six breedings, and at the twenty-first day for all breedings. Each first-generation animal was examined externally each week, and all observations obtainable were recordedo Special attention was paid to respiratory condition, tumors, and abscesses. Blood cell counts were made on one male and one female in each of the three dietary groups in each of the five replicates (i.e., 30 animals of the total colony of 150). Blood was obtained from the tail vessels. These counts were performed during the fourth, tenth, and eighteenth months of experiment. All first-generation animals, whether found dead, sacrificed because of moribund condition or sacrificed to terminate the experiment, were subjected to complete necropsy. The tissues were submitted for histological examination to members of the Department of Pathology of The University of Michigan Medical School. Also included were members of the second generation which died or were sacrificed before the end of their second breeding period. 6

IIIo RESULTS Ao GROWTH Growth curves for the first-, second-, and third-generation males and females fed the diets containing nonirradiated ("OX") potatoes and potatoes irradiated at two levels ("lX" and "2X") are shown in Fig. 1. Body-weight averages for the first generation are extended beyond the growth and reproduction phase to about 70 weeks, at which time the mortality complicates the significance of the Dierenes between the curves would not be significant for females after the onset of pregnancy. The three diets resulted in uniform growth, except first-generation females fed the "lX" diet, which showed a slight but statistically (P < o01) significant superiority in body weight. Table II shows means and standard deviations for first-generation animals up to 36 weeks, and shows the small but significant increase in weight of 1X females but of no other groups. B, FOOD EFFICIENCY The average gram body-weight gain per gram diet solids consumed for the 25 animals in each of the 6 groups is shown in Fig. 2 for the first 12 weeks following weaning. After 8 weeks, the fraction of food contributing to body-weight increase is small and normal fluctuations in growth rate lead to large differences in food efficiency. For the first 8 weeks, however, -there is apparently no difference in the ability of each diet to furnish nutrition for optimum growth. C. REPRODUCTION Table III shows the reproductive performance for the first-, second-, and third-generation animals through the first and second breeding. Differences can be found between animals in the three dietary groups but these are not consistent. For example, apparent pregnancy was lowest for 1X females through all six breedings, and in the three instances where one group showed a relatively poor birth rate, this was always the 1X group. The average number of pups born per litter was uniformly good. The average number of pups surviving to one day after birth was lower for first breedings than the second, and it was especially low during the first breedings of the first and third generations; but there were no differences between dietary groups in this respect. The number of pups surviving to weaning was variable from breeding to breeding, particularly poor during the first breeding of the second and third generation, but again all groups behaved in a similar fashion. In. both the first and second breedings of the third generation, however, the average weight of pups in the 2X group was somewhat less than that for the other groups. 7

KW OF EXPERIMENTATFIRST GENERATION > 100 - -Fig 1 G c u r v f fi- — r- -.-c — — n Thr-gna a aredIXi IX Fig. 1. Growth curves for first-, second-, and third-generation male and female rats fed diets containing irradiated potatoes. 400 350 - 300 250 200 150 -- 100 of age n members 50 6 97 98 99 100 101

TABLE II FIRST-GENERATION MALES AND FEMALES UP TO THIRTY-SIX WEEKS MEAN BODY WEIGHTS AND STANDARD DEVIATIONS FOER Mean Body Weig ht, Grams, and Standard Deviation Week Male Groups Female Grou s OX IX 2X XL OX 1X 2X XL 0 49.0 + 4 236 + 8 376 + 12 441 + 16 473 t 20 526 + 24 554 + 28 548 + 32 565 + 36 586 + 0.4 4 6 7 7 8 8 11 10 10 48.4 + 241 + 377 + 447 + 480 + 513 + 546 + 542 + 560 + 580 + 0.5 2 5 7 7 8 7 9 9 8 48.3 + 242 + 384 + 442 + 476 + 516 + 549 + 546 + 564 + 587 + 0,5 3 6 7 9 9 10 11 11 11 529 + 4 393 + 4 442 + 5 475 + 5 498 + 6 508 + 6 521 + 8 47.7 + 168 + 227 + 257 + 275 -+ 307 + 314 + 327 + 350 + 542 + o.4 2 4 5 5 6 6 6 7 6 47.7 t 175 + 2357 267 + 284 + 521 + 333 + 345 + 364 + S68 + 0.5 2 3 4 4 5 5 6 6 8 47.5 + 172 + 231 2+ 258 +2 274 ~ 311 + 512 + 324 + 541 ~ 340 + 0.5 2 4 4 7 5 6 9 6 214 + 2 254 + 5 258 + 5 270 + 4 275 + 4 283 + 4 290 ~ 5 _ ,, _.,, _,_,_,.,,~.-,__ --- —----- -— C~C=ilbD~DI- — i- -~ -~- -~ -— i- uq2=rr I-.~~LPe-L-t.PI.CL _C-ZCii~;t~Y~dU-T —ilf PI

() -J 20.7 z In N LL w 0 w L) z 0 0. 0 o 0.. 0. 3 U) 0 cn 0 U) r:O.I (. I OX diet IX 2X \\ \, MALES m FEMALES.0 I I I I I I I I I I I 1 2 3 4 5 WEEKS 6 AFTER 7 8 9 10 II 12 WEANING Fig. 2. Efficiency of utilization of food for growth by males and females of the first generation fed diets containing irradiated potatoes ("1X" and "2X") relative to those fed the control diet ("OX"). 10

TABLE III REPRODUCTIVE PERFORMANCE OF FIRST-, SECOND-, AND THIRD-GENERATION ANIMALS First-Generation Animals Second-Generation Animals Third-Generation Animals Breeding: First Second First Second First Second Diet: OX IX 2X OX IX 2X OX iX 2X OX IX 2X OX iX 2X OX IX 2X 1. Number of females bred 20 20 19 20 20 19 20 19 20 19 18 18 20 20 20 20 20 20 2. Number of males used 20 20 20 20 20 20 10 10 10 10 10 9 20 20 20 20 20 20 3. Percent of females bred which appeared to become pregnant 90 90 95 85 75 84 100 84.2 100 94.7 77.7 94.4 100 95 100 100 95 100 4. Percent of apparently pregnant females which gave birth 83 83 100 82 67 81 100 87.5 95.0 100 85.7 100 95 100 95 100 100 100 5. Average number of pups born per litter 11.2 9.6 10.7 10.4 11.9 10.5 10.9 10.3 11.1 9.8 9.2 9.8 10.1 10.4 10.7 9.9 9.6 9.5 H H 6. Percent of pups born surviving birth and the first day after birth 7. Percent of pups surviving the first day which reached 21 days (weaning) 8. Average body weight of pups at 7 days 9. Average body weight of pups at 14 days 10. Average body weight of pups at weaning 80 78 77 86 92 95 91.53 92.4 90.0 93.8 92.7 97.6 78.5 77.8 68.3 90.1 90.2 91.6 93 86 92 86 86 84 57.0 62.6 65.4 83.2 71.8 72.5 58.0 64.3 52.1 93.3 80.1 90.2 -- -- -- -- -- -- -- -- -- 15.0 14.5 15.1 14.3 14.3 13.2 16.0 15.9 14.9 -- -- -- -- -- -- -- -- -- 30.4 30.6 31.7 29.9 28.3 26.4 30.7 30.3 28.7 51.4 53.7 52.9 50.1 47.0 48.1 47.3 49.6 44.9 51.1 53.5 54.5 49.1 48.8 44.3 50.4 51.0 45.7

Do BnLOOD CELL COUNTS Table IV, presents three sets of blood cell counts made on a male and female representative from each of -the 15 groups:'Blood cell counts were made on these same animals at 4, 10, and 18 months of age, although. by the last set of data some of the original members of the group had been sacrificeed, In addition, blood cell counts for all parert animals surviving at 19 months are showno What variations existed among animals on the different diets did not persist over the period studied, and nearly without exception every:range of values for any one count overlapped every other one: E MORT"ALMTY[ Mortality cu:rves for first" and second-generation males fed each of the three diets are shown in Fig. 3 and for females,..n Figo 4~ Each animal is represented by a horizontal bar, the length of the bar corresponding to the animal's age at death Those animals wh:ich died or became moribund after termination of the experiment was begun are indicated by a daggero There is, of course, no significance to the mortality curve after this point. The mortality of second-generation an i mals was due principally to a vascular disease (see below), and those remaining after 16 months, although in apparent good health, were sacrificed to complete an evaluation of the extent of the lesions in the entire colony. They had been continued beyonad 50 weeks of age on the control diet, as they were not intended to have been fed the experimental diets beyond this time. (The same practice was followed with the third generation, but there was no mortality before the thirtieth week of age, when they were all given the con trol diet, and shcwed lttle mortality fcor several months thereafte:r The fourth-generation members were transferred to the control diet shortly after weaning, anrd, likeewwise, had nc si.g:..ifican.t mortalityo ) The one important difference a-ong animals on the -various dietary groups appeared in the survival rate at 21 mon.-ths fcr first-generation members, and at 16 months for second-generation animals~ Theese results are shown in. Table Vo In the first generation, the mortality was twice as great among males on the LX and 2X diets as among those on the control diet, but there was virtually n.o difference among the female groups. The Student-Fisher t-test applied to the mortality:rate of the male groups revealed no statistically significant difference between any two of them. In the second gerneration, most of the mortality occurred after the animals has been given the "OX" dieto Before the change, however, only one control had succumbed, while three had succumbed from the 1X and three from the 2X groupso Altogether, after 16 months, the difference between the female groups was rather marked, whle there was less difference among the male groupso 12

TABLE IV BLOOD CELL COUNTS OF REPRESENTATIVE ANIMALS AT FOUR, TEN, AND EIGHTEEN MONTHS OF AGE (Each value is an average for 5 animals; figures in parentheses are minimum and maximum values in the group of 5 animals) eato- Heogobin, Corpuscular White Blood Differential Count: Group Hemat Heogr bi Hemoglobin, Cells, Thou- % of White Cell Count arn t, grams a ~*_ _. sands per mm3 P(l) SL(2) LL(3) M(4) E(5) Four Months MALES OX (5) 48.9 16.0 32.7 15.0 19 74 2 3 2 IX (5) 47.9 15.9 33.2 13.3 14 79 3 3 1 2X (5) 47.6 15.5 32.6 14.0 18 76 2 3 1 Pellet (5) 48.3 16.2 33.5 14.7 13 80 3 3 1 Ten Months OX () 50.0 15.9 13,150 22 65 8 2 3 (14.8-17.4) 3 (9,800-18,400) (17-30) (63-73) (2-15) (1-3) (2-5) 16.5 12,110 26 60 8 4 2 1X (5) 49.8 (15.3-17.4) 3-2 (8,900-15,400) (23-29) (56-67) (4-12) (1-7) (1-4) 2X (5) 48.9 15.1 30.9 13,100 24 62 9 4 2 (13.7-16.3) (8,800-19,000) (11-40) (55-70) (3-15) (2-8) (1-4) Eighteen Months 16.0 15,500 42 39 7 9 4 OX (3) 49.6 (15.1-17.0) (13,550-17,650) (30-55) (25-54) (3-9) (7-12) (0-5) 16.5 9,000 35 51 7 8 1 1x (2) 52.9 (16.4-16.5) (5,700-12,200) (31-39) (49-52) (6-7) (5-10) (0-1) 15.9 22,300 51 31 9 9 1 X (2) 48.9 (15.3-16.4) (10,350-34,250) (41-61) (21-40) (8-10) (8-9) (0-2) OX (10) 17.6 15.1 15,200 40 41 13 5 2 (9.4-17.4) (7,750-27,700) (29-59) (23-53) (5-27) (1-10) (0-3) 15.5 10,750 34 46 15 5 1 1X (4) 48.o (14.2-16.2) (5,800-22,700) (29-37) (38-50) (9-19) (3-8) (0-3) x (6) 6.9 15.0 15,900 38 46 10 5 2 (12.6-18.3) (9,750-20,350) (22-60) (30-66) (2-21) (0-8) (0-5) 14.6 17,800 36 40 13 9 2 XL (8) 6.1 (8.4-16.6) (12,150-23,450) (20-54) (22-55) (9-19) (5-15) (0-4) Platelets B(6) 0 0 0 0 0 Adequate 0 Adequate 0 Adequate 0 Adequate 0 Adequate O Adequate 0 Adequate O Adequate O Adequate O Adequate O Adequate O Adequate O Adequate O Adequate FEMALES Ox (5) Ix (5) 2X (5) Pellet (5) ox (5) Ix (5) 2X (5) ox (4) IX (4) 2X (4) ox (10) IX (8) 2X (11) XL (10) Four Months 46.1 14.6 31.6 10.4 19 74 3 3 1 48.1 15.4 32.0 10.2 12 80 3 3 2 47.9 15.5 31.9 9.0 17 77 2 2 2 46.4 14.7 31.7 14.9 16 77 2 2 3 Ten Months 15.1 6,280 32 58 6 2 2 46.6 (14.4-16.3) (4,000-9,200) (12-49) (37-81) (4-8) (1-3) (0-4) 15.4 6,480 20 65 9 4 2 47-4 (14.6-172) 32. (4,400-9,100) (12-31) (51-78) (3-14) (0-7) (0-5) 14.7 5,850 25 65 8 1 2 45.9 (12.7-15.7) 21 (3,600-8,300) (11-32) (55-74) (1-19) (0-2) (0-3) Eighteen Months 15.7 7,000 46 33 13 6 3 49.3 (14.6-16.6) (4,750-12,100) (29-69) (14-46) (3-17) (1-12) (2-4) 15.4 6,500 34 49 11 6 1 1 (15.1-15.8) (5,400-7,500) (31-36) (46-53) (8-15) (3-9) (0-2) 14.9 6,900 34 43 17 5 2 47.6 (14.7-15.3) (5,750-7,800) (31-38) (36-48) (8-27) (2-7) (0-3) 15.1 9,400 42 36 12 82 46.7 (13.8-16.7) (7,000-16,500) (31-66) (14-51) (4-27) (4-16) (1-4) 14.7 8,200 39 41 11 7 3 45.9 (10.8-15.6) (5,650-15,600) (19-59) (26-61) (4-19) (3-10) (0-5) 15.7 7,600 35 42 15 7 2 1 (14.9-17.8) (3,700-11,550) (26-52) (25-60) (8-29) (2-11) (0-7) 15.6 10,900 39 34 15 5 6 47.8 (.14.5-16.4) (7,050-29,550) (28-51) (15-47) (6-25) (3-10) (0-25) 0 0 0 0 O Adequate O Adequate O Adequate 0 Adequate 1 (0-1) Adequate 0 Adequate O Adequate O Adequate O Adequate 0 Adequate (1) Polymorphonucleocytes (2) Small leucocytes (3) Large leucocytes (4) Monocytes (5) Eosinophils (6) Basophils 15

t- Animal Died (or Required Sacrifice) During Termination Period H I F I. p.J Age in Weeks Fig. 3. Survival curve of first- and second-generation males fed irradiated potato diets.

t -Animal Died (or Required Sacrifice) During Termination Period L s I 1! I J I I FIRST GENERATION I I I on. 1 — r-n-~ --- I,I I - I i I I LI I.I. —,-.l'......... I I - 1i- _ I 7I 1 I I NA 15 IC All 2nd Gen. Animals Fed Control Diet I I -It.1+ I i! I aI I I I 1 — 4 9 I I 1, 7- 1 1 1 t I I I f I — L Routine I Termination - L I- Begun — t Lt ' 'it I -- - ^ tlt SECOND GENERATION L mI i I1 OX Diet (Control) --- IX Diet --. 2X Diet I i71 _I _1 2 1 I rIz: L I I I t i tft _____tIII II F_ —' __ ____ ___ _=EL I I I I *L II it I li.......I It- i i i.- i_ -' I I I I i I A 0 20 40 60 80 100 Age in Weeks Fig. 4. Survival curve of first- and second-generation females fed irradiated potato diets.

TABLE V MORTCAL7T. DAA FOR. FRST AND SECOND-GENERATION MALES AND FEMALES DURING PERIODS SPECIFIED Percent Mortality of Animals at Time When Termination Began Dt % Decay First Generation Second Generation Diet in Diet;s* Male s, Females, Males, Females, 89 Weeks 95 Weeks 69 Weeks 68 Weeks OX 0 9 32 36:X 2o4 64 4o 2X 3.9 64 4o OX 0 1 30 20 ix - 0lO 50 45 2X 0.1 4o0 50:During fifteernth to twenty-f:ifth. week of life for first-generation animals and not at all during the life of second-generation animals. F.o GROSS TI.SS'UE WEIG.THS As part of the necropsy procedure, routine weights were taken of the opened heart, spleen, kidneys, lungs, liver, and, from males, testeso In Table VI the results appear for each group as the mean percentage of organ to body weight with standard dev. at ion; data from the "XL" group are includedo In the male groups, there appeared to be a significantly lower ratio of liang to body weight:r the controls than with males fed the 2X diet (statistically signif:icra.nt to the 5% level)o There are no other important differences, even. between animals fed the potato diets and the Purina Lab Chow diet. _n the case of the females, the ratio of spleen and lurg to body weights appea:red significantly lower in the lX females than. with either the control females or those fed the 2X diet. Again, there were no important differences between animals fed the -two types of diets. The only exceptions to this were that the heart and:idney weights of the females fed the Purina Lab Chow were lower than those of the OX females, but not of the lX and 2X females. 16

TABLE VI MEAN PERCENTAGE OF ORGAN TO BODY WEIGHT OF SIX TISSUES FROM FIRST GENERATION ANIMALS FED THE POTATO DIETS Diet Heart Spleen Kidneys Lungs Liver Testes Males ox OX 2X XL.371 + 017.397 +.021. 394 +.018.367 + 016.245 +.028.212 +.013.229 + o013.207 +.013.459 + o042.534 +. 056.491 +.032.421 +.036 o550 + o043.76o0 o l6.840 +.145.653 +.092 35.4 +.170 3.43 ~.170 3.45 ~.170 3.72 +.220.295 + o351.304 +.014 o288 ~.033.299 t.017 H Females OX IX 2X XL.462 ~.047.387 t.039.401 +.023.326 +.013.278 +.211 +.294 +.261 +.031.015 o04.040.393 +.025.412 +.055.365 +.023.350 +.012.712 +.097.473 t.027.626 +.077.693 +.120 5.20 +.160 5.12 +~.180.536 +.180 5.05 +.110

IVo PATHOLOGIC CHANGES Necropsy on each animal in this study included examination of cranial contents, cervical, thoracic, and abdominal viscera, and the extremities, The heart, lungs, liver, spleen, kidneys, and testes were weighed routinelyo These data have been presented above with other quantitative data. A minimum of 30 to 40 tissue sections was examined from each animal, with the exception of those of the XL group, in which tissues from only 18 animals were submitted for complete microscopic examinationo From the remaining animals of this group, those sacrificed at the termination of the study, only sections of kidney, adrenal gland, and pancreas were studied microscopicallyo This was done to reveal the presence of the necrotizing arteritis described belowo Therefore the incidence of other diseases encountered in the XL group is useful in a qualitative rather than strictly quantitative sense. All tissues were fixed in 10% formalin, embedded in paraffin, and the sections stained with hematoxylin and eosino Frozen sections of heart, lung, liver, kidney, and adrenal gland stained with Sudan IV were routinely processed. During the course of this study, microscopic examination of tissues was performed by several members of the Department of Pathology. The data were summarized by one of us (GDA); the original slides were reviewed when necessary to confirm the comparability of diagnoses from the several pathologistso In summarizing the data on the kidneys and neoplasms, all the original sections were reviewed, The summary of pathological findings presented below includes information concerning only first-generation animalso In keeping with the design of the experiment, of the 120 animals of the second generation, only the 7 that died prior to completion of the second breeding were submitted for microscopic examination. In each case, the major finding was the presence of the necrotizing arteritis described below. In general, the pathological processes encountered in this study were similar to those that have been reported in other long-term studies of this species. The diseases observed in any one dietary group did not differ qualitatively from those encountered in the other groupso The incidences of the more frequently encountered and major diseases in the four groups are presented below in tabular form, while the pathologic changes in these diseases and the less common conditions are discussed under headings of organ. systems involved. A BRAIN Significant lesions of the brain were uncommon in all groups. Cerebral edema of varying degree, and mild focal leukocytic infiltration of the meninges were encountered in several animals of each group. In one animal of group 1X, 18

a large brain abscess was associated with suppurative otitis media. The brains of two other animals of this group were infiltrated diffusely by a malignant glioma. The brain of one animal of group 2X was involved by direct spread of a neoplasm originating in the hypophysis. B. CARDIOVASCULAR SYSTEM 1o Heart.-The cardiac lesions observed in the several groups were similar in type. Sclerosis of coronary arterial branches was noted in all groups and, with somewhat greater frequency in the 1X and 2X groups (Table VII). These figurTs, however, are not based upon a systematic grading and specific comparison of the vessels and the differences, therefore, may not be significant. Small foci of chronic interstitial inflammation occurred in the myocardium with a frequency roughly parallel to that of arteriosclerosis (Table VII)o A peculiar focal endocarditis characterized by marked fibroblastic proliferation and infiltration of chronic inflammatory cells was found in five animals, three of group OX, one of group 2X, and one of group XLO Cartilaginous metaplasia in the region of cardiac valves was encountered in three animals, two of group IX and one of group XL. Intracardiac mural thrombi were present in three animals of group OX and in two animals of group 2X, Some degree of degenerative fatty infiltration of myocardial fibers occurred in slightly less than one-half of the members of each group, and was unrelated to other cardiac lesions. TABLE VII INCIDENCES OF THE MOST COMMON NON-NEOPLASTIC PATHOLOGICAL PROCESSES IN THE FIRST-GENERATION COLONY Incidences apply to 50 animals (25 males and 25 females) except for XL's as noted Dietary Group OX IX 2X XL Sclerosis of the coronary arteries 6 8 15 3* Focal interstitial myocarditis 7 13 14 1* Necrotizing arteritis Males 18 16 17 8 Females 12 15 9 2 Total 30 31 26 10 Severe bronchiectasis with associated pneumonitis and abscesses 5 1 0 5 "Vascular nephropathy" 19 11 12 7 Otitis media 13 16 15 3* Incidence applies to a total of 18 rather than 50 animals. 19

2. Plood Vesselso-The freqbuent occurrence of a necrotizing arteritis in all the groups virtually oversh.adowed the other diseases encountered in this studyo The presence of -the disease was usually heralded upon gross examination by the tortuosity andc nodularity of the arterial branches in the region of pancreas and mesentery, the site of most frequent involvement~ The presence of lesions elsewhere in the -cardiovascular system usually was not suspected grossly. However, subsequent microscopic examination revealed widespread lesions, of var'iable occurrence, in the intrinsic arteries and arterioles of heart, gastrointestinal tract, liver, kidneys, gonads, genitourinary tract, and more rarely, other areaso MiWc:roscopi.cally, `the changes in the large pancreatic and mesenteric arteries resembled lesions which have been called "periarteritis nodosao" Varying stages of the process, from acute necrosis to advanced scarring, were observed. Characte:ristically, in an active lesion, there was subendothelial deposition of a "fibrinoid" materia:l, extensive destruction of the media, with fibroblastic and angioblastic ingrowth, dense leukocytic infiltration, and adventitial and peri.arterial involvement by the inflammatory process. Thrombi were frequently present in affected arteries, Destruction of the wall often resulted in aneurysmal dilatation of the affected segmento Rupture of such an. aneurysm rarely resulted in massive intraperitoneal hemorrhageo In small arteries and arterioles in the splanchnic area and in distant organs, a somewhat different process characterized by fibrinoid necrosis and slight, if any, peri.vasc-ular fibro'blastic proliferation and infiltration of chronic inflammatory cells was encountered. As seen in i Table 'j'I, the inci.dence of necrotizing arteritis was approximately equal in the groups receiving potato diets, whether irradiated or not, and was markedly lower in the control group receiving a "stock" diet- An apparent predilertion for males was evidento This tabulation includes under the same heading all stages and degrees of severity of the process, and a variable pat-tern of organ, invoAiement. The detailed analysis of this arteritis is beyond the scope of the present study, and will be presented in a later publicationo1 Co RESPIRATCORY SYSTEM The well-known s.sceptibility of rats to respiratory infection was reflected in the hi-gh inin-.deTnfe of these changes in each of the experimental groups. Most frequently encountered were vary inrg degrees of chronic tracheobronchiti s often accompanied by slight dilatation of bronchi, and patchy pulmonary emphysema and atelectasis.o n a number of animals of each group, greater in the 1X and 2X groups (Table V-11), extensive destruction of pulmonary parenchyma had resulted from progression of the intflammatory process. The lungs of these latter animals were often noted at necropsy to be firm, nodular, and extensively consolidatedo Microscpic. examination revealed severe bronchiectasis with associated abscesses and foci of acute and bchronic pneumonia. Proliferation of columna:r epithelium 20

was prominent, and squamous metaplasia was occasionally encountered. Often, little functional lung tissue remained in such areas. Significant acute inflammation of the respiratory tract was relatively infrequent. Focal purulent bronchopneumonia was observed, apparently as an incidental terminal event in an occasional animal of each group. D. DIGESTIVE SYSTEM 1. Salivary Glands o-Significant alterations were idnrequent in the salivary glands of any group. Sclerosis or fibrinoid necrosis of small intersititial arteries was seen occasionaly in animals with necrotizing arteritis iz other organs. One female or group IX was found to have a malignant mixed tumor, or carcinosarcoma, apparently arising in parotid gland. 2o Gastrointestinal Tracto,-The most frequently observed lesions of the gastrointestinal track consisted of sclerosis and fibrinoid necrosis in small intramural arteries and arterioleso These lesions occurred in all experimental groups as part of the syndrome of necrotizing arteritis described aboveo Despite the severity of the vascular lesions, they were not associated with infarction of the bowel. In a single animal with arteritis, a focal area of gangrene of small intestine was foundo However, in another animal with no demonstrable arteritis, focal hemorrhagic infarction of small bowel occurredo One instance of segmental infarction of colon, apparently due to mesenteric venous thrombosis, was foundO In two animals, one a male of group OX and the other a male of group XL9 malignant neoplasms of apparent gastric origin were found. The former was a highly undifferentiated neoplasm, apparently a carcinoma, while the latter was a well-differentiated adenocarcinomao A spindle cell sacroma arising in colonic wall occurred in a male of group OXo 53 Liver.-Lipid was demonstrable in the livers of over 80% of the animals of each group, No systematic attempt was made to grade the degree of lipidosis which was quite variable, and most frequently minimal. The lipid was deposited for the most part in the form of minute droplets in tne cytoplasm of parenchymal cells, and occasionally Kupfer cells, with a tendency to concentrate in central lobular areaso Sclerosis and, less often, necrosis of small hepatic arterial branches were noted occasionally in animals having necrotizing arteritis in other organs, Two primary hepatic neoplasms were encountered, both in females of group IX, One tumor was a hepatoma, the other a cavernous hemangiomao 4. Pancreas,-The most prominent pancreatic lesions, occurring in all groups, consisted of periarteritis nodosa-like lesions of larger arteries, and 21

fibrinoid necrosis of smaller arteries and arterioles as outlined above, Closely associated with these lesions were foci of chronic interstitial pancreatitiso Islet cell adenomas were discovered in six animals, one of group OX, four of group 1X, and one of group 2X. None of these neoplasms produced metastaseso E. GENITOURIVNARY SYSTEM lo Kidneys.-A distinctive type of nephropathy was encountered in animals of each. of the dietary groups, and was found to be related to the presence of necrotizing arteritis in these animalso At necropsy, the affected kidneys were found to be somewhat enlarged and mottled. Their cortical surfaces were pebbled or granular due to the presence of contracted scars alternating with microcystic areas. Microscopically, these contracted areas consisted of scarred glomeruli, atrophic tubules, increased interstitial connective tissue, and focal lymphocytic infiltrates. Small intrarenal arteries and arterioles were sclerotic and fibrinoid necrosis was not infrequently encountered. Tubules were widely dilated, contained abundant proteinaceous material, and the epithelium exhibited various retrogressive changeso Hemosiderin was deposited in the epithelium of occasional tubules. When the sections of kidneys of all animals in the study were collectively reviewed, this "vascular nephropathy" was found with the incidence given in Table VIIo With but one exception, this renal abnormality was associated with necrotizing arteritis elsewhere in the body, and even in this single animal, there was necrosis in a few small pancreatic arteries which, however, were surrounded by infiltrating carcinoma of gastric origin, rendering the significance of the necrosis uncertaino For purposes of this study, lesser degrees of neph:rosclerosis were not systematically ranked. A detailed analysis of these lesions will be included with the subsequent report of the vascular disease in these animals. A variety of renal lesions was encountered independently of the vascular disease. Minor degrees of focal chronic inflammation were present in animals of each groupo Degenerative fatty infiltration of tubular epithelium occurred in approximately one-half of the animals of each group, with no constant relation to other renal abnormalityo Focal calcification of calyceal mucosa or actual calculi were encountered twice in group OX, in three animals of group 1X, once in group 2X, and twice in group XL. A single renal neoplasm, a spindle cell sarcoma was discovered in a female of group OXo 2. Reprcduetive Organs. -The most prevalent abnormality of gonads and genital tracts of both sexes consisted of fibrinoid necrosis of small arteries and arterioles seen in all groups as a feature of the syndrome of necrotizing arteritis described above. Chronic endometritis was encountered in several females of each group, and suppurative inflammation of preputial or bulbovestibular glands was fould with like frequency, 22

35 Mammary Glandso -Significant lesions of mammary glands were limited to the females, and consisted almost entirely of adenofibromas. The incidence of these neoplasms was approximately equal in the various groups (Table VIII)o The histologic pattern varied widely, from predominantly fibrous tumors to almost purely adenomatous growths, many of which exhibited marked secretory activity. The neoplasms were histologically benign, except one tumor from an animal of group OX, in which malignant change in both components had occurred, producing a carcinosarcomao Metastases were not observed, TABLE VIII INC IDENCES OF NEOPLASMS IN THE FIRST-GENERATION COLONY Incidences apply to 50 animals (25 males and 25 females) except for XL's where the incidence applies to only 18 animals. Dietary Group OX 1X 2X XL Pituitary tumors 6 8 7* 1 Adrenal tumors 4 6 2* 2 Thyroid "adenomas" 6 6 5 1 Islet cell adenomas 1 4 1 0 Mammary adencfibromas 9* 11 8 3 Miscellaneous additional neoplasms Benign 1 2 0 0 Malignant 3 6 1 1 Total neoplasms Benign 26 37 21 7 Mal:ignant 4 6 3 1 *1 malignant Fo ENDCRCF.-NE GLANDS Epithelial neoplasms of the various endocrine glands constituted a frequent finding in animals of both sexes in each groupo Several animals were found to harbor two or more of these endocrine adenomas simultaneously. The incidence of these neoplasms (Table VZII) was approximately equal in groups OXX,, and 2X, and while the data are incomplete for group XL, similar neoplasms were found in that group. Adenomas cf the anterior lobe of the hypophysis varied from microscopic foci to large growths replacing almost the entire glando These neoplasms were sharply circumscribed though not encapsulated, and frequently compressed the adjacent glandular cells. The component cells were larger than those in the adjacent nor 23

mal gland and had pale cytoplasm. Special stains for differentiation of cell types were not performedo These neoplasms were benign, except one in a female in the group 2X in which there was local infiltrative growth. Of 14 adrenal neoplasms, nine were considered to be medullary in origin. Although the distinction was not based upon appropriate special stains, five neoplasms were thought to be cortical in origin. In, only one animal, a female of group 2X, was there metastasis from an adrenal tumoro Independent of the neoplasms, foci of hemorrhage and necrosis were encountered in the adrenals of approximately 10% of the animals of each groupo A peculiar epithelial proliferation in the thyroid gland was encountered with approximately equal frequency in groups OX, IX, and 2X (Table VIII), In these glands, large, pale-staining cuboidal or polyhedral cells, growing in a medullary or slightly aveolar pattern extended between and around thyroid follicles in foci of varying sizeo Although pleomorp.hism was present and division figures were encountered, none of these growths extended beyond the involved thyroid lobeo They were, therefore, tabulated as benign "adenomaso" Varying degrees of hyperplasia of parathyroid glands, possibly related to renal lesions or to neoplasms of other endocrine glands, were noted in animals of each group. The occurrence of pancreatic islet cell adenomas has been described above. Go BONE MARROW, SPEEN, AND LYMPH NODES The degree of cellularity of the bone marrow was somewhat variable in all groups. Likewise, varying degrees of lymphoid hyperplasia were encountered in speen and lymph nodeso Deposition of hemosiderin in spleen and lymph nodes was a prominent feature in each groupo No abnormalities more specific than these were encounteredo H. SKIN Chronic, nonspecific cutaneous ulcers and sirius tracts were observed occasionally on the extremities. A few epidermal inclusion cysts were encountered. Three cutaneous neoplasms appeared, a fibroepithelial papilloma in a female of group OX, a squamous cell carcinoma in a female of group IX, and a low-grade sarcoma in a male of group 2Xo I. EARS Chronic purulent otitis media was common in each of the groups (Table VII)o Although generally of apparently incidental importance, otitis media was associated with brain abscess in one animal,

J. EYES Significant ophthalmic disease was infrequently encountered. Corneal vascularization or scarring was found in one or two animals of each group, and early cataractous changes were present in a like number. Suppurative panophthalmitis was present in a single animal of group 1X. K. NEOPLASMS A total of 105 neoplasms was encountered in 74 animals. With the exception of an adenocarcinoma of uncertain origin in an OX female and a thoracic fibrosarcoma in a 1X female, the neoplasms have been described above. Together the adenofibromas of mammary gland and the neoplasms of endocrine glands accounted for approximately 90% of the total number, The simultaneous occurrence of neoplasms of various endocrine glands and of adenofibromas with neoplasms of endocrine glands accounted for the majority of instances of multiple neoplasms in a single animal, The total incidence of benign and malignant neoplasms in the several groups is given in Table VIII. V. DISCUSSION One disadvantage of irradiation is its enhancement of the rate of decay of foodstuffs which normally spoil by enzymatic rather than microbial actiono When the potatoes for this experiment were obtained and irradiated, the conditions under which irradiation of potatoes could be conducted without appreciable decay occurring were not appreciated. These conditions are: (1) delay irradiation as long as practicable after harvesting to permit suberization to heal harvest injuries; (2) use a minimum dose, e.g., 5000 rads which inhibits all but a tolerable amount of sprouting; and (3) minimum handling of the potatoes after irradiation because suberization and periderm formation are also inhibited by irradiation. The potatoes used in this experiment were irradiated in December, about three months after harvest. A longer pre-irradiation storage period would have permitted maximum suberization and an opportunity to cull damaged potatoeso The dose used was several times that necessary for sprout control. The high dose, however, did serve as a safety factor for the wholesomeness test. Of all the factors, however, the one which most seriously compromised the experiment was the excessive amount of shipping and handling to which these potatoes were subjected after irradiation, as mentioned early in the report, Whether or not the high mortality rate of first- and second-generation animals stemmed from the conditions of potatoes fed will be discussed below. The sub-optimum condition of the potatoes, however, did not appear to affect the nutritional value of the diet. Growth, food efficiency, and reproduc25

tive performance were optimum., Witn minor exceptions, no differences in these criteria could be traced to irradiationr of the potatoes in the diet. The firstgeneration females fed the "1X` dievt^ appeared to attain a significantly higher body weight than did females fed the other two diets, but this effect did not appear with the females of the second and third generation fed the same diet. Nothing appeared from the periodic blood cell counts which revealed an important difference among the various groupso Practically all ranges of values overlapped. The males fed the IX diet maintained a lower white blood cell count throughout most of this period, but considering the small numbers and the variations involved, this is not of st atistical significanceo With regard to organ weight-body weight ratios, there was only the higher lung weight in the males fed the irradiated diets, and this may have been influenced by the increased incidence of respiratory infection in these groups, as noted later. With regard to the fact that the mortality rate of two generations of both male and female rats fed the irradiated potato diets was consistently, if in some cases only slightly, higher than those fed the control diets, a conclusion of unwholesomeness of such potatoes cannot be necessarily drawn. Irradiation promoted decay in the potatoes used in. this experiment, and some of this was present in the diet early in the experiment. Delayed effects of this decay may account for the higher mortality of the males fed the irradiated diets. Although the effect was not proportional to the amount of decay included, and although there was little if any effect on the females, this factor cannot be ruled out. This factor cannot explain the second.-generation mortality since the practice of removing all decay from iThe potatoes fed started before the second-generation was borno There may, howe-eer, be another reason for that mortality pattern, This appears in Table IX', whiich shows the relat;ion of mortality of secondgeneration animals to that of their parents by the nineteenth month of the experiment At this time, the mortality in the second-generation control group reached 7%, that in the IX group, 27%, and. that in the 2X group, 23%o The table TABLE IX NUMBER OF SECOND-GEtNE-RATIO' ANTMIALS (LEFT OF HYPHEN) AND NUMBER DEAD AS OF 15 OCTOBER 1957 (RIHiT OF HYPEHN) ACCORDING TO WHETHER NEITHER, ONE, OR BOTH PARENTS HAD DIED BY SAME DATE, BY DIETARY GROUP.... _ Parents Dead. _Diet Neither One Both OX 18-1 12-1 0-0 iX 7-0 17-6 6-2 2X 4-o 22-5 4-2 26

shows that, considering all groups together, the mortality rate of second-generation animals, both of whose parents were still living, was only 3%, of those of whose parents one had died, 32%, and of those of whose parents both had died, 40o% In the control group where one parent had died, it was usually the mother; in the 1X and 2X groups it was usually the father, a point ruling out a predominant effect of maternal diet upon the survival of offspring. If it is reasonable to assume that early offspring mortality is related in some way to early parental mortality, then it should also be noted from the table that more than half (60%) of the second-generation animals in the control group happened to have come from parents both of which were still living, and none from parents both of which had diedo In the groups fed the 1X and 2X diets, however, only 23% and 13%, respectively, had come from parents both of which were living, and 20% and 135, respectively, had come from parents both of which had died. The parent-generation males and females were random-mated, and it was a matter of chance which particular males and females became parents. It thus appears that second-generation animals fed the irradiated potato diets quite by accident had a larger share of parents whose survival or resistance to disease was low. Whether the mortality in both generations was primarily due to the vascular disease which in turn was under genetic influence is the subject of a 11 later papero The question of what accounted for the apparently accelerated mortality of first-generation males in groups 1X and 2X after about 16 months is significant0 While it is difficult to designate an immediate mechanism of death in an older animal with long-standing disease, the major pathological findings at necropsy must be construed to be of ultimate causal significance. These findings have been analyzed for those males already dead at a time when the differences in the mortality rate of groups 1X and 2X from the others had become clearly established. By this time, about 16 months, 42 of the 75 males had died or were sacrificedo The major findings are presented in Table X. In spite of the higher mortality of the males fed the irradiated potato diets, similar to that shown in Table VII for 19 months, there is no great difference in the incidence of the arteritiso In some animals it was difficult to assign precedence to the respiratory infection or the arteritis, but there was a considerably greater incidence of major respiratory infection in the IX and 2X animals relative to the controls than there was of the arteritiso Since twothirds of the males fed each of the potato diets, irradiated or not, were susceptible to some degree of necrotizing arteritis, it appears that the more frequent presence of severe respiratory infection in the 1X and 2X males may have been a factor accounting for the additional mortality. With regard to the pathological findings in general in this study, the diseases encountered in each group were of the same type as encountered in the other groups although of somewhat different incidence in some instances. No qualitative differences in the pathological findings could be attributed to irradiation of the diet. However, several disease processes stand out by virtue of their 27

TABLE X MAJOR PATHOLOGICAL FINDINGS IN THE FIRST 42 FIRST-GENERATION MALES WHICH DIED OR WERE SACRIFICED (BY ABOUT 16 MONTHS) Dietary Group OX iX 2X XL Number of deaths 6 14 13 9* Number in which the following was the major pathological finding Arteritis 4 4 6 2 Arteritis with respiratory infection 1 4 4 0 Respiraratory infection alone 0 2 2 1 Miscellaneous 0 1 0 2 No cause of death evident 1 3 1 4* Three were sacrificed in apparent good health for comparative purposes only. frequency in all groups, or by virtue of a somewhat higher incidence in the 1X and 2X groups. The high incidence of a severe and widespread necrotizing arteritis in groups OX, 1X, and 2X all but eclipsed other pathological findings. The incidence of this disease in the three groups was approximately the same, and thus irradiation of the potatoes in the diets of groups 1X and 2X cannot be considered to be the etiologic factor. A similar process has been described in this species under various conditions. 14 The much lower incidence of this disease in animals of group XL and the fact that they were affected much later in life than animals in the other three groups suggest that the high incidence of the disease may be related to factors in the potato diets unrelated to irradiation. The detailed analysis of this arteritis, since unrelated to irradiation, is beyond the scope of this study, and will be considered in a subsequent publication. The great susceptibility of this species to respiratory disease is well known.l5 As shown in Table VII, severe bronchiectasis was common in all groups and was somewhat greater in the 1X and 2X groups. As pointed out above, severe respiratory disease may have been a factor in the apparently increased early mortality of the males of group 1X and 2X. However, the occurrence of respiratory disease in general in the rat is extremely frequent, and our tabulation takes into account only the "end stage" of respiratory disease, and not the frequently observed lesser degrees of inflammation. These facts must be considered when evaluating the significance of the apparently increased incidence of respiratory disease in the 1X and 2X males. 28

Middle ear infections were extremely common in all groups, regardless of diet (Table VII). This incidence is comparable to that reported from other colonies,1 and there was no evidence suggesting any effect of irradiation of the diet in this regard. The high incidence of neoplasms in this study was of interest, but certainly not unprecedentedo17918 The frequent occurrence of adenofibromas of mammary gland17-19 and of neoplasms of endocrine glands20 has been reported previously; these two groups of neoplasms account for most of those encountered in this study. There was no suggestion of any signif:iant difference in the incidence of neoplasms related to irradiation of dietary componentso VI. REFERENCES lo Sparrow, Ho H., and Christensen, Fo, "Improved Storage Quality of Potato Tubers After Exposure to Cobalt-60 Gamma," Nucleonics, 12, 16 (1954). 2. Brownell, L. E., et al., Utilization of the Gross Fission Products, Univ. of Mich. Eng. Res, Insto Report 1943-17-P, Ann Arbor, Mich., November, 1954. 35 Brownell, L. E., et al., "Storage Properties of Gamma-Irradiated Potatoes," Food Technology, 11, 306 (1957). 4. Lehman, A. J., et al., "Procedures for the Appraisal of the Toxicity of Chemicals in Foods, Drugs, and Cosmetics," Food Drug Cosmetic Law Journal, 10, 679 (October, 1955). 5. Cfo Progress Reports to Uo S. Army Quartermaster Food and Container Institute, 1819 Pershing Road, Chicago, Illinoiso 6. Method for specific gravity obtained from Dro Donald Islieb, Farm Crops Dept., Michigan State Univ., East Lansing, Michigan. 7. Sherman, Henry C., Chemistry of Food and Nutrition (The MacMillan Co., New York, 1946), p. 629. 8. Waibel, P. E., Bird, H. R., and Baumann, C. A., "Effect of Salts on the Instability of Thiamine in Purified Chick Diets," J. Nutrition, 52, 273 (1954). 9. Lutz, R. E., J. Indo Hyg., 8, 177 (1926). 10o Brenchley, W. Eo, Ann. Appl. Biol,, 26, 671 (1978). 11. Brownell, Lo Eo, Burns, Co Ho, and Abrams, Go Do, in preparation, 29

12. Wilens, S. L., and Sproul, E. E., "Spontaneous Cardiovascular Disease in the Rat. II. Lesions of the Vascular System," Am. J. Path., 14, 201-216 (1938). 13. Ingle, D. J., and Baker, B. Lo, "A Consideration of the Relationship of Experimentally Produced and Naturally Occurring Pathologic Changes in the Rat to the Adaptation Diseases," Reco Prog. Horm, Res., 8, 143-169 (1953). 14. Masson, GoM.Co, Hazard, J. B., Corcoran, A. C., and Page, Io Ho, "Experimental Vascular Disease due to Desoxycorticosterone and Anterior Pituitary Factors," Arch. Path., 49, 641-664 (1950). 15. Ratcliffe, H. L., "Spontaneous Diseases of Laboratory Rats," in: The Rat in Laboratory Investigation, Farris, E. J., and Griffith, Jo Qo, Jr. (Eds.), J. Bo Lippincott Co., Philadelphia, 1949, 2nd ed., ppo 519-520. 16. Nelson, J. B., and Gowen, J. W., "The Incidence of Middle Ear Infection and Pneumonia in Albino Rats at Different Ages," J. Infect. Dis., 46, 53-63 (1930)o 17. Davis, R. K., Stevenson, Go T., and Busch, K. A., "Tumor Incidence in Normal Sprague-Dawley Female Rats," Cancer Reso, 16, 194-197 (1956), 18. Crain, R. C., "Spontaneous Tumors in the Rochester Strain of the Wistar Rat," Am. J. Patho, 34, 311-336 (1958)o 19o Wright, Ao Wo, Klinck, Go H., and Wolfe, J. M., "The Pathology and Pathogenesis of Mammary Tumors Occurring Spontaneously in the Albany Strain of Rats," Am, Jo Patho, 16, 817-834 (1940), 20. Gilman, J., Gilbert, Co, and Spence, I., "Phaeochromocytoma in the Rat; Pathogenesis and Collateral Reactions, and Its Relation to Comparable Tumors in Man," Cancer, 6, 494-511 (1953). 30

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