Carcinogenic Action of Some Substances Which May Be a Problem in Certain Future Industries

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ATOMIC ENERGY COMMISSION CARCINOGENIC ACTION OF SOME SUBSTANCES WHICH M A Y BE A PROBLEM IN CERTAIN FUTURE INDUSTRIES Argonne National Laboratory Published for use within the Atomic Energy Commission. Inquiries for additional copies and any questions regarding reproduction by recipienb of this document may be referred to the Technical Information Division, Atomic Energy Commission, P. 0. Box E, Oak Ridge, Inasmuch as a declassified document may differ materially from the original classified document by reason of deletions necessary to accomplish declassification, this copy does not constitute authority f o r declassification of classified copies of a similar document I which may bear the same title and authors. Date of Manuscript: Unknown This document consists of 13 pages.

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CARCINOGENIC ACTION OF SOME SUBSTANCES WHICH MAY BE A PROBLEM By Austin M. Brues, Hermann Lisco, and Miriam Finkel The work to be presented was a part of the program of the Health Division of the Manhattan Engineer District, Metallurgical Laboratory, and was performed at the University of Chicago. It was necessitated by the nature of the work done in developing the atomic bomb. In the course of manufacturing plutonium in a chain-reacting pile, products of uranium fission a r e evolved; although these atomic split-products a r e relatively small in terms of total mass, they a r e largely radioactive inasmuch as nuclear fission produces many isotopes of normal elements, and an almost imperceptible amount of chemical material may therefore emit an amount of radioactivity which is lethal to a large animal. These products must be separated from the pile uranium as part of the plutonium separation process. In addition t o the radioactive fision products, it was necessary to investigate the toxicity of plutonium itself, which gives off alpha particles and about which literally nothing was known at t h e time these studies were initiated. Another industrial hazard is the radiation given off from the pile in the course of its operation. Dr. Paul Henshaw will discuss the latter problem in the following paper. Our particular assignment was to investigate the late or chronic effects of exposure to the fission products and plutonium. This was, of course, an especially important part of the toxicity studies, since it is a relatively simple matter t o determine the acute toxic doses for such material and to institute measures protecting personnel from such amounts It is well lmown, however, that chronic lethal effects can be produced by amounts of radium much smaller than those required to produce acute damage to tissues and organs, and this might be true of any radioactive material which was retained in tissue and which gave off radiation over a long period, as is the case with radium, The Manhattan District ,was determined not to encounter a delayed surprise such as that which greeted the radium painting industry when a high incidence of bone sarcoma began to appear in its workers years after exposure. Certain characteristics of the fission products a r e worthy of mention. They occupy the middle of the periodic table and thus include most of the r a r e earths, with the addition of iodine, barium, and strontium. The half-life of these radioactive substances is widely variable, from a fraction of a second to many years. Those of very short half-life a r e to be considered relatively unimportant in t e r m s of chronic toxicity hazard, since their radioactive energy will have be%omedissipated in the course of the “cooling” period before pile uranium is subjected to chemical separation. The effects may be expected to depend on the half-life, therefore, (determining the duration of tissue exposure) and also upon the nature and energy of the radioactive emanation, and on the metabolism and distribution of the element itself, inasmuch as the latter factors will determine the distribution of irradiation damage in the body. To cite extreme illustrative examples, one would expect sodium24, with its almost uniform distribution in extracellular water and its high-energy gamma-ray emission, to subject the body to an almost uniform flux of radiation with effect

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presumably nearly identical with those of total body external gamma or X-radiation; while plutonium, with a short-range alpha particle and discrete tissue distribution, may be expected to distribute its radiation in the body in a highly non-uniform pattern resulting in extreme exposure of certain cells, relative to the total radiation received by the whole With these considerations in mind, experiments were set up to observe the effects of radioactive strontium, cerium, yttrium, zirconium -columbium mixture, and plutonium. Although there is no public information, of course, as to the total amounts of these substances which may have been produced, attention can be called to the table of fission products published in Science for June 14, 1946, in which it is shown that Lrtdividual investigators may expect to obtain the fission products named in batches of one curie each for investigative purposes. This should give an impression of the extent of the hazard encountered in work involving chain reactors. The major part of the experiments was done with strontium89 and plutonium, with pilot experiments set up to gain information regarding the effects of the other substances. Radium was also utilized in order to make possible comparison with previous work on animals and observations on humans, The Sprague-Dawley strain of r a t s was used in all of the rat experiments; the mice used were Carworth CF-1 strain and ABC hybrid mice obtained from the Roscoe B. Jackson laboratory. These mice were chosen because of their availability in large quantities, and because many of the standard strains of mice have special tumor incidence characteristics making them undesirable for work designed primarily to gain information as to the nature of a general tumor hazard, not knowing 5 priori where the chips might fall, The ABC mice, as judged by information obtained froin Dr. Arthur Cloudman, fulfilled this criterion well, and those used had all been bred in such a way as to eliminate the milk factor of the parental and grandparental A strain. The CF-1 mice, more uniform genetically, were believed to be relatively free of milk factor and to have no particular tumor bias, although we could obtain no data as to exactly what might be expected of these mice if maintained over a long period. For purposes of a special problem, a few high-lymphoma C58 mice were obtained through the kindness of Dr. MacDowell; in addition, several hundred rabbits and a few dogs have been utilized. No important observations on chronic toxicity to dogs have yet emerged. The rats and mice were housed in an animal f a r m air-conditioned to permit regulation of temperature and humidity, and triethylene glycol vapor was used to minimize the possibility of cross-infection on an air-borne basis. Of the total of 30,000 mice and rats used in toxicity studies at the Metallurgical Laboratory, about one-third were observed over an extended period and constitute the chronic toxicity study to date. The first of these experiments was begun_850 days ago. This consisted of a s e r i e s of 350 mice injected with by Dr. C. L. Prosser. A t the time when it became possible t o set up a much larger series of mice injected with this isotope, 40 weeks later, it had already become evident that we might expect a very high incidence of bone tumors in those m b ? receiving high (close to lethal) doses, and it became desirable to discover at what lower dose bone tumors no longer occurred. For this reason the experiments were

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designed in such a way that pragressively l a r g e r numbers of animals received progressivelv smaller doses, until the largest number of mice fell in the control group. Most of the other experiments were carried out in the same fashion. Once an experiment was set up, its component groups of animals continued to be caged together until the end; animals in a given e’veriment and their controls were housed in the same room, with a single exception; in certain large experiments a part of the control mice were housed in a room f r e e f r o m treated animals in order to free them from the minimal amount of radiation they might receive in the vicinity of treated mice. This was probably an unnecessary precaution, since there has been no evidence that this homeopathic exposure caused any difference in the results. A l l animals were observed daily; certain animals showing tuniors. pallor, emaciation, listlessness, or other signs of disease were observed with particular c a r e and sacrificed when 9 extremis. Complete autopsy (with the exception of the central nervous system) was done on all animals, sections were taken whenever indicated, and the carcasses preserved in 4% formaldehyde. Just before death hemoglobin and white cell count were determined and blood films were made; roentgenograms were taken on all animals. Since many animals a r e still living and since pathologic diagnosis is inconiplete in the case of many others, this is to be considered a progress report on the The acute lethal dose (LD50 at 30 days) for Sra9 in mice is 8 microcuries per gram body weight. The highest dose used in chronic experiments was accordingly 5 microcuries per gram. A parallel series was used in which injections were given each month in order to maintain a level of dosage, since decay of the isotope and excretion result in 80% loss of activity within the month. The design of the largest strontium experiment is given in the accompanying table. DESIGN OF LARGE MOUSE EXPERIMENT WITH sra9 Single Injection Monthly Injections Number of Mice Number of Mice

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These mice were divided equally between CF-1 female, ABC female and ABC male mice. The testiness and belligerency of C F - 1 male mice as they approached the end of the first year of life, T.$lth resulting perineal trauma and urogenital infection, made them undesirable for lifetime e‘xperiments. Strontium, because of its :lose chemical similarity to calcium, is mainly deposited in bone, as is also true in the case of radium. The energy of beta particles of Sra9 is such that its energy is largely dissipated within 1/2 mm Gf the site of deposition. Thus bone, bone marrow, and the immediately surrounding tissues a r e t h e site of virtually all of the Although it is the main purpose of this paper to discuss carcinogenesis, certain other observations on results of reiatively high dosage may be of interest. Most of the pigmented mice receiving vieri 1ri;crc;curie pr gr zm showed greying of the hair over the skull and spine, apparently where the hair follic!es were within range of the radioactivity. Leukopenia, lymphopenia, and hypochromic anemia were frequently seen, while myeloid metaplasia and tiyperplxjitl uf lymphoid tissues have also appeared with considerable frequency in relation to thc higher dwes. Mammary Cumors.--- Bot!l strains uf inice have very low incidences of mammary tumors. The overall incidence in this esperiment at the present time (500-600 days of age) is 1.7 per cent in CF-1 females and 0.9 per cent in ARC females. No evidence has yet appeared suggesting ihat thr: trcatrrrect influences the incidence of these tumors. origin. These include icmlized lyniphosarcomas and lymphoblastomas of any or all of the lymphoid tissues, whether o r not a leukemic blood picture is present. Somewhat less than half of the animals m t h this group of diseases shows a blood picture suggestive of leukemia, although mild anenlid is oiten present. It has Seen impossible to distinguish the leukemic cases on the basis of gross autopsy findings. Lymphoma is a common disease of both strains, and its incidence appears to increase with advancing age. There is some evidencc! that radioactive strontium has a positive effect on lymphoma incidence, although much less strikingly than total body radiation. Table 2 s h w s the incidence of lyniphoma in rolation 1-0 treatment at 450 days. INCIDEXCE OF LYMPHOMA IN CHRONIC Sr8’ MICE Treatment Number Lf Mice Number With Lymphoma P e r Cent Lymphoma All low doses

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These figures have statistical significance, but it is too soon to determine whether the increased incidence is due to acceleration of the normal rate of lymphoma development o r to induction of lymphoma de novo. Bone Tumors. Radioactive strontium is par excellence a producer of bone tumors. Under the conditions of the experimental work in our laboratory, it has proved to be a much more effective agent to this end than radium, whose effect in this regard is well known. The tumors a r e largely (about three-fourths) osteogenic, the remainder being fibrosarcomas. Metastases have frequently occurred, but are more often seen in rats than i n mice. The sites most frequently encountered are the femora, humeri, and tibiae, 70 per cent of the bone tumors being in the extremities and the rest in the jaw, pelvis and spine. The Incidence of bone tumors in relation to dosa?e is shown in Table 3. INCIDENCEOF BONE TUMORS FROM sra9 Figures show per cent of animals surviving the t i r e of appearance of the first tumor which developed bone sarcoma up to the present writing (400 to 500 days) 8ex influence. It might appear from Table 3 that there is a stronger tendency to bone tumor development in ABC females than in males. In all groups With tumors, there are 155 males with 36 tumors and 134.females with 2 1 tumors. This difference lacks tumor development is influenced only moderately by the dose, and that when the latent period is ended, tumors appear with considerable rapidity of succession. Time of tumor development. This is strikingly shown in Table 4. It is seen that time of

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TIME FROM FIRST TREATMENT TO DEATH WITH BONE TUMOR First tumor Mean of tumors No. with tumors Effect of age on bone tumor development. This was looked for in a small series of mice which were first injected with Sr89 at about 7 1/2 months of age. Heavy mortality due to salmonella infection has prevented the survival of a significant sample of these mice beyond 200 days, but the one surviving mouse developed a tumor at about the expected time. In another experiment, pregnant female mice were treated late in pregnancy and amounts were transferred to fetuses comparable to those given the large series. Although this experiment is not complete, it does not appear that there h a s been any acceleration of bone tumor development where the bones received their agent during the period Of most, rapid growth. Many of the mice which thus received SrB9 at about the time of birth, and contained at birth about 5 mc/kilo, have shown stunting of growth and bending of the atrophied long bones. Other s ecies A few rats and rabbits have also been observed after single injections injection of 1.6 mg/kilo and ten of 19 rats given 0.6 mg/kilo. They have also occurred …