Document 2JkgGZbr7KyBMBjLmq4pV8prp

2/11/75 Copied for : KWNelson MJMessel FLBickel/GEFox/LRoberts jRCarpenterMOVarner From JPSieverson I thought you would bs interssted in ths conclusions of this study. Ingested Mineral Fibers Do They Penetrate Tissue or Cause Cancer? Paul Gross, MD; Russell A. Harley, MD; Layinka Margaret Swinburne, MRCP, MRC Path; John M. G. Davis, PhD; William B. Greene ?* Three different laboratories Indepen dently Investigated the ability of Ingested mineral fibers to penetrate tissues In rats. All concluded that there was no evidence of tissue penetration by ingested mineral fibers. These experimental observations are supported by the findings in coal and hard-rock miners who swallow, during their lifetime, nearly 100 times the amount ol dust that is stored In their lungs. The Intestinal wall or mesenteric lymph nodes of these people show no evidence of stor age of the Ingested dust particles. AnlrnalsJetlasbestos over much of their lifetime and allowed lo live to the age qf cancer production, failed tn provide evi dence ol a cancerogenlc effect. Submitted for publication May 31, 1974; ac cepted Aug 9. 1974. From the Medical University of South Caro lina, Charleston (Drs. Gross, Harley, and Mr. Greene). St. James's Hospital, Leeds, England (Dr. Swinburne), and the Institute of Occupa tional Medicine, Edinburgh (Dr. Davis). Reprint requests to the Department of Pathology, Medical University of South Carolina, 80 llarre St. Charleston. SC 29401 (Dr. Gross). Because of the association of peri toneal mesotheliomas with asbes fibers of submicronic dimensions oc cur naturally in river water as it tos exposure, it was suggested thaterodes serpentine and amphibole out inhaled asbestos fibers cleared from croppings. Increased industrial appli the lungs and swallowed, migrated cations of mineral fibers may have through the gastrointestinal (GI) wall accelerated this ingestion somewhat. to the peritoneum there to initiate Nevertheless, the world literature the cancer. The more recent discovery does not suggest that an alarming in - of submicronic mineral fibers in crease in GI inflammatory disease or beers, wines, and drinking water has cancer has occurred, or that the inci resulted in publicized expressions of dence is higher in regions where as fear that a health hazard exists. bestos fibers are present in natural A recent investigation1 suggests waters. that the lungs of city dwellers not oc Because of the publicly expressed cupationally exposed to asbestos may fears that ingestion of beverages or contain many millions of submicron food containing submicronic mineral sized fibers. A percentage of these fi fibers may expose large populations bers have been identified as chryso- to a cancer-producing hazard, respon tile. Amphibole asbestos fibers have sible public officials arc demanding also been identified in the lungs of that the scientific community confirm city dwellers but their amount or con or refute the assertion that a health centration has not been determined.5 hazard exists. The present report is Since only about 1% to 2% of the intended to be a response to this de dust inhaled during a lifetime is re mand. A portion of the work here re tained in the lungs, the other 98% ported was begun nine years ago, to 99% being transported via the pul long before the possible consequences monary clearance mechanism to the of asbestos ingestion became of wide pharynx,3 it is probable that the GI spread interest. However, because of tract will be the recipient of much their negative nature, the results of the inhaled and subsequently in were not published. Two other labora gested dust particles, nonfibrous as tories more recently did publish nega well as fibrous. tive results of asbestos feeding stud Ingestion of asbestos fibers has un ies: one, as an abstract or short note'; doubtedly been in progress in some and the other, as part of a discussion countries for centuries, inasmuch 4$ on the dangers of nitrites in food.'1 Arch Environ Health/Vol)29, Dec 1974 Ingested Mineral Fihers/rirncc *! 141 HER 0001298 When it was learned recently that three laboratories were either en gaged in a study on asbestos inges tion or had completed it, agreement was reached to pool the results of these studies in the present joint paper. Taconite tailings from an iron mine, containing submicronic mineral fibers have been piped into Lake Su perior for nearly 20 years. Because some ultramicroscopic mineral fibers have been discovered recently in the drinking water from this lake, dust from this mineral is included in these studies. MATERIALS AND METHODS Chrysotile Feeding Ground laboratory chow was intimately mixed with ball-milled chrysotile asbestos (consisting of the whole spectrum of fiber lengths and diameters) so that the latter constituted 5% by weight of the mix. This was supplied along with water ad libitum to ten male rats for 21 months at which time all were killed. There was no time pe riod between the asbestos feeding and death to allow the GI tract to rid itself of the ingested asbestos. Five laboratory con trol rats of the same age as the asbestosfed animals were killed at the same time as the latter. The GI tract of the animals was care fully examined for lesions and random blocks of the tissue were removed for par affin impregnation and subsequent sec tioning. Blocks of tissue were also removed from the thoracic and other abdominal or gans. The remainder of these tissues was placed together in a jar of formaldehyde solution for possible future study. Because no thought was given at that time to the possibility of transmigration of fibers, tis sue from the GI tract was not isolated from other tissues and no attempt was made to keep the formaldehyde solution that had been contaminated by asbestoscontaining. fecal material from contami nating the other tissues with asbestos fibers. Paraffin sections, cut at 6pm and stained with hematoxylin-eosin, were made of the various tissues. One-millimeter blocks were cut from the fixed tissue of the GI tract after nine years sojourn in formalde hyde solution and embedded in epoxy resin for electron microscopic examination. Weighed portions of the large and small intestines, and of the mesentery as well as of the other organs were processed as de scribed below for determination of their mineral fiber content Administration of Fibrous Minerals by Gavage Amosite and taconite mine tailings were finely ground in a mill (Spex) so that an aqueous suspension of either dust could be ejected through an 18-gauge needle. Un anesthetized rats were given 1 ml of sus pension containing 400 mg of the dust by gavage, using an 18-gauge blunted needle. Because some of the animals struggled during the gavaging, a certain amount of trauma occurred. Ten rats were given amosite and ten, taconite. Five rats of the same age were left untreated as controls. Two gavaged rats from each series and one control animal were killed at 24 and 48 hours, and one, two, and three weeks later. At autopsy, the GI tract was carefully removed so that the remaining tissues did not become contaminated with the content of the GI tract Weighed portions of the various tissues were processed in a manner similar to that of the preceding study and according to the method to be described. Oleomargarine Feeding Because of the likelihood of traumatic hematogenous introduction of mineral fi bers during the gavaging--a possibility recognized belatedly--a new series of ani mals was initiated. For this series, finely ground amosite and taconite tailings were each intimately incorporated into oleo margarine to obtain a 10% amosite content or a 20% taconite content. Rats were housed in individual cages (ten for each mineral) and deprived of all food except for 50 gm of the dust-containing oleo. Dur ing this time (six days), the dust consump tion per rat ranged from 2.2 gm to 5 gm amosite and from 6.0 to 10 gm taconite. Ten control rats received no dust. One, two, three, four, and five weeks af ter completion of the dust-feeding period, two rats from each series were killed. Two control rats were killed at the third, fourth, and fifth week, and one at the first week (three other control rats had died during the night). At autopsy, weighed portions of the GI tract and of other organs were pro cessed according to the method described below. Blocks of intestinal wall fixed in formaldehyde, 1 mm in diameter, were re moved for electron microscopic study. Butter Feeding In the second laboratory, three ex periments were conducted: a preliminary short-term study, and two long-term ex periments. The animals were SPF rats of the Wistar strain, initially about 12 weeks old. The asbestos was sieved to a fiber length up to 100pm and was incorporated in butter so as to result in either a 0.4% or 0.2% mixture. 1. In the preliminary study, a small number of rats were given a single dose of either 20 or 40 mg crocidolite asbestos. Fe cal pellets were examined by optical mi croscopy and after monatomic ashing at 39.7 mm Hg, at 200 w, from between 15 minutes to one hour, at intervals from six hours to one week after feeding. The ani mals were killed from between 1 week to 19 days and were dissected so as to avoid contamination by gut contents. The gut and other tissues were fixed in formalde hyde solution. Paraffin sections (6pm thick). were examined by optical microscopy and samples of tissue were also subjected to monatomic ashing as described for fecal pellets. 2. Groups of rats were fed according to the following schedules once weekly for 16 weeks: 31 rats--10 mg Rhodesian chryso tile; 33 rats--5 mg crocidolite of unknown origin; and 34 rats--10 mg crocidolite of the same unknown origin. On the same sched ule a control group of 24 rats was given the butter only. All rats were fed a normal diet of standard pelleted food in addition. 3. A group of 35 rats was fed once weekly for 18 weeks 10 mg of North West Cape crocidolite, and another group of 28 rats received 10 mg of Transvaal crocido lite on the same schedule. There was also a butter-fed control group of 24 rats. All of these rats were also additionally fed-a nor mal diet of standard pelleted food. A few animals from each group of each experiment were killed in the first six months to assess any possible intermediate pathological changes in the abdominal re gion or elsewhere. The remainder were al lowed to survive until they became ill or died a natural death. At autopsy, any exudate present in the body cavities was aspirated and examined microscopically. Where no exudate was present, the cav ities were washed out with a small volume of normal saline and also examined for the presence of cells or asbestos fibers. The lungs were inflated in situ with formalde hyde saline and removed intact. All tissues suspected of neoplastic change were examined histologically. In addition, the major organs, abdominal wall, and chest wall, and lymph nodes were examined in a proportion of animals from each batch (53 in all). Samples of this tissue were also subjected to monatomic ashing to facilitate the recognition of as bestos residues. Long-Term ad Libitum Feeding of Asbestos in Butter These studies, in the third laboratory, on the penetration and transmigration of in gested asbestos fibers have not been com- 342 Arch Environ Health/Vol 29, Dec 1974 Ingested Mineral Fibers/"'*- al j HER 0001299 plcted but the data that have been ob tained on a few animals are included here because they complement and supplement the information obtained by the other two laboratories. Cbrysotile and crocidolite, properly ground, were intimately mixed with butter to make two batches, each containing 5 mg/gm of butter of one or the other type of asbestos. Two groups of 12 rats each were fed one or the other type of asbestos ad libitum. Animals were killed one and three months after the start of ingestion and sections of the esophagus, stomach, small intestine, cecum, colon, mesenteric lymph nodes, liver, spleen, and lung were prepared for light microscopy and trans mission electron microscopy. Preparalion of Tissue Digests (First Labo ratory)---Weighed amounts of tissue gener ally between 1 and 5 gm were added to about 20 ml of 5% sodium hypochlorite (commercial bleach) and allowed to be di gested. In order to accelerate the digesting process, the mixture was placed in an oven at 50 to 60 C. It often became necessary to add new hypochlorite solution to the tissue in order to carry the process to completion. When a clear solution was obtained, it was centrifuged and the sediment washed three times with large amounts of unfil tered tap water. Care was taken during the washing so as not to lose any sediment. The latter was finally made up to a volume of 2 ml with distilled water and 20/d of the well-mixed suspension was placed on an electron microscope grid. Photographs were taken of all structures that had a greater than 1:3 aspect ratio. Differentiation of Electron MicroscopeSize Fibers (First Laboratory).--Some prepa rations initially were not satisfactory and resulted in enigmas that were resolved when structures that had been interpreted as fibers by light microscopy were found to be needle-like crystals. These readily dis solved and disappeared when the suspen sion was acidified. Digestion of Tissue vs Tissue Section: Relative Sensitivity of Methods.--Consid ering the weight of tissue in a section on an electron microscope grid to be about L5x 10 mg and the amount of digested mesenteric tissue represented in a 20/U drop on an electron microscope grid to average about 23 mg, then the latter grid represents nearly 19,000 times as much tis sue as the grid containing a section of tis sue 1-mm square and 1pm thick. In other words, if only one fiber were found on a grid containing a digest of mesenteric tis sue, it would have been necessary to exam ine approximately 19,000 sections in order to find that one fiber in the same tissue. Although the digestion method is ca- pablc of sampling amounts of tissue many thousand limes greater than can reason ably be sampled with the method using sections 1-mm square and 1pm thick, it also has the disadvantage of being suscep tible to give false-positive results. These may be caused by incomplete digestion of tissues giving rise to fibers in approxi mately the same size range of those in gested, but distinguishable from the latter in other respects. Another, more trouble some, cause of false-positive results is con tamination. The contamination could theo retically be avoided by careful technique during the autopsy of the animal, the re moval and weighing of the tissues, and by conducting the digestion and the prepara tion of the digests as well as of the grids in a clean room supplied with filtered air. It would also be necessary to use only water and reagents that had been passed through a Millipore filter. RESULTS Chrysotile-Fed Rats (First Laboratory) Although they had ingested a large amount of asbestos each day for al most two thirds of their lifetime, the rats had thrived and appeared healthy. There were no deaths in this group. At autopsy, no lesion could be found by gross examination in the GI tract of these animals. Random par affin sections of the GI tract likewise revealed no important abnormality microscopically. Sections of the other organs showed no evidence of dam age. As might have been expected, the digests of the tissues of the GI tract that included the mucosa and its covering material, when examined with the electron microscope, con tained chrysotile fibers. However, no fibers were found in the sections of intestines examined with the electron microscope. What appeared at first glance to be of considerable importance was the finding of fibers in the digests of mes enteric tissue of three rats. In one, a chrysotile fiber was found. In the other two, the fibers were nonchrysotile. In a fourth rat, two nonchrysotile fibers were identified in a digest of a mixture composed of cardiac, pulmo nary, renal, and splenic tissues. However, two control rats were al so found to have chrysotile fibers in the tissue digest of the GI tract One of these two controls also had a non- chrysotile fiber in the same digest. All fibers were, of course, submicronic in dimensions. Asbestos and Taconite Dosage by Gavage (First Laboratory) One rat, gavaged with amosite and killed 48 hours later was found to have a lacerated and perforated esoph agus. Much of the amosite was extravasated into the paravertebral muscles in the form of a streak paral lel to the esophagus. The digests of the mesentery', kidney, and lung of this animal contained fibers consis tent with amosite as well as other fi bers. This is the only rat out of eight gavaged with amosite and examined that showed fibers in the digest of tis sues outside of the GI tract. (Two other rats gavaged with amosite had died overnight and were discarded.) In only one out of ten rats gavaged with taconite were fibers found in a digest of tissues other than those of the GI tract. In the digest of lung tis sue from this animal, three fibers were found. Six of eight rats given amosite showed fibers in the digest of GI tract tissues and similar findings were made in the digests of the GI tract tissues of two out of ten rats given taconite tailings. No digest of tissues from the GI tract of rats killed after the 48-hour period contained fibers. A chrysotile fiber was found in the di gest of GI tissue of a rat gavaged with taconite tailings. Of the five control rats killed with this series of gavaged animals, uric was discarded because it died during the night. The digest of mesenteric tissue of one control contained two fi bers. A fiber was also found in lim di gest of kidney from another coi'tn.l rat. Feeding Wilh Oleomargarine (First Laboratory) The digests of the GI tr.v t * . animals fed with margari".- tained a few fibers that wer- . -- tent with those incorporated oleomargarine. A chrysotile : .- >found in the digest of a rat -i ... . site. A single fiber was found in Arch Environ Health/Vol 29, Dec 1974 "I : ' Ingested Mineral Fibers/Gross et 243 HER 0001300 gest of mesenteric tissue of one taco- C'snite tailings-fed rat. On the other hand, the digest of the mesentery of an amosite-fed rat contained a chiysotile fiber and in two other amosite-fed rats the digests of mesenteric tissue contained two fibers each. A fiber with the splayed end typical of as bestos was found in the digest of lung tissue from a rat fed amosite and killed two weeks later. The digests of the tissues of the other animals inclu sive of those of the controls contained no fibers. Electron microscopic study of the sections of intestinal wall re vealed no fibers. The findings so far enumerated are summarized in Table L It should be emphasized that all of the fibers found were of ultramicroscopic dimensions. Butter Feeding (Second Laboratory) Preliminary Experiment.--After a single dose of crocidolite, light micro scopic examination showed abundant fibers in fecal pellets after 24 hours. Examination after monatomic ashing , showed smaller numbers of fibers in ( 48-hour pellets; but thereafter none were detected by either method. Ex amination of the gut and other or gans showed no fibers even after a 40 mg dose. Long-term Experiments.--Study with the light microscope of the tissues of the animals as well as of the aspirates and washings of the serous cavities, revealed no evidence of transmigra tion of asbestos fibers. No mesothe lioma was encountered in these ani mals. Although two breast cancers and one lymphoma of axillary nodes were noted in one asbestos-fed group, similar cancers were seen in the con trol group, three breast cancers, one lymphoma, and one sarcoma of the thigh (Table 2). No lesions of any kind were seen that could be related to the ingested asbestos. Long-Term ad Libitum Feeding ol Asbestos in Butter (Third Laboratory) Light microscopic examination of all tissues of animals killed one and three months after the start of as bestos ingestion showed no asbestos fibers that had penetrated into the tissues. Electron microscopic exami nation of the small intestine and mes enteric lymph nodes is complete for the animals killed after one month, and far advanced for the rats killed after three months. No sign of pene tration by asbestos fibers has been seen. COMMENT Chrysotile Feeding Inasmuch as the rats in this study had been on the asbestos diet up to the time of death, the surface of the gut could be expected to be covered by a fiber-containing film of mucin. These fibers would and did become manifest in the digests of these tissues. Although the presence of the fibers in the other tissues could conceivably be explained on the basis of trans migration, a more probable explana tion would be that the surface of these tissues became contaminated with fibers while in the same jar with the cut segments of the GI tract. The presence of fibers in the tissues of control animals can be explained only on the basis of several embar rassing circumstances. These are con cerned with contamination: one, from the ambient air; another could have occurred when the technician was weighing the tissues. The weighing pan could have been covered by fibercontaining moisture derived from the GI tract of a previous weighing. A third source of contamination could have been the unfiltered water and reagents used in the preparation of the digests. The following conclusions can safely be drawn from these results: 1. Twenty-one months of contact with high concentrations of chrysotile fibers caused no gross or microscopic lesion of the intestinal wall. Inas much as lung cancers" and other chest cancers' induced by asbestos in rats have made their appearance from 16 to 17 months after the first exposure, this 21-month exposure to extremely high dosage of asbestos dust should have been adequate for a cancerogenic action to become manifest if such a potential were present in this mode of asbestos exposure. 2. Electron microscopic examinat tion of the gut wall disclosed no evi dence of transmigration of fibers. 3. Although the method of exam ining the sediment of digested tis sues for the presence of fibers allows for the scanning of the equivalent of a volume of tissue many thousand times greater than can be examined on an electron microscope grid, the former has not yielded incontrover tible proof that transmigration of fi bers occurs. Only one chrysotile fiber was found in the digests of tissues other than those of the GI tract and this, in only one animal out of ten. Furthermore, the importance of this finding becomes doubtful in view of the fact that tissue digests of two control rats also were found to con tain chrysotile fibers, and in the tis sue digest of one of these controls, a nonchrysotile fiber was found. Administration of Fibrous Minerals by Gavage The widespread dissemination of amosite fibers in tissues outside of the GI tract of the rat in which the perforation of the esophagus and ex travasation of amosite into para vertebral muscles occurred is reminis cent of the experiment of Pontefraet and Cunningham." These writers per formed laparotomies on rats and in jected asbestos dust through the gas tric wall. Here, the needle tore through tissues, opening vessels in the path of the needle. Furthermore, the needle track, remaining open for a time, allowed leakage of fibers into the abdominal cavity and onto the cut edges of the laparotomy. When these workers concluded that asbestos fi bers transmigrated from the GI tract to practically all tissues of the body inclusive of blood, they ignored the artifacts inherent in their technique. It is surprising that, with the po tentially traumatic technique of gavaging, only one of 18 rats gave evidence of the widespread dissem ination of fibers characteristic of the Pontefract and Cunningham tech nique and that in another gavaged rat, three fibers were found in the di gest of lung tissue. In spite of the indication that falsepositive evidence of transmigration could have been obtained (as indi- 344 Arch Environ Health/Vol 29, Dec 1974 Inaested Mineral Fihpre/firncc t al j HER 0001301 cated by the presence of a chrysotile liber in the digest of GI tissue of a rat given taconite tailings, and the pres ence of a fiber consistent with either amosite or taconite tailings in the di gest of mesenteric tissue of a control rat), it is noteworthy that following the administration of a single large dose (400 mg) of either amosite or taconite, no evidence of trans migration of fibers was observed in 16 of the IS rats gavaged. Oleomargarine Feeding The results of this portion of the in vestigation indicate that if trans migration of mineral fibers from the GI tract occurs at all, it must be a rare event since only five fibers con sistent with those ingested were found in the digests of mesentery of three rats and four such fibers were found in the lung digests of two other rats (Table 1). Other fibers (five in all) found in the digests of mesenteric tissue of three rats probably repre sented contaminations-such as the chrysotile fiber in the digest of the rat fed amosite. The importance of the nine fibers (Table 1) found in the tissue digests of five of the 37 rats fed or gavaged with mineral fibers must be evaluated in the perspective of the billions on billions of fibers that had been in troduced into the GI tract of these animals. If transmigration of fibers through the GI wall were a fact, one would logically expect more than one to three fibers out of many billions to accomplish this feat, and this phe nomenon should be demonstrable in more than five out of 37 rats. The dose of asbestos ingested by the rats is equivalent to a consumption of 1.1 to 2.5 kg asbestos by a 75-kg man in six days, and double this amount in terms of taconite tailings. The impor tance of the nine fibers found is re duced to a negligible level by the finding in four rats of five fibers that are obvious contaminants, thus point ing to a very high probability that the nine fibers in question are also con taminants. As indicated previously, all fibers were ultramicroscopic in di mensions, and could have been de rived from the air of the laboratory, as well as from the unfiitered water and the technique used (weighing of the tissues) in the preparation of the study samples. Butter Feeding The failure to detect penetration of the GI mucosa or transmigration of optically visible fibers in this study is important since asbestos fibers in this size range are known to be patho genic, and there is a growing body of evidence to indicate that asbestos fi bers in the submicronic size range are not pathogenic. The fact that no can cer of the GI tract or mesothelium de veloped after long-term exposure of the GI mucosa to large numbers of as bestos fibers is highly significant. Long-Term ad Libitum Feeding of Asbestos in Butter The lack of success in the third lab oratory using optical as well as elec tron microscopy in finding evidence of penetration of asbestos fibers after one and three months of ingestion of these mineral fibers respectively, adds weight to the importance of these negative findings in the other two laboratories. GENERAL COMMENTS The method of digestion used in this investigation by one laboratory is faulted by the possibility of chance contamination that may he difficult to evaluate and thereby give rise to false-positive results. The possibility of false-negative results also exists. For instance, fibers may be lost by in complete digestion of the tissue, by incomplete sedimentation during cen trifugation, and by being covered with partially digested material on the electron microscope grid. How ever, in view of the ratio of the amount of tissue examined by the di gestion method as compared with that examined by the electron micro scope section method (many thou sands to one), the chance of a nega tive result with the former when the latter is positive would appear to be negligible. Although transmigration of large objects such as bullets from one por tion of the body to another has been documented, this transmigration is concerned with foreign materials that had already achieved penetra tion into the body interstitium. The penetration of inhaled dust particles through the alveolar membrane and the subsequent lymphatic transport to satellite nodes has been recognized and studied experimentally for more than a century but the mechanism by which this penetration takes place is still unknown. Drinker and Field10 did determine that particles may pene trate the alveolar membrane and ap pear in the lymphatic fluid within minutes after their insufflation into the lung of a dog. This rapid pene tration of the alveolar membrane by Table 1.--Tadulation of Fibers Found in Digests of Tissues* Fibers Found Tissue Digest Mesentery Lung or kidney * Total Dust Amosite 7 rats Taconite tailings 10 rats Control (no dust) 4 rats Amosite 10 rats Taconite tailings 10 rats Control (no dust) 7 rats 48 rats No. Week-Ratt 2 2 wk 2 5 wk 1 1 wk t 2 wk (lung) 3 2/7 wk gavago (lung) 9 * Electron microscopic examination, t Week-rat refers to the animal killed, at specified time. Contaminating Fibers (Chrysotile and Others) No. 1 1 wk (chrysotile) 1 2 wk (chrysotile) GI tract 2 1 (kidney) 5' Arch Environ Health/Vol 29, Dec 1974 Ingested Mineral Fibers/Gross et ai 345 HER 0001302 * Types of Tumor Mesothelioma Carcinoma Sarcoma Lymphoma No. of animals Table 2.--Tabulation of Tumors Found in Rats Fed Asbestos Chrysotile, 10 mg 0 2 (breast) 0 0 31 Crocidolite, 5 mg 0 0 0 0 33 Crocidolite, 10 mg 0 0 0 1 (axillary nodes) 34 Control 0 3 (breast) 1 (thigh) 1 NW Cape Crocidolite, 10 mg 0 0 0 0 24 35 Experiment 3 Transvaal Crocidolite, 10 mg 0 0 0 0 28 Control 0 0 0 0 24 particles was confirmed by Ham burger and Robertson'1 using a dif ferent technique. There are, however, important dif ferences between the alveolar mem brane that protects the lung interstitium from intrusion by foreign material and the GI mucosa that, among other functions, also performs a similar guard duty. Whereas the al veolar membrane is largely composed of thin squamous cells, the intestinal mucosa is composed not only of mu cin-covered tall columnar epithelium ut also by a substantial tunica propria mucosae. The GI mucosa con stitutes, therefore, a barrier against the intrusion of foreign materials that is many times more formidable than the alveolar membrane. Penetration of the alveolar mem brane, as an accomplished fact, is best demonstrated by the presence of en larged, discolored, and often stonyhard satellite lymph nodes. No such involvement of mesenteric lymph nodes has been described in coal min ers or silicotic hard-rock miners even though they had ingested during their lifetime many times the amount of dust than finally remained stored in their lungs and satellite nodes. Consideration should be given to the fact that the dust stored in the lungs is estimated to represent only 15o to 2% of the dust that had been inhaled and, after clearance from the lungs, swallowed. If penetration of finely divided for eign particles through the GI mucosa were a common event or even a rela tively uncommon event, over the life time of an individual, the submticosa of the GI tract and the mesenteric lymph nodes would be the repositories for a considerable accumulation of a wide variety of materials other than those cleared from the lungs. One need only consider the wide variety of insoluble materials, mostly cellulosic, that are part of many foods. Some, like pepper, can be very irritating when in contact with unprotected tis sue and would cause a severe in flammatory reaction. Furthermore, if penetration of the GI mucosa by par ticulates did occur, the amount of stored material in the intestinal sub mucosa and in mesenteric lymph nodes would increase with age. The fact, however, is that storage of such foreign material has not been seen in either the intestinal wall or the mes enteric nodes. The failure to find fibers by elec tron microscopic examination in sec tions of intestinal wall of rats fed asbestos or taconite tailings is con sonant with the failure to find evi dence of transmigration in the di gests of other tissues of these animals. SUMMARY The more important conclusions can be briefly summarized as follows: 1. Twenty-one months of intimate contact of rat GI mucosa with very high concentrations of chrysotile as bestos failed to produce cancer or any other kind of lesion. These results are highly important inasmuch as in ear lier work different jnodes of asbestos exposure have resulted in cancer pro duction within 16 to 17 months. 2. Twenty-one months of high-dosage-chrysotilc feeding of rats re sulted in no evidence of penetration of GI mucosa by the mineral fibers and no incontrovertible proof of transmigration as judged from elec tron microscopic studies. 3. Administration of a single dose of 400 mg of either amosite or taco nite tailings by gavage provided no evidence of transmigration of fibers even though the method of examina tion used was many thousand times more sensitive than the method of ex amining tissue sections. 4. The voluntary intake by rats within six days of 2 to 10 gm of amo site or taconite tailings mixed in oleo margarine likewise provided no evi dence of transmigration of mineral fibers, although a total of nine fibers was found in all of five rats that could have been considered evidence for transmigration, except for the proba bility that they were contaminants. 5. Short-term and long-term feed ing (up to IM years) of chiysotile and crocidolite by a second laboratory re sulted in no tumor production in the GI tract or mesothelium during the lifetime of the animals. Light micro scopic studies provided no evidence that penetration of tissues by in gested asbestos fibers occurred. 6. A partially completed light mi croscopic and electron microscopic study in a third laboratory has pro vided data that support the results and conclusions of the other two labo ratories with regard to the failure of ingested asbestos fibers to penetrate tissues. 7. The penetration of the pulmo nary alveolar membrane and trans migration of inhaled dust particles are facts readily demonstrable by the presence of enlarged,.discolored, min eralized tracheobronchial lymph nodes. In contrast to this, the GI mu cosa, constituting a much more formi- 346 Arch Environ Health/Vol 29, Dec 1974 Inges'ari Mineral Fibers/ Gross et al HER 0001303 dable barrier, does not permit the penetration of insoluble particulates as is evidenced by the lack of similar involvement of mesenteric lymph nodes in miners who, during their lifetimes, ingest nearly 100 times the amount of dust stored in their lungs and satellite lymph nodes, plus the considerable amount of insoluble par ticulates in the daily food intake. Nei ther is evidence of penetration or storage of inhaled or ingested foreign material to be found in the intestinal wall of these miners. 1. Gross P, ct al: Mineral liber content of hu man lungs: A comparison of the counts obtained from the lungs of people of Pittsburgh, Pennsyl vania, with those from the lungs of people of Charleston, South Carolina. Am Ind Hyg Assoc J, to be published. 2. Longer AM: Discussion in Longer AM, et al: Inorganic fibers, including chrysotile, in lungs of autopsy: Preliminary report, in Walton \VH (cd): Inhaled Particles: III. Unwin Brothers, Limited, The Gresham Press, Old Woking, Surrey, En gland, 1971, vol 2, p 693. 3. Policard A, quoted by Gordonoff T: Epuration pulmonaire, in Compte Rervhts des Jmirnea Frangaises de Pathologic Miiidre, Paris, Charbo- References nage de France, Oct 27-28,1960, p 165. 4. Bonscr GM, Clayson DB: Feeding of blue asbestos to rats, in the i5th Annual Reports of the British Empire Cancer Campaign. 1967, p 242. 5. Smith WE: Asbestos, talc and nitrites in re lation to gastric cancer. Am Ind Hyg Assoc J 34:227-223, 1973. 6. Gross P, cl al: Experimental asbestosis: The development of lung cancer in rats with pulmo nary deposits of chrysotile asbestos dust Arch Environ Health 15:343-355, 1967. 7. Gross P, Harley RA Jr. Asbestos-induced inlrathoracic tissue reactions. Arch Pathol 96:245-250, 1973. 8. Pontefract RD, Cunningham HM: Pene tration of asbestos through the digestive tract of rats. Mature 243:352-353, 1973. P. Gross P: Is short-fibered asbestos dust a bio logical hazard? Arch Environ Health 29:115-117, 1974. 10. Drinker CK, Field ME: Lymphatics. Lymph and Tissue Fluid. Baltimore, Williams & Wilkins Co, 1933. 11. Hamburger M, Robertson OH. unpublished experiments quoted by Robertson OH: Phagocy tosis of foreign material in the lung. Physiol Her 21:112,1941. MYTHOLOGY Index of Gods, Etc The Triple Gods The triple gods of hypertension And other osclerosis-- Sir William Osier is Authority for this recension-- The triple gods, I meant to say, Were Bachus, Vulcan, Venus. Sir William's counsel is hygienous And surely non-sclerotic, though it may Be justa nute restrictive; I remark The lethal one of these is work. Charon I sing of Charon and his ferry-- The price is right. Short is the tlight, But the journey is dismal--very. --Rusticatus Arch Environ Health/Vol 29, Deo 1974 ( HER 0001304 Ingested Mineral Fibers/Gross et al 347