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3 100 80 60 40 20 0 0.01 GRUNERITE (NON FIBROUS) ~ PMP , AMOSITE (UICC-S. AFRICA) I 1 1 111111 1 1 1 111111 1 1 1 111111 1 I t I°II1I 0.05 0.10 0.20 0.50 1 2 5 CONCENTRATION (mg/ml) Figure 14. Cytotoxic effect caused by various minerals when exposed to rabbit alveolar macrophages. For a proper comparison, a standard reference sample of fibrous grunerite (UICC amosite) containing particles of mixed sizes, and another sample specially prepared with short fibers will be used. Since relatively short fibers are observed in Lake Superior, the information obtained from these fibers will be useful. Fibrous cummingtonite with a high magnesium content from several geological local- ities will also be studied for comparison to determine if different processing methods may alter surface properties and, in turn, affect the biological properties. In addition, minerals of known biological properties, such as UICC anthophyllite, UICC chrysotile A, chrysotile RG 144, UICC crocidolite, Indian tremolite, UICC actinolite, antigorite, fibrous glass, and quartz will be studied for comparison. Several assays will be employed to evaluate the biological properties of the inerals. The direct toxicity of the particles will be tested by hemolysis of sheep red blood cells, viability of rabbit alveolar macrophages, human lung fibroblasts such as strain WI-38 and perhaps the mouse ascitis tumor cell line P3881. The possible mutagenic effects of these materials will be evaluated in well-established mutagenesis test systems, such as the Ames test and the L5178Y mouse lymphoma cell assay. Neoplasm induction will be tested by the use of tracheal transplants, as well as transformation of Syrian hamster embryo (SHE) cells, or mouse fibroblasts, such as the C3H 10T>f or BALB/c 3T3 celi lines. 173

Conclusion Preliminary In vitro tests show that both fibrous grunerite and PMP I amphibole are lytic to sheep erythrocytes and depress the viability of rabbit alveolar macrophages, while non-fibrous grunerite is inactive in both systems. The biological significance of these studies Is at this time unclear. Hopefully the proposed investigation will contribute sufficient information to correlate mineral properties to health hazards associated with inhalation and/or ingestion of minerals other than the known commercial asbestos. Mineralogical characterization was done by Illinois Institute of Technology, Chicago, Illinois. Contract #68-02-2451. Discussion C. COOPER: I want to congratulate Dr. Palekar for the description of what is getting under way, and the great care that has been taken apparently to obtain test materials that at least resemble some of the fibers in the taconite areas. I think an important question is how representative this is of the entire Mesabi range and I personally don't have figures available to me as to whether or not the size distributions found at the Peter Mitchell pit are representative of a larger area. I wonder if anybody in the audience here, or Dr. Palekar herself, have data on other areas in the Mesabi range to answer the question as to whether or not 15 percent, approximately, of the fibers are longer than 5 micrometers in length, because the representativeness of this sample is going to be, I think, an important issue in the future, and I wonder if anybody could address themselves to that? L. PALEKAR; I don't have a clear-cut answer to your question, but if somebody in the audience wants to answer that... A. LANGER: You mean the representativeness of the Peter Mitchell fibers? COOPER: Yes. LANGER: It's unlike anything in the rest of the Mesabi. COOPER: Are there air samples in other areas with this same distribution? LANGER: No there are not. Unfortunately for the Reserve Mining Company, the situation at the Peter Mitchell pit is unique for the Mesabi range. The mineral fibers have been originated through contact metamorphism with the Duluth Gabbro, which metamorphose the pre-existing materials here. Now Malcolm Ross is here, who has done work on the amphiboles in the area. He knows a great deal about the geochemistry of the amphibole/pyroxene phases; this is a high temperature metamorphic assemblage, while the rest of the Mesabi range, the rest of the Biwabik iron ore formation, are generally considered to be low temperature iron silicates. They do have problems with fibers, but these may not be as important biologically as the asbestiform amphiboles in the Peter Mitchell pit. This is just unique for that particular area. W. NICHOLSON: In looking at the fiber distribution in other than the Reserve Mining areas, they are of a smaller size distribution and tend, rather than being regular fibers, (that is with collinear sides) to be chips of fibrous length. They are irregular fragments rather than the natural fibers that we've been hearing of, and they are in general of a size distribution somewhat smaller than that which has been described here, but there are many fibers (that is defined by a 3 to 7 length to width ratio) that are present in other areas. P. GROSS: I would like to comment on the two microphotographs of tissue which Dr. Palekar showed. I was most interested in the visualization of fibers at that magnifi- cation, which indicated that the fibers were quite long, much longer than 5 microns. As a matter of fact, one of the fibers that I saw, where one of the giant cells was, was as 174

long as a giant cell, which probably was in the neighborhood of 100 pm in length. Also the photomicrograph of the bronchial-polyp, this sort of picture has been produced in my laboratory with long fibers of any kind: glass, silicon carbide, aluminum silicate, as well as asbestos. Again, it suggests the presence of a fairly considerable number of long fibers, and it seems to me that may be a reflection of an exceedingly high dosage administered even though your long fibers were less than 15 percent of the total. PALEKAR: Yes sir, we administered the highest tolerable dose. The animals received twelve weekly injections of 1 mg. A. WILEY: Since there seems to be a good deal of controversy about what's a fiber and what's not a fiber, I was interested in your characterization of a grunerite sample as non-fibrous and I'd like to know what you meant by it. PALEKAR: The particles are not completely characterized at this time and it was presumptuous on my part to present the data. This is really a very preliminary study and no conclusions can be drawn at this time. We have asked our colleagues from IITRI to analyze this properly. Thus far I have just taken their word for the non-fibrous nature of the particles. G. NORD: Yesterday we saw a great deal about the mineralogy of amphiboles. One of the things that was brought up was the defect structure of amphiboles. Amosite has a very high defect density; it's polysynthetically twin on a unit cell scale. The grunerite that you used, or I should say the minerals that you used from the Mesabi range sample, may have an entirely different defect population. Is there going to be any attempt to characterize this defect population? That could also go for the characterization of the samples discussed by the previous speaker. I have one other comment: It's not enough to characterize a fibrous mineral strictly by an energy-dispersive analysis. You cannvt tell the difference between a low calcium pyroxene and a low calcium amphibole. It tis not enough to characterize a low calcium amphibole merely by knowing its chemistry. It also has a different structure; you have orthorhombic amphiboles and you have monoclinic amphiboles. Grunerite/cummingtonites are monoclinic. You also have anthophyllites which are orthorhombic. If one is to characterize these samples adequately so one can separate out the very small differences, perhaps in the experimental data, you will have to do a great deal more work. PALEKAR: Well, this paper is by no means the entire story. I never said that this is it, that this is the only thing we are going to do. We are open to ideas and we are going to characterize many more minerals more thoroughly; this is just the beginning and we intend to do further analyses. B. SMITH: Dr. Palekar, I believe you said that the EM measurements that you had on a standard reference sample of amosite, UICC amosite, was showing about 87 percent of the particles shorter than 5 pm, and that the measurements that you had on the preparation, the PMP preparation that you made from taconite rock, showed about 85 percent of fibers running below 5 pm. Now, as I looked at the photographs you showed, the photographs of the taconite preparation had a micron scale on them, so we were looking at fibers that were being compared with a 1-micron scale. They didn't seem to be more than, or only a little bit more than the scale. They looked to me about 2 or 3 times the size of the scale, so I guess they were fibers that were about 2 or 3 Nm long. In comparison, the photograph you showed of the UICC amosite was fitted with a 10-micron scale and there were an enormous number of fibers visible in that photograph that were much longer than the 10- micron scale. This presents a problem that has puzzled me many times in samples that I've looked at, where we've gotten electron microscopy measurements that are telling us that two samples really are about the same as far as the mean fiber length is concerned. When I look at them with an optical microscope, it's very apparent to me that there are an enormous number of long fibers that I can easily see at say 400X in one sample, and with the other sample that electron microscopy figures are telling me is about the same, I have a tough time seeing any fibers. Now how do we get around this problem? PALEKAR: Yes, I agree with you wholeheartedly and I had the same questions to our mineralogist. The electron micrographs of the fibers are not representative. It is known that there is a tremendous variation between samples. One must make an effort to use the 175 2063104971

same sample for mineralogical analysis and biological evaluations to establish a proper relationship between the two. 0. WALIA: I don't have a question but I'd like to address myself to some of the comments regarding the preparations and characterizations that we did for Or. Palekar. The comment that electron microscopy is not the only criteria to distinguish one fiber from another is true, and we did not depend only on that. Instead we picked up the fibers from the filter samples, mounted them on glass fibers, and then performed x-ray diffraction studies on them. We then compared the data with the known fibers from the taconite mines, and also with the ASTM standards, and from that we were able to identify or pinpoint their identity as to the mineral species. Second, regarding the size distribution comments, if you remember the tables Dr. Palekar showed, in the case of UICC amosite, where we have compared our size distribution data, which is both by diameter and by length, you get a comparison within ±6 percent. I believe this is a good comparison and from the table you see that UICC amosite has fibers which are as long as 200 pm. When we look at the taconite samples, which we have prepared, and the size distribution data, you see that there is no fiber greater than 20 pm. To my knowledge, from all the taconite rock samples I've seen, I've never come across any mineral fiber which, even using this ambiguous three-to- one aspect ratio criteria, that I can say is 200 pm in length. Another comment I'd like to address myself to is about the non-fibrous grunerite we used. This non-fibrous grunerite, which has some preliminary results that Or. Palekar showed, was the one we got from bawabush iron ore formations in Canada, and the non-fibrous nature of this is based on the lack of flexibility of the fibers which you commonly see in UICC amosite type materials. NOTE: The following notes were sent following the meeting and were not part of the verbal discussion at the end of the session. GROSS: Dr. Palekar's description of the bronchial lesions that develop in animals following the intratracheal injections of long-fibered asbestos as "polyps" deserves explanation. A polyp is generally conceived to be a tumor - a neoplasm. The intrabronchial lesions developing in animals after intratracheal injections of asbestos are not tumors. The lesions are composed of inframmatory tissues that surrounds impacted, aggregated asbestos. The inflammatory tissue extends (often in a finger-like manner) into the bronchial lumen and, in time, becomes covered by normal-appearing bronchial epithelium - hence its resem- blance to a polyp. R. BLEIFUSS: The reports submitted by the Illinois Institute of Technology Research Institute (IITRI) regarding the origin of the sample materials to be used in these biological studies indicates that the source material represents an unusual situation within the Peter Mitchell Pit (PMP) of Reserve Mining Company. The original sample material represents a unique occurrence within the PMP in the same sense that the PMP may be said to be unique to the rest of the Mesabi Range. IITRI personnel collected more than 100 samples in their initial survey an which they carried out extensive mineralogy studies to characterize the ore. Based on this initial information the sample location from which they extracted the fibers for the biological study was selected as described below.i "On October 2, 1975, approxiwtately 750 lbs of high fibrous content ore were located and collected. It was found that the ore containing rich fibrous veins was a very localized phenomenon. Such samples were avail- able only near the incursion of the Duluth Gabbro and occurred only in two very localized areas within approximately 100 m of each other." lIITRI Report No. C6321C02-11, Final Report, Contract No. 68-02-1687, "Amphibole Mineral Study to Complement the Ongoing Characterization of Finely Particulate Environmental Conta.inants for Biological Experimentation." 176

"Fibers were separated from fiber-rich rocks using several methods. Both hand and vibratory cobbing were used to separate fibrous material (~1.5 kg) in veins. Several rocks were found to consist almost entirely of soft, light green or brown fibrous material. These rocks were crushed, ground, and sieved (<35 mesh) to produce a material (~3 kg) with a high fibrous-to-non-fibrous ratio." "These separated fibrous materials are not necessarily representative in all respects of the majority of the fibers in the ore in the Reserve Mine or In the tailings from the magnetite extraction at Silver Bay, Minnesota. However, this method was used as large quantities of materials with a large fibrous fraction could be produced more easily than by separating fibers from the ore or the tailings." The mineral composition of the sample prepared from this "high fibrous" ore, which has been encapsulated for the biological studies, was determined by x-ray diffraction. The minerals "definitely present" include cummingtonite, riebeckite, and rich(t)erite. Minerals "present as trace material" were tremolite and crocidolite. However, the basic mineralogy studies on the 100 original samples include no mention of riebeckite, richterite, or crocidolite. Both the riebeckite2 and crocidolite3 have been described in the literature and are present only in trace amounts in the Peter Mitchell Pit. The sodium in these two minerals is considered to be of metasomatic origin. Richterite was not reported by previous workers in the area which suggests that it may be the result of local hydrothermal activity. Thus the sample prepared for these biological studies contains three minerals which were either unreported or considered to be present in trace amounts by previous authors. These minerals are all commonly reported to be of inetasomatic origin, meaning that some of the critical elements (sodium) for their formation was introduced from outside the iron forma- tion. The occurrence of these minerals in veins further suggests that they are related to metasomatism. The sample which was finally selected and processed to produce the fibers for'biologi- cal studies appears to have a unique metasomatic origin, or at least some of the minerals in that sample are related to metasomatism. The sample is certainly not representative of the potential tailings from the PMP. It cannot be classified as typical since three of the finer most important mineral components are certainly atypical in the PMP area. The sample was selected to provide a high "fibrous" to "non-fibrous" ratio that was unobtainable from representative taconite samples. Biological experiments on this sample will contribute little to the resolution of the problem pertaining to the possible carcinogenic nature of taconite tailings. The argument that it is a means of establishing a bridge between a known carcinogen (amosite) and a possible, or suspected carcinogen (cummingtonite in taconite tailings) is not realistic. The direction of the sampling program was to obtain a fibrous sample as analogous to amosite as possible. In so doing it is so far removed from being representative, or typical, of taconite tailings as to make the final outcome essentially meaningless. aGundersen, J. N. and Schwartz, G. M., The Geology of the Metamorphosed Biwabik Iron- Formation, Eastern Mesabi District, Minnesota. Geological Survey Bulletin No. 43, 1962. 3White, D. A., The Stratigraphy and Structure of the Mesabi Range, Minnesota, Minnesota Geological Survey Bulletin No. 38, 1954, 92 pp. 177 2063104973