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CS National Bureau of Standards Special Publication 506. Proceedings of the Workshop on Asbestos: Definitions and Measurement Methods held at NBS, Gaithersburg, MD, July 18-20, 1977. (Issued November 1978) EPA STUDY OF BIOLOGICAL EFFECTS OF ASBESTOS-LIKE MINERAL FIBERS D. L. Coffin EPA Health Effects Research Laboratory Research Triangle Park, North Carolina 27709 and L. D. Palekar Health Effects Research Laboratory Northrop Services, Inc. Environmental Sciences Group Research Triangle.Park, North Carolina 27709 Abstract A large amount of the earth's crust is composed of rock containing mineral fibers which resemble asbestos to varying degrees in their physical and chemical properties. Consequently, such materials are likely to be encountered inadvertently during the extraction of various ores, the extraction of rock for commercial purposes, and even from rock moving operations encountered during highway construction, and the like. Because the air and water may become contaminated by these fibers, it is of interest from the standpoint of environmental protection to know how the biological effect of such material compares with that of asbestos. Consequently, a study has been instituted by EPA to investigate the relative biological potency of such materials. The project is being approached on both in vivo and in vitro levels. The minerals being studied at the outset are fibrous ampriboles from a taconite mine, but it is the intent to broaden these studies as soon as possible. The animal studies are being conducted in pathogen-free rats by intratracheal instillation (with and without interacting organic carcinogens) and by intrapleural injections. The end points are tumor induction and other chronic diseases. Attention is also being given to early pathogenic sequences. The in vitro studies consist of red cell lysis, pulmonary macrophage systems, and~various biological and chemical studies connected with the influence of these agents on cell membranes and interaction with mutagens and carcinogens. The prime objective is to compare the biological effect of the minerals studied to the corresponding asbestos species to determine the comparative influence of such co-variables as fiber length, trace element content, surface area, zeta potential, and the like, on the biological outcome. Thus, the study will relate biological activity to mineralogical characterization so that generalization can be made on the basis of such factors. Key Words: Alveolar macrophages; hemolysis; intrapleural injections; intratracheal instillation; multinucleated giant cell; PMP I; PMP II; Polyp. Preceding page blank 163 L

The hazards for human health associated with the extraction and handling of various members of the commercial asbestos series are now well known. However, a new issue has recently come to the forefront of environmental toxicology concerning the possible health hazard from inhalation or ingestion of fibrous silicate minerals, not asbestos per se, that contaminate the air and water. Such silicate materials are ubiquitous in the earth's crust where amphibole-bearing rocks may serve as a potential source for a number of mineral species, for example, fibers from the cummingtonite-grunerite series, hornblende, etc. When the above-mentioned facts became known, there was a tendency to class all of these materials as "asbestos" and to try to make inferences concerning their potential health effects in man merely on the basis of supposed analogy to commercial asbestos. We know now, however, that there is an enormous variation in these materials; some closely resemble the corresponding asbestos, and others do not. It would be folly, therefore, to base the threat to human health solely on such a crude determinant. This is particularly true since, despite the great number of epidemiological and biological studies carried out with asbestos, much remains to be learned concerning the exact causal mechanisms of the various lesions attributed to such exposure. For instance, one cannot safely postulate a common etiological mechanism for the usual lesions of asbestos exposure such as pulmonary fibrosis, carcinoma of the lung, and mesothelioma, and the possible role of asbestos for tumors in other locations which at this time is largely unexplored. Because of these issues, the Environmental Protection Agency (EPA) has taken the initiative to study these matters to determine if a threat to health exists from non- asbestos minerals, and if it does, by means of its quantification, to determine how best to control it on the basis of health benefit versus cost. EPA is conducting a study of the relative pathogenic potential of such minerals compared to asbestos, silica, and other particulate substances of known toxicity. The prime purpose of these experiments is to relate biological effects to the physiochemical properties of the minerals. Beginning with the convening of an advisory committee, the following approach evolved, which includes mineralogical as well as biological studies. Mineralogical Studies Intensive study was made from 50 large rock specimens removed from a taconite mine. After preliminary lithological examinations, two of these were selected for employment in biological experiments, which are designated as PMP I and PMP II. Fibers were separated from the rock by such means as mechanical vibration, hand cobbing, air jet milling, spinning, and riffling. The final specimens were subjected to a detailed analysis by means of optical and electron microscopy, x-ray emission spectroscopy, and x-ray diffraction. Computations of surface area and determination of extractable organics were made. Comparisons were also made an the basis of the above parameters with UICC amosite (fibrous grunerite) and airborne material collected in the vicinity of the mine and the ore processing plant. On the basis of the above measurements, a decision was made to prepare a large amount of this material suitable for biological experimentation. Figures 1 through 8 and Tables I-III illustrate various mineralogical characteristics of the samples chosen from the mine for biological studies, as well as samples from the airborne material in the vicinity of the mine and ore-processing area. Figures 1 and 2 represent electron micrographs of air samples from mine and processing areas respectively. The chemical analysis of air samples revealed that in addition to magnetite and quartz particles there were predominantly two other types of minerals in both areas. The electron microscope x-ray analysis revealed the presence of Mg, Si, Ca, Mn, and Fe in one sample (fig. 3), whereas the second sample contained only Mg, Si, and Fe (fig. 4). Data from a careful analysis of size distribution of the air samples are presented in Table 1, showing two samples from each of the processing and mine areas. The majority of the particles in both areas were found to be less than 5 pm in length and less than 1 pm in diameter. A small percentage of particles were between 5 and 10 pm in length, with varying diameters. Air samples from the processing areas contained 66 to 70 percent fibers with diameters less than 0.5 pm as compared to 52 to 55 percent in the mine area. This may suggest that further fibrillation of the rock occurs during the processing. 164

. Figure 1. Air sample from mine area showing long and straight fibers (10,000x). Figure 2. Air sample from the area of processing plants also showing long and straight fibers (10,000x). z /z ® ® En 00170 Ca Q17O3! 165 Figure 3. Electron microscope x-ray spectra of air sample indicating the presence of Mg, Si, Ca, Mn, and Fe.

Figure 4. Electron microscope x-ray spectra of air sample indicating the presence of Mg. Si, and Fe. Table 1. Sumnary data of size distribution of mineral fibers in ambient air samples. - - - - - - - - - Lengths by Percent Number in Microns - - - - - - - - - <1 1-5 5-10 >10 Total Air Sample No. 1 <0.50 9 71 5 0 0.51-1.00 0 12 2 0 >1.00 1 0 0 0 Total 10 83 7 0 100 -93% 7% + - Air Sample No. 2 <0.50 8 66 2 1 0.51-1.00 0 13 1 0 >1.00 0 2 6 1 Total 8 81 9 2 100 89% . 11% 1 f . Air Sample No. 3 <0.50 5 55 2 0 0.51-1.00 0 21 4 0 >1.00 0 4 8 1 Total 5 80 14 1 100 85% . 15% Air Sample No. 4 <0.50 9 52 5 0 0.51-1.00 0 21 3 0 >1.00 0 3 5 2 Total 9 76 13 2 100 85% 15% Below 5 um Diameter by Percent Number in Microns 166 Above 5 Wn

CS Figure 5. Electron micrograph of PMP I showing long and straight fibers with acicular particles (1000x). Figure 6. Electron micrograph ~of PMP II indicating long and straight fibers and particles (1000x). The electron microscope x-ray emission spectra of the fibers collected from the two rock samples revealed the presence of Mg, Si, Ca, Mn, and Fe on PMP I (fig. 7); and Mg, Si, and Fe on PMP II (fig. 8). The size distribution of the samples is given in Tables 2 and 3. The data indicate that the majority of the fibers are less than 5 pm in length and less than 0.5 pm in diameters in both samples. Figure 7. Electron microscope x-ray spectra of PMP I showing the presence of Mg, Si, Ca, Mn, and Fe. N Q a 167 ~ g ~ w

Figure 8. Electron microscope x-ray spectra of PMP II showing the presence of Mg. Si, and Fe. Table 2. Size distribution of PMP I sample. - - - - - - - - - - - - - Lengths in Microns (lun) ------------- 0.00 - 0.50 0.51 - 1.00' 1.01 - 5.00 5.01 - 10.00 10.01 - 25.00 Total 0.00 - 0.50 1.47 8.09 68.38 2.94 0.73 81.61 0.51 - 1.00 0.00 0.00 5.88 2.94 0.00 8.82 1.01 - 2.00 0.00 0.00 4.41 0.73 0.73 5.87 2.01 - 5.00 0.00 0.00 0.00 0.00 2.94 2.94 5.01 - 10.00 0.00 0.00 0.00 0.00 0.73 0.73 88.23 --• 11.74 99.97 Below 5 um Above 5 ym Diameter by Percent Number in Microns Table 3. Size distribution of PMP-2 sample. Lengths by Percent Number in Microns <1 1- 5 5- 10 10 - 15 0.00 - 0.50 27.06 41.53 0 0 0.51 - 1.00 0 18.01 5.50 1.80 >1.00 - 10 0 0.80 3.90 1.80 27.06 60.34 9.40 3.60 .-- 87% -. «-~ 13% -+ Be1ow 5 um Above 5 1¢a Diameter by Percent Number in Microns 168

Figure 9. Fibrous grunerite (UICC amosite) showing the general shape of the particle which is long and straight (1000x). Sim-e-the air samples and the rock samples seem to be representative of the grunerite Tamiiy, a fibrous grunerite, namely UICC standard reference amosite, with known biological properties, was selected as a possible control for the studies, and characterized. The electron microscope x-ray analysis of amosite indicates the presence of Mg, Si, and Fe (fig. 10). Size distribution data for this material are presented in Table 4. Eighty- seven percent of the fibers were found to be less than 5 Nm in length and 1.5 Nm in diameter. I V Figure 10. Electron microscope x-ray spectra of fibrous grunerite (UICC amosite) indicating the presence of Mg, 51, and Fe. En Qi.IU Ca =7Q39 Table 4. Size distribution data of UICC amosite by IITRI method. - - - - - - - Lengths Distribution (by percent number), in Microns - - - - - - - 0.2-0.5 0.5-1 1-2 2-5 5-10 10-25 25-50 50-100 100-200 Total 0.00-1.10 15.90 3.48 1.64 1.80 0.57 0.20 -- -- -- 23.39 0.10-0.40 8.69 13.49 18.24 16.40 5.16 1.68 0.41 0.20 0.01 64.28 0.40-1.50 -- -- 2.54 4.75 1.31 1.84 2.69 0.20 -- 12.93 87% 7% Diameter by Percent Number in Microns 169 ~ 6%

The air samples, ttie fibers obtained from rocks, and amosite fibers were examined by electron microscope for their general shape. All samples contained straight and long fibers and acicular particles (figs. 5, 6, 9). These photographs are not representative of the size distribution. Biological Studies Toxicity evaluations are proceeding both in vivo and in vitro. Whole animal experi- ments are being carried out to determine the comparative ef e~ ct of the above-mentioned mineral fibers in inducing lesions such as pulmonary fibrosis, lung cancer, and pleural mesothelioma. Basically, a comparison between a test amphibole of the cummingtonite- grunerite family, UICC amosite, and an inert particle is intended. These studies are being conducted in Fisher 344 pathogen-free rats during their life span. The particles are administered to the animals by intratracheal instillation and intrapleural injections. In vitro studies are conducted on sheep blood erythrocytes and rabbit alveolar macropFages. The cytotoxicity is evaluated by quantitation of red cell hemolysis and cell death respectively. - In Vivo Studies The doses for the intratracheal instillations were determined by an initial range- finding study. Several doses of the particulates were administered to the animals and the highest tolerated dose was determined. Two series of intratracheal studies are planned. Innoculation of the animals in the first series is complete. The second series will be initiated in the near future. Chronic Intratracheal Testing of PMP Amphibole The first series will determine whether the particles alone cause significant toxicity to animals. The regimen for this series is as follows: Series I: Unknown Sample - PMP I Amphibole ........600 animals Asbestos Control - UICC Amosite..........200 animals Negative Control - Saline and Gel ........ 200 animals Chronic Interaction Studies by Intratracheal Instillations The purpose of the second series is to determine whether the particles will interact with a known carcinogen to produce a higher incidence of tumors. A knpwn amount of benzo(a)pyrene (BaP) will be coated on the particles to compare the synergistic effect of the carcinogen with amosite, the test amphibole, and hematite. The regimen of this series is as follows: Series II: PMP I Amphibole + Bap ....................300 animals UICC Amosite + BaP .......................300 animals Iron Oxide + BaP .........................300 animals PMP I Amphibole ..........................200 animals Iron Oxide ...............................200 animals BaP ......................................200 animals Chronic Intrapleural Testing of PMP Particles Intrapleural studies employing 20 mg of particles injected once into the pleural cavity are being carried out as follows: Series III: Unknown Sample - PMP I Amphibole......... 150 animals Asbestos Control - UICC Amosite.......... 150 animals Negative Control - Saline ................150 animals 170

Ip addition to the lifetime experiments, exploration of the pathological sequences induced by these materials in the lung is in progress by experiments in which sequential sacrifices are being carried out. Figures 11 and 12 demonstrate epithelial polyps and fiber-containing giant cells observed in the parenchyma of rats treated with 12 weekly Injections of 1 mg of amosite or the test sample PMP I, 50 days after the last innocula- tion. The polyps essentially consist of several multi-nucleated giant cells covered with columnar epithelium. Figure 11. Epithelial polyps observed in the bronchi (250x). Figure 12. Multinucleated giant cell containing fibers (1000x). N ~ 171 W ~ ~ J

In Vitro Studies The second part of the biological studies consists of in vitro investigation to determine cytotoxicity of the particles. Two techniques are empTed, namely, sheep erythrocyte hemolysis and rabbit alveolar macrophage destruction. A comparison was made between several commercial asbestos samples of known biological properties, PMP I and non- fibrous grunerite. The data presented in figure 13 suggest that the amphiboles are not as hemolytic as chrysotile fibers, requiring large doses to achieve 50 percent hemolysis. Among the amphiboles, anthophyllite, PMP I, and tremolite are similar in their effect. Crocidolite and amosite seem to be less hemolytic. In contrast, non-fibrous grunerite is non-hemolytic. In the rabbit alveolar macrophage study, amosite and PMP I caused marked depression of cellular viability, whereas non-fibrous grunerite showed no significant change in cellular viability (fig. 14). The sample PMP II is not yet tested. A second advisory committee was convened to consider further investigations to increase our understanding of the mechanisms of mineral interactions with the biological systems. It was the opinion of the committee that the comparative study of minerals should be started as soon as possible. On the basis of the existing data, produced by different laboratories throughout the world, the problem of contamination of the environment with inorganic fibers may pose a significant health threat. Indeed, it may shed significant light on existing problems, e.g., asbestos in potable water supplies, asbestos released from degraded asbestos cement water pipes, natural sources, etc. The selection of minerals and bioassays are as follows: fibrous and non-fibrous grunerite will be collected from different geological localities and their biological properties will be compared. The careful mineralogical analysis and bioassays may indicate whether there is some influence in terms of the crushing process that may create new fiber surfaces not present when communiting materials from other areas. 100 N } 80 QJ 2 = 60 ~ 40 20 0 0 CHRYSOTILE • CROCIDOLITE p (RHODESIA-UICC) TREMOLITE (INDIA) V (S. AFRICA-UICC) GRUNERITE • PMP (NON FIBROUS) O ANTHOPHYLLITE (S. AFR ICA-UICC)  AMOSITE (S. AFRICA-UICC) I I p....q_..~. ~-..q 0.01 0.02 0.05 0.10 0,20 0.50 1 2 5 CONCENTRATION (mg/ml) 10 20 Figure 13. Hemolysis of sheep erythrocytes by various minerals. 172

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