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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