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FILE NAME: Talc (TALC) DATE: 1984 DOC#: TALC205 DOCUMENT DESCRIPTION: Journal Article - Evaluation by Electron Microscopy Techniques of Asbestos Contamination in Talcs  T^c REGULATORY TOXICOLOGY AND PHARMACOLOGY 4, 222-235 (1984) Evaluation by Electron Microscopy Techniques of Asbestos Contamination in industrial, Cosmetic, and Pharmaceutical Talcs L . P a o l e t t i,* S. C a ia z z a ,* G . D o n e l l i* a n d F . P o c c h ia r i *Laboratorio di Ultrastrutture, Istituto Superiore di Sanit, Viale Regina Elena 299, 00161 Roma, Italy Received December 9, 1983 Talc powders from national and international markets were analyzed in order to assess their fiber contents and the proportion of asbestos in the fibrous material. Samples of talc powders used as excipients in pharmaceutical and cosmetic preparations demonstrated fiber contents up to 30% of total particles. About a half of the talc powders revealed the presence of asbestos: in five samples chrysotile (a serpentine asbestos) was present, in the other ones tremolite and anthophyllitc (an ampbibole asbestos). The amounts of asbestos vary up to 90% in the different samples of the fibrous fraction. About 75% of observed asbestos fibers were thinner than 0.4 pm, i.e., below the resolving power of light microscopy which until now was the most utilized technique for evaluating the environmental pollution due to asbestos. INTRODUCTION Talc is a Mg silicate with a particular lamellar (sheet) structure in which each lamella consists o f one sheet of Mg (O H ^ between two layers o f S i04 (1). In natural deposits Mg is often substituted in talc crystals by other cations such as Fe, Ni, Cr, M n, etc. Deposits are not generally monommeralic, but, since they result from geologic processes which caused the form ation o f several different mineral phases, they are heterogeneous as far as the kind and relative am ount of the minerals (2). Thus, it is not surprising that materials known as talc powders may contain less than 50% o f talc (3). The most common minerals that may be found mixed with talc in m ineral deposits are listed in Table 1. Among them, two fibrous kinds of amphibole, trem olite and anthophyllite, and a fibrous kind of serpentine mineral, chrysotile, constitute some of the best known varieties o f asbestos (2, 4, 5). Talc powders are widely employed in a num ber of industrial processes and in commonly used products (i.e., in the manufacturing o f pottery articles and insulating materials, in paper manufacturing, as additives in asphalts, in pesticides, in cosmetic products, and as excipients in pharmaceutical preparations). It is well known that professional exposure to talc may cause the kind of fibrosis known as talcosis. Moreover, a large number o f epidemiological data point out the 222 0273-2300/84 $3.00 Copyright O 1994 by Academic Press. Inc. All rights o f reproduction in a n y form reserved. ASBESTOS CONTAMINATION OF TALCS 223 TABLE 1 Minerals Commonly Associated with Talc in Natural Deposits Carbonates Calcdte, dolomite, magnesite Amphiboles Tremolite, anthophyllite Serpentines Chrysolite, antigorite, lizardite Others Quartz, mica, chlorite, rutile, pyrophyllite risk of cancer connected with the fibrous components o f talc powders, i.e., talc fibers and fibrous impurities. "Asbestos bodies" in the lung tissue of workers exposed to talc have been reported several times in the literature (7-11). An epidemiological study on workers in talc deposits has demonstrated a threeto fourfold increase in cancer risk as compared with the risk of the population in general (12). Moreover, the same histological lung alterations are shown by workers exposed to talc powders and by workers exposed to asbestos (13). Recently, a connection between ovarian cancer and the use of asbestos containing talcs has also been suggested (14, IS). However, there is a considerable lack o f analytical data about the fibrous components o f talc. In 22 talc powders examined, values ranging from 8 to 30% o f fibrous particles were reported in 1968 by U. S. authors (16). A similar study carried out by NBS investigators (17) evidenced percentages from 2 to 30% of fibrous particles in all the analyzed samples. In both cases it was not specified what fraction of such percentages was constituted by asbestos. Studies carried out in the United Kingdom on talc powders used for different purposes revealed that 3 out of 24 specimens contained trem olite (18). More complete data concerning 20 talcs for cosmetic use and 1 for pharmaceutical use purchased in the New York City area from 1971 to 1975 have been published (19): 10 among the cosmetic talcs examined contained, from 1 to 14% (w/w), trem olite and antho phyllite, and two of them also contained traces of chrysotile. Taking also into account the lack of data on the Italian situation, a systematic analysis of talc powders employed or at least marketed in Italy was carried out in our laboratory to determine the percentage of fibrous particles and the am ount of asbestos in such powders. Moreover we analyzed 14 talc powders provided by the European Pharmacopoeia from various geographical areas and to be utilized for different purposes. In this study we utilized electron microscopy (EM) and associated analytical tech niques such as electron diffraction and X-ray microanalysis which allow the mor phological and structural characterization as well as the elemental analysis of particles at high levels of resolution. MATERIALS AND METHODS A small am ount o f talc obtained from the original package without mixing and homogenizing it, in order to avoid any fragmentation of particles, was weighed and 224 PAOLETTI ET AL. suspended in a solution o f 0.5 to 0.8% Formvar in dichloroethane at a concentration of 0.02 to 0,05 g/m l talc. After air insufflation by a Pasteur pipet to avoid sedimentation, two or three drops of the suspension were put on a just-cleaved mica sheet and rubbed with another mica sheet. After solvent evaporation, the film including talc particles was separated from the mica sheet on the surface o f twice distilled H20 and collected on 200-mesh copper grids. A thin carbon film was then evaporated on the grids. The samples were observed by a TEM Siemens Elmiskop 102 at 10,000 magnification and 100 kV HT. To obtain information on the crystalline structure of mineral particles, selected-area electron diffraction (SAED) was utilized. Chemical elements present in the mineral particles were detected by X-ray microanalysis, equipped with a wave dispersion spectrometer. Statistical Evaluation o f Data We checked the statistical compatibility of the results o f replicate determinations of fibrous fractions for the same talc specimen. The x 2test was utilized to check the hypothesis that the differences in the values o fthe fibrous fractions were due to chance (21). For each kind of talc the average value of the percentages o f fibers and of nonfibrous particles were calculated. The numbers o ffibers and o fnonfibrous particles measured in each determination have been utilized as elements o fa contingency table and x2was calculated comparing the numeric values obtained and the ones expected (i.e., the corresponding average percentages). At a significance level of a = 0.05, the values obtained for x2were not generally significant enough [xP< xz(0.95)] to reject the initial hypothesis. However, in some cases we obtained comparatively high x2values [xp > x2(0.95)]. Careful reexamination of the specimen characteristics and the counting methods evidenced that the less reliable results concerned counts performed on fields where large clusters of particles were observed or where, due to their num ber, many particles were in contact. We realized also that the wide range of the counting results could be related to the lack of a defined threshold for the minimum size required for a particle to be counted. After stating new and more strict counting criteria (particle clusters and particles smaller than 0.2 were not included in the counts), we reexamined the same talc specimens and obtained a very good statistical compatibility with the initial hypothesis. In Table 2 the values of x 2 we obtained and the ones corresponding to a 95% probability at the same degree of freedom are listed. To check whether the method o f sample preparation could cause an increase or a decrease in the fibre percentage, for each kind o f talc powder we analyzed the correlation between the total num ber of particles per unit area o f the film (the area of one mesh of the grid) and the percentage o f fibrous particles on such a surface. This correlation was studied both on different Formvar films and on different areas of the same film. In all cases the correlation parameter r 2 (21) between the particle number and the fibrous fraction in the unit area resulted in values very close to zero, as shown in Table 3. ASBESTOS CONTAMINATION OF TALCS 225 TABLE 2 x 2Test to Check the Reproducibility in the Evaluation of the fibrous Fraction in Talc powders Sample X2 Observed X2 (0.95) A 9.9 16.9 B 1.61 5.99 C 10.6 11.1 D 7.58 9.49 E 3.65 7.81 F 2.95 7.81 G 0.37 3.84 H 2.34 5.99 I 7.58 9.49 J 3.65 7.81 K 2.95 7.81 L 4.32 5.99 M 1.39 9.49 Statistical Criteria fo r Evaluating the Fiber-Particle Ratio in Talc Samples A binomial distribution is expected for the values of the fibrous fraction of a given number of particles and for the percentage of asbestos fibers in a given num ber of fibers. This allows calculation o f the number o f particles to be observed in order to obtain the required accuracy o f m easurem ent For a binomial distribution the relative standard deviation (as percentage o f the mean value ft) calculated by means o f the a priori probability P o f observing a fiber and by means of the number N of observed particles is <rJft = l ( \ - P ) / ( N - P ) Y a The total num ber N of particles to be observed in order to evaluate the fibenparticle ratio may be calculated by means of this equation according to the required accuracy of measurement. Table 4, in which the standard deviation is reported as a function o f P and N, shows that, in order to evaluate the fiber concentration with a relative standard deviation o f less than 10-15%, ATmust be about 103, even for concentrations between 5 and 30%, and must increase by at least of a factor 10 for lower concentrations. TABLE 3 CORRELATION BETWEEN PARTICLE NUMBER TOR U N IT AREA AND FIBROUS PARTICLE PERCENTAGE Sample A B C D E F G Correlation parameter, r2 0.22 0.13 0.03 0.13 0.002 0.4! 0.06 226 PAOLETTI ET AL. TABLE 4 Relative Standard Deviation for a Binomial D istribution p 2V= 100 N = 1000 N = 10,000 0.01 99% 31% 0.05 43% 14% 0.10 30% 9% 0.15 24% 7% 0.20 20% 6% 0.25 17% 5% 0.35 15% 5% 10% 4.3% 3% 2.4% 2% 1.7% 1.5% N ~ total number of particles to be observed. P * probability of observing one fiber. Since some talc specimens may contain very few asbestos fibers, and therefore are barely detectable, it is necessary to define an upper lim it for the pollution level, in case that no asbestos fibers should be detected in a given specimen. Even in such a case we cannot infer that the talc specimen is completely asbestos free. We can simply calculate the probability P of finding at least one asbestos fiber among a num ber N of fibrous particles as a function of the ratio v between the number of asbestos fibers and total num ber of fibers IP = 1 --(1 --u)*]. From Table 5, in which the probability is reported as a function of N and v, we infer, for instance, that for very small concentrations (t> 4 10-4), the probability of observing one asbestos fiber is very close to zero for samples containing 102--103 fibrous particles. To fix an upper Emit for the estimated asbestos concentration in the sample we chose a value o f the probability P of singling out at least one fiber of asbestos in the sample o f fibers observed: in order to obtain the results we are looking for, we set a value of P = 0.9 (90% of probability). Then we calculated the concentration that, for the num ber of fibers observed, provided such a probability (P = 0.9). This value TABLE 5 Probability o f Observing at Least One Asbestos FIber among N Fibrous Particles as a function of the Ratio v between Asbestos Fibers and Total Fibers N 50 100 200 500 1000 2000 IO '4 0.005 0.01 0.02 0.05 0.10 0.18 X 10-4 0.025 0.05 0.10 0.22 0.39 0.63 10-3 0.05 0.10 0.18 0.39 0.63 0.86 x io-3 0.10 0.18 0.33 0.63 0.86 0.98 X 10'3 0.14 0.26 0.45 0.78 0.95 1.00 X 10'3 0.22 0.39 0.63 0.92 0.99 1.00 X 10'3 0.30 0.50 0.75 0.97 1.00 1.00 10~2 0.39 0.63 0.87 0.99 1.00 1.00 X 10~2 0.64 0.87 0.98 1.00 1.00 1.00 x 10"5 0.92 1.00 1.00 1.00 1.00 1.00 10'1 1.00 1.00 1.00 1.00 1.00 1.00 ASBESTOS CONTAMINATION OF TALCS 227 of asbestos concentration in the sample has been considered as the upper lim it since a higher concentration would provide a probability higher than 90%, that is, practically the certainty, o f observing one asbestos fiber. RESULTS Twenty-nine different samples of talc for industrial, cosmetic, and pharmaceutical uses have been analyzed: 15 from the Italian market and 14 provided by the European Pharmacopoeia from the international market and from various geographic areas. The powder samples prepared by the Formvar film method showed to be suitable for studies by electron diffraction and X-ray microanalysis and for further observation by light microscopy. Moreover the samples showed a considerable degree o f stability, even after repeated observations. The statistical analyses showed the reliability and reproducibility of the results obtained for powder samples prepared by the above-described method. According to the criteria accepted by the Council o f European Communities (32), particles have been considered as fibrous when having a lengthiwidth ratio greater or equal to 3 and a width less than 3 fim. Fibers respectively greater and less than 5 pm in length have been considered separately, according to the above-mentioned criteria, which consider as more haz ardous, because o f their biological effects, the fibers longer than 5 fim. The counts for the evaluation o f fibrous particle percentages have been performed on a total number of particles ranging from 2 X 103 to 10 X 103 for each kind of talc, so in general the resulting error in the percentages (standard deviation calculated by the expression given in the statistical criteria) was not greater than 10%. To evaluate the pollution due to asbestos in the studied talcs, the fibrous kinds of amphiboles, tremolite and anthophyllite, and the fibrous kind of serpentine, chrysotile, have been investigated (2-4). The electron diffraction patterns of the various mineral phases in the samples are not straightforward (27); nevertheless the patterns show peculiarities which allow determ ination, with certainty, the presence o f serpentine asbestos (Fig. 1) or amphibole asbestos (Figs. 2 and 3) (23, 28, 29) among the particles which constitute the talc powders. The identification of the kind of mineral in amphibole asbestos may be confirmed through the identification of the characteristic chemical elements by means of the electron microprobe. Table 6 reports the concentrations of such elements in the most common kinds of asbestos. The identification of the kind of amphibole present in the talcs may be performed by means of the evaluation of the Ca (tremolite) and Fe (anthophyllite) concentrations in the mineral. For each kind of talc powder, a sample o f 100 random fibrous particles has been considered. We studied the electron diffraction pattern o f each sample and its Ca and Fe contents, by means o f X-ray microanalysis. As reported in Tables 7-10 the fibrous particle percentages ranged from 2 to 30% in all of the talc powders analyzed. As reported in Tables 7-9, in 8 out o f 15 Italian talcs the presence o f asbestos fibers has been revealed, in 7 samples there were fibers o f trem olite, and in 1 sam ple there were fibers o f chrysotile. 228 PAOLETT ET AL. Fig. 1. Electron diffraction pattern of a chrysotile fiber. Fio. 2. Electron diffraction pattern of an anthophyllite fiber. asbestos contamination of talcs 229 Fig. 3. Electron diffraction pattern of a tremolite fiber. The presence of asbestos fibers was revealed in 6 out of 14 talcs from the European Pharmacopoeia: in 3 samples there was chrysotile, in 2 samples the amphiboles tremolite and anthophyllite were present, and in 1 sample there was trem olite and chrysotile. In the 2 talc samples containing amphibole asbestos, trem olite and an thophyllite were present in great percentages, reaching in both cases about 20% of the entire particulate. Figures 4 and 5 show a few typical fibers o f such mineral varieties observed in some of the samples examined. interestingly, about three-fourths of the asbestos fibers observed in each sample had a diam eter smaller than about 0.4 /xm, that is, below the resolving power of TABLE 6 Chemical Composition in Oxide Percentage of the Most Common Varieties of Asbestos Chrysotile Crocidolite Amosite Anthophyllite Tremolite Si02 MgO FeO FejOj A1i0 3 CaO KjO Na20 HjO 41.8-42 41.8-42.8 0.1--1.6 0.2-1.3 0.1-0.5 0-0.1 0-0.1 -- 13.6-14 49-53 0-3 13-20 17-20 0-0.2 0.3-2.7 0-0.4 4-8.5 2.5~4.5 49-53 1-7 34-44 -- -- -- 0-0.4 -- 2.5-4.5 56-58 28-34 3-12 -- 0.5-1.5 -- -- -- 1-6 55-60 21-26 0-4 0-0.5 0-2.5 11-13 0-0.6 0-1.5 0.5-2.5 230 PAOLETO ET AL. TABLE 7 Percentage of Fibrous Particles and of Asbestos Fibers in Some Industrial Talcs % Fibers in the particulate % Fibers > 5 itm in the particulate % Asbestos fibers versus total fibers % Asbestos fibers in the particulate A 26.5 1.7 B 5.7 0.3 C 4.7 0.7 D 2.6 0.3 2.8 0.6 0.8 0.1 0.5 0.2 0.6 0.1 <2 8 + 2.7 4 1.9 <2 <0.5 0.5 + 0.2 0.2 + 0.1 <0.05 Variety of asbestos _ Tremolite Chrysotile -- phase-contrast microscopy, until now the most widely used technique for the obser vation of particles and mineral fibers. For the talc samples in which the presence of asbestos fibers was not detected, the upper lim its chosen for the asbestos content were those evaluated by the abovementioned criteria (90% probability criteria). Such upper limits are listed in Tables 7-10. It must be pointed out that in all talc powders analyzed, quite frequently, fibrous talc particles have been observed morphologically to be very sim ilar to amphibole fibers (Fig. 6) but easily recognizable by their typical electron diffraction patterns (Fig. 7) (19-23). In the examined samples, particles of minerals usually found together with talc (Table 1), in particular Ca and Mg carbonates, have been frequently observed; as already mentioned, until now no attempts have been made to evaluate quantitatively their content in talc samples. DISCUSSION It is well known that occupational exposure to talc is associated with a diffuse interstitial lung-scarring talcosis. Moreover, many experimental and epidemiological data show that the fibrous fraction of talc powders is more hazardous than the platy one because of presence of asbestos fibers which contam inate natural talc deposits. The aim of this study is the evaluation of the percentage o f fibers and, in particular, of asbestos fibers present in talc powders. TABLE 8 Percentage o f Fibrous particles and o f asbestos fibers in Some Pharmaceutical Talcs % Fibers in the particulate % Fibers > 5 un in the particulate % Asbestos fibers versus total fibers % Asbestos fibers in the particulate Variety of asbestos A 5.4 + 0.8 B 8.7 1.0 C 4.5 + 0.6 D 3.1 0.4 E 3.7 + 0.4 1.1 + 0.4 3.1 0.6 0.4 + 0.2 0.9 0.2 1.0 0.2 8 + 2.7 <2 14 3.4 21 4 17 3.7 0.4 + 0.2 <0.2 0.6 0.2 0.7 0.2 0.6 0.2 Tremolite -- Tremolite Tremolite Tremolite / ily JCS --' tett seSWS moiae -- emolite remol" " ' ' ASBESTOS CONTAMINATION OF TALCS 231 TABLE 9 Percentage of fibrous Particles and o f Asbestos fibers in Some Cosmetic Talcs % Fibers in the particulate % Fibers > 5 pm in the particulate % Asbestos fibers versus total fibers % Asbestos fibers in the particulate Variety of asbestos A 6.1 0.9 B 21.6 1.6 C 11.1 1.1 D 4.9 0.5 E 10.3 0.7 F 5.1 0.6 1.6 0.5 5.0 0.9 3.2 0.6 0.7 0.2 3.2 0.4 1.8 0.4 <2 <2 <2 32 4.7 <2 10 3 <0.1 <0.4 <0.2 1.6 0.3 <0.2 0.5 0.2 . -- -- Tremolite -- Tremolite By means o fthe method described above, it is not possible to obtain a good estimate of the percentage by weight of other minerals present in talcs. In some samples, a very high level of asbestos contam ination was revealed, possibly \ related to risk for people in contact in various ways with talc powders. This underlines \ the need of a deeper knowledge o f the characteristics o f talc currently in use and of \ suitable regulations, should the results of this study be confirmed on a vast scale, \taking also into account the lack o f national and international legislation about talc powder characteristics. The sole regulations concerning the am ount o f fibrous am phiboles in talc were issued in 1976 by the CTFA: It was stated that talc powders tilized in cosmetics and in toiletries should contain at least 90% talc and no revealable foestos fibers. \ TABLE 10 \ Percentage of Fibrous Particles and of Asbestos fibers in Talcs Provided \---------------- by the European Pharmacopoeia \ Fibers in % Fibers > 5 pm % Asbestos fibers % Asbestos fibers particulate in the particulate versus total fibers in the particulate Variety of asbestos i 0.3 "% 1.1 ! \ 0.24 1 0.35 : 0.37 ,0.48 .25 1.23 >,87 \ V\ 8 \\ 2.1 0.2 6.39 0.6 0.83 0.13 1.43 0.25 1.9 0.2 2.8 0.3 0.56 0.13 0.32 0.1 7.7 0.57 2.0 0.3 1.8 0.27 1.8 0.26 1.5 0.26 2.3 0.32 <1 87 1.2 <1 <2 <2 <1 <2 <2 92 1.3 <2 3 1.2 4 1.4 <2 3 1.4 <0.04 21.7 1.0 <0.03 <0.06 <0.07 <0.07 <0.05 <0.03 18.7 0.8 <0.07 0.13 0.05 0.15 0.05 <0.08 0.15 0.07 Tremolite 88% Anthophyllite 12% Traces of chrysotile -- -- -- -- -- Tremolite 95% Anthophyllite 5% -- Tremolite 50% Chrysotile 50% Chrysotile -- Chrysotile 232 PAOLETTl ET AL. Fia. 4. Tremolite fiber associated with talc particles. Fig. 5. Chrysotile fibers associated with talc particles. ASBESTOS CONTAMINATION OF TALCS 233 Fig. 6. Talc fibrous particle. Fig. 7. Electron diffraction pattern of a talc fibrous particle. ASBESTOS CONTAMINATION OF TALCS 235 26. Stewart, I. M. (1978). Transmission electron microscopical methods for the determination ofasbestos. NBS Spec. Publ. (U. S.) 506,271-280. 27. SlUKNE, M. I,, Talbot, J. M., and Rendal, R. E. G. (1971). Electron diffraction patterns of U.I.C.C asbestos samples. Environ. Res. 4, 141-145. 28. Rund, C. O., Barrett, C. S., Russel, P. A., and Clark, R. L. (1976). Selected area electron diffraction and energy dispersive X-ray analysis for the identification ofasbestos fibres, a comparison. Micron 7, 115-132. 29. Mueller, P. K., and Aloocer, A. E. (1975). Asbestosfiber atlas. Technical Report Data EPA 650/ 2-75-036, State of California Department of Health. 30. Hall, T. A. (1971). The microprobe assay of chemical elements. In Physical Techniques in Biological Research (G. Oster, ed.), pp. 158-266. Academic Press, New York. 31. Chandler, J. A. (1977). X-RayMicroanalysis in theElectronMicroscope. North-Holland, Amsterdam. 32. Council o f European Communities (1983). Council Directive on the Protection o f Workersfrom the Risks Related to Exposure to Asbestos at Work. 33. Cosmetic, Toiletry and Fragrance Association (CTFA) (1976). CTFA Specification: Cosmetic Talc. CTFA, Washington, D. C.