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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 REGULATORY TOXICOLOGY AND PHARMACOLOGY 4 222 2315984 Evaluation by Electron Microscopy Techniques of Asbestos Contamination in Industrial Cosmetic and Pharmaceutical Talcs L. PAOLETTI S. CAIAZZA G. DONELLI AND F. POCCHIARI 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 anthophyllite an amphibole 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 ...mi.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 of one sheet of Mg OH between two layers of SiO4 ) In natural deposits Mg is often substituted in talc crystals by other cations such as Fe Ni Cr Mn etc. Deposits are not generally monomineralic but since they result from geologic processes which caused the formation of several different mineral phases they are heterogeneous as far as the kind and relative amount of the minerals 2 Thus it is not surprising that materials known as talc powders may contain less than 50 of talc 3 The most common minerals that may be found mixed with talc in mineral deposits are listed in Table 1. Among them two fibrous kinds of amphibole tremolite and anthophyllite and a fibrous kind of serpentine mineral chrysotile constitute some of the best known varieties of asbestos 2 4 5 Talc powders are widely employed in a number of industrial processes and in commonly used products i.e. in the manufacturing of 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 of epidemiological data point out the 222 0273-2300 3.00 Copyright '1984 by Academic Press Inc. All rights of reproduction in any form reserved ASBESTOS CONTAMINATION OF TALCS TABLE 1 MINERALS COMMONLY ASSOciated with Talc in Natural Deposits Carbonates Calcite dolomite magnesite Amphiboles Tremolite anthophyllite Serpentines Chrysotile antigorite lizardite Others Quartz mica chlorite rutile pyrophyllite 223 risk of cancer connected with the fibrous components of 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 three- to 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 15 However there is a considerable lack of analytical data about the fibrous components of talc In 22 talc powders examined values ranging from 8 to 30 of 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 tremolite 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 tremolite 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 amount 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 techniques such as electron diffraction and 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 amount of 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 of 0.5 to 0.8 Formvar in dichloroethane at a concentration of 0.02 to 0.05 ml talc After air insufflation by a Pasteur pipet to avoid sedimentation two or three drops of the suspension were put on a 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 of twice distilled H2O and collected on 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 electron diffraction SAED was utilized Chemical elements present in the mineral particles were detected by ray microanalysis equipped with a wave dispersion spectrometer Statistical Evaluation of Data We checked the statistical compatibility of the results of replicate determinations of fibrous fractions for the same talc specimen The x2 test was utilized to check the hypothesis that the differences in the values ofthe fibrous fractions were due to chance 21 For each kind of talc the average value of the percentages of fibers and of nonfibrous particles were calculated The numbers offibers and ofnonfibrous particles measured in each determination have been utilized as elements ofa contingency table and xwas 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 xwere not generally significant enough xxp < 0.95 to reject the initial hypothesis However in some cases we obtained comparatively high xvalues xxp > 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 number 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 ...mwere 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 xwe obtained and the ones corresponding to a 95 probability at the same degree of freedom are listed To check whether the method of sample preparation could cause an increase or a decrease in the fibre percentage for each kind of talc powder we analyzed the correlation between the total number of particles per unit area of the film the area of one mesh of the grid and the percentage of 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 r21 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 TABLE 2 xTEST TO CHECK The ReprodUCIBILITY IN THE EVALUATION OF THE FIBROUS Fraction in Talc PowdeRS Sample ABCDEFOILSMAN ABCDEFOILSMAN ABCDEFOILSMAN ABCDEFOILSMAN ABCDEFOILSMAN ABCDEFOILSMAN ABCDEFOILSMAN ABCDEFOILSMAN ABCDEFOILSMAN ABCDEFOILSMAN ABCDEFOILSMAN ABCDEFOILSMAN ABCDEFOILSMAN xObserved 9.9 1.61 10.6 7.58 3.65 2.95 0.37 2.34 7.58 3.65 2.95 4.32 1.39 x0.95 16.9 5.99 11.1 9.49 7.81 7.81 3.84 5.99 9.49 7.81 7.81 5.99 9.49 225 Statistical Criteria for Evaluating the 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 number of fibers This allows calculation of the number of particles to be observed in order to obtain the required accuracy of measurement For a binomial distribution the relative standard deviation as percentage of the mean value calculated by means of the a priori probability P of observing a fiber and by means of the number N of observed particles is = ( N - 1/2 N The total number of particles to be observed in order to evaluate the particle 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 of P and N shows that in order to evaluate the fiber concentration with a relative standard deviation of less than 10-15 N must be about 10even 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 PER UNIT Area and Fibrous ParticLE PERCENTAGE Sample Correlation parameter ABCDEFG 0.22 ABCDEFG 0.13 ABCDEFG 0.03 ABCDEFG 0.13 ABCDEFG 0.002 ABCDEFG 0.41 ABCDEFG 0.06 226 PAOLETTI ET AL TABLE 4 RELATIVE Standard Deviation for a Binomial DisTRIBUTION P 0.01 0.05 0.10 0.15 0.20 0.25 0.35 N 100 99 43 30 24 20 17 15 N 1000 31 14 % % % % % N = 10,000 10 4.3 % 2.4 % 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 limit 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 number N of fibrous particles as a function of the ratio u between the number of asbestos fibers and total number of fibers P = 1 - ( - 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 v < 10-4 the probability of observing one asbestos fiber is very close to zero for samples containing 10 fibrous particles To fix an upper limit for the estimated asbestos concentration in the sample we chose a value of the probability P of singling out at least one fiber of asbestos in the sample of 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 number of fibers observed provided such a probability P = 0.9 This value TABLE 5 PROBABILITY OF OBSERVING AT LEAST ONE ASBESTOS FIBER AMONG N FIBROUS Particles as A FUNCTION OF THE RATIO V BETWEEN ASBESTOS FIBERS AND TOTAL FIBERS N U 1074 5 ^ 10 10-3 ^ 10 3 ^ 10-3 ^ 10 ^ 10 10-2 ^ 10-2 ^ 10-2 10-1 50 0.005 0.025 0.05 0.10 0.14 0.22 0.30 0.39 0.64 0.92 1.00 100 0.01 0.05 0.10 0.18 0.26 0.39 0.50 0.63 0.87 1.00 1.00 200 0.02 0.10 0.18 0.33 0.45 0.63 0.75 0.87 0.98 1.00 1.00 500 0.05 0.22 0.39 0.63 0.78 0.92 0.97 0.99 1.00 1.00 1.00 1000 0.10 0.39 0.63 0.86 0.95 0.99 1.00 1.00 1.00 1.00 1.00 2000 0.18 0.63 0.86 0.98 1.00 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 limit since a higher concentration would provide a probability higher than 90 that is practically the certainty of observing one asbestos fiber RESULTS Twenty 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 ray microanalysis and for further observation by light microscopy Moreover the samples showed a considerable degree ofstability even after repeated observations The statistical analyses showed the reliability and reproducibility of the results obtained for powder samples prepared by the described method According to the criteria accepted by the Council of European Communities 32 particles have been considered as fibrous when having a length ratio greater or equal to 3 and a width less than 3 m Fibers respectively greater and less than 5 ...min length have been considered separately according to the mentioned criteria which consider as more haz- ardous because of their biological effects the fibers longer than 5 ...mThe counts for the evaluation of fibrous particle percentages have been performed on a total number of particles ranging from 2 ^ 10to 10 ^ 10for 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 % To evaluate the pollution due to asbestoisn 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 determination with certainty the presence of serpentine asbestos Fig ) 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 of 100 random fibrous particles has been considered We studied the electron diffraction pattern of each sample and its Ca and Fe contents by means of 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 of 15 Italian talcs the presence of asbestos fibers has been revealed in 7 samples there were fibers of tremolite and in 1 sample there were fibers of chrysotile 228 PAOLETTI ET AL FIG 1. Electron diffraction pattern of a chrysotile fiber FIG 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 tremolite and chrysotile In the 2 talc samples containing amphibole asbestos tremolite and anthophyllite were present in great percentages reaching in both cases about 20 of the entire particulate Figures 4 and 5 show a few typical fibers of such mineral varieties observed in some of the samples examined Interestingly about fourths of the asbestos fibers observed in each sample had a diameter smaller than about 0.4 ...mthat 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 SiO2 MgO FeO Fe O Al O3 CaO KO Na O HO 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 04 0-0.5 0-2.5 11-13 0-0.6 0-1.5 0.5-2.5 230 PAOLETTI ET AL TABLE 7 PERCENTAGE OF FIBROUS PARTICLES AND OF ASBESTOS FIBERS IN SOME INDUstrial Talcs % Fibers in the particulate % Fibers 5 m in the particulate % Asbestos fibers versus total fibers % Asbestos fibers in the particulate ABCD 26.5 -1.7 ABCD 5.7 -0.3 ABCD 0.7 ABCD 2.6 -0.3 2.8 -0.6 0.8 0.1 0.5 0.6 -0.1 < 8 2.7 1.9 < 0.5 0.5 -0.2 0.2 0.05 Variety of asbestos ~~ Tremolite Chrysotile ~ 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 limits 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 similar 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 ) 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 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 contaminate natural talc deposits The aim of this study is the evaluation of the percentage of fibers and in particular of asbestos fibers present in talc powders TABLE 8 PERCENTAGE OF FIBROUS PARTICLES AND OF ASBESTOS FIBERS IN SOME PharmaceuTICAL TALCS % Fibers in the particulate % Fibers > 5 m 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 0.4 E 3.7 -0.4 1.1 0.4 3.1 -0.6 0.2 0.9 -0.2 1.0 0.2 - 2.7 < 3.4 21 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 I emolite remolite ASBESTOS CONTAMINATION OF TALCS 231 TABLE 9 PERCENTAGE Of Fibrous PartICLES AND OF ASBESTOS FIBERS IN SOME COSMETIC TALCS % Fibers in the particulate % Fibers...m = % Asbestos fibers % Asbestos fibers in the particulate versus total 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 < < < 32 -4.7 < 10 0.1 0.4 0.2 1.6 0.3 0.2 0.5 -0.2 _ ~ - Tremolite ~ Tremolite By means of the 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 contamination was revealed possibly related to risk for peoplein contact in various ways with talc powders This underlines 'P, the need of a deeper knowledge of the characteristics of talc currentlyin use and of _ suitable regulations should the results of this study be confirmed on a vast scale taking also into account the lack of national and international legislation about talc characteristics The sole regulations concerning the amount of fibrous am- powder in talc were issued in 1976 by the CTFA It was stated that talc powders hibolesin tilized in cosmetics andin toiletries should contain at least 90 talc and no revealable bestos fibers % TABLE 10 i PERCENTAGE OF FIBROUS PARTICLES AND OF ASBESTOS FIBERS IN TALCS PROVIDED . BY THE EUROPEAN PHARMACOPOEIA \ Fiberisn % Fibe > r 5 sm% Asbestos fibers % Asbestos fibers particulate in the particulate versus total fibers in the particulate 1 0.3 1.1 2.1 0.2 6.39 -0.6 < 87 -1.2 0.04 21.7 -1.0 -0.24 : 0.35 0.37 0.48 0.25 0.25 9.23 9.23 87 8 \} 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 A A A A A A 92 1.3 < 1.2 1.4 < 1.4 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 Variety of asbestos - Tremolite 88 Anthophyllite 12 Traces of chrysotile ~ _ - Tremolite 95 Anthophyllite % - Tremolite 50 Chrysotile 50 Chrysotile - Chrysotile 232 PAOLETTI ET AL FE um Re sagt UN Ne a 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. SKIKNE 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 ray analysis for the identification ofasbestos fibres a comparison Micron 7 115 132 29. MUELLER P. K. AND ALCOCER 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 26A6cademic Press New York 31. CHANDLER J. A. 1977 Ray Microanalysis in theElectron Microscope Holland Amsterdam 32. Council of European Communities 1983 Council Directive on the Protection of Workers from 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.