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FILE NAME: Colgate (COL) DATE: 2014 DOC#: COL040 DOCUMENT DESCRIPTION: Journal Article - Asbestos in Commercial Cosmetic Talcum Powder as a Cause of Mesothelioma in Women Asbestos in commercial cosmetic talcum powder as a cause of mesothelioma in women Ronald E. Gordon1, Sean Fitzgerald2, James Millette3 'Department of Pathology, Icahn School of Medicine at Mount Sinai, New York, USA, 2SAI Laboratory, Greensboro, NC, USA, ' MVA Inc., Duluth, GA, USA Background: Cosmetic talcum powder products have been used for decades. The inhalation of talc may cause lung fibrosis in the form of granulomatose nodules called talcosis Exposure to talc has also been suggested as a causative factor in the development of ovarian carcinomas, gynecological tumors, and mesothelioma Purpose: To investigate one historic brand of cosmetic talcum powder associated with mesothelioma in women Methods- Transmission electron microscope (TEM) formvar-cated grids were prepared with concentra tions of one brand of talcum powder directly, on filters, from air collections on filters in glovebox and simulated bathroom exposures and human fiber burden analyses The grids were analyzed on an analytic TEM using energy-dispersive spectrometer (EDS) and selected-area electron diffraction (SAED) to determine asbestos fiber number and type Results: This brand of talcum powder contained asbestos and the application of talcum powdei released inhaladle asbestos fibers. Lung and lymph node tissues removed at autopsy revealed pleural mesothelioma Digestions of the tissues were found to contain anthophyllite and tremolite asbestos Discussion: Through many applications of this particular brand of talcum powder, the deceased inhaled asbestos fibers, which then accumulated in her lungs and likely caused or contributed to her mesothelioma as well as other women with the same scenario. Keywords: Asbestos, Talcum powder, Chamber test, TEM, SEM, EDS, SAED, Mesothelioma Introduction Malignant mesothelioma occurs m both the perito neum and m the lung pleura.1 Mesothelioma cases have been attributed to direct occupational exposure, indirect exposure and secondary exposure.1A highei rate of `'idiopathic" mesothelioma has been reported in women, as no link between asbestos exposure and patients has been identified." Previous research suggests that ovarian cancer and peritoneal mesothe lioma may be directly attributed to the use of talcum powder contaminated with asbestos or from exposure to partners occupationally exposed to asbestos.'"7 Using talcum powder m closed spaces may increase the likelihood of inhaling the powdei laced with asbestos. Repeated applications increase the oppor tunities for inhalation and the asbestos could become concentrated in the peripheral airways and alveoli of the lungs of the talcum powder users This has been supported by the presence of granulomas in the lungs of some talcum powder usersR Correspondence to R E Gordon, Department of Pathology, Icahn School of Medicine at Mount Sinai, 1 Gustave L Levy Place, New York 10509, USA Email Ronald Gordonmountstnai org In 1976, Rohl and Langer tested 20 consumer products labeled as talc or talcum powder, including body powders, baby powders, facial talcums, and a pharmaceutical talc.6 Of the 20 products tested, 10 were found to contain tremolite and anthophyllite, principally asbestiform The product with the highest asbestos content was the same product tested in this study. Both asbestiform anthophyllite and asbesti form tremolite were found m the Rohl and Langer tests Given that asbestos has been determined as the primary cause of mesothelioma, it is important to note that cosmetic talc contained asbestos in the past.6 The contamination results from the mining process, since ore specimens taken directly from the mines have repeatedly been tested and shown to contain asbestos, most often anthophyllite and tremolite but also serpentine chrysotile asbestos,6,9'10 In part from the review of corporate documents and the sworn testimony of those responsible for the sourcing of talc used in the products studied here, it was determined that three mines provided the raw material for use as talcum powder The talc used by this cosmetic company that manufactured and W $ . Maney & Son Ltd 2014 DOI 10 1179/204939674Y 0000000081 International Journal of Occupational and Environmental Health ?014 \cx 000 r o 000 1 Gordon et ai Asbestos in commercial cosmetic talcum powder distributed the talcum powder was from three distinct regions, the Willow Creek mine in Southwest Montana, the Regal mine near Murphy, North Carolina, and imported talc from the Val Chisonc region of the Italian Piedmont.11-16 The specific geology of talc is an important indicator of whether a talc source may be contaminated with asbestos These three mines all contained asbestos fibers, anthophyllite, and tremolite.11-18 The Val Chisone talc from Italy was studied by Pooley in 1972.18 Mine sample had intergrowths with serpcntinc-type, chrysotile asbestos along with tremolite and anthophyllite asbestos The talc from Italy was named `American Ground Italian' and designated as AGI 1615.19 21 This talc was diluted with a talc from another source to make it acceptable based on X-ray diffraction (XRD) protocols. How'ever. it contained asbestifoim tremolite and anthophyllite 22 In this study, three laboratories analyzed a specific brand of talc from more than 50 containers of this cosmetic talcum powder product of different sizes and colors, produced over a 50-yeai time span to determine the presence of asbestos. The authois conducted independent product testing m unassociated laboratories in North Carolina, Geoigia, and New York. A fourth laboratory, which also tested this product, will herein be referred to as Laboratory D The lung and lymph node tissues from a woman who died from mesothelioma and testified to only using this specific brand of talcum pow'der were analyzed for the presence of asbestos and talc This is the first report that explores the hypothesis that a specific brand of talcum powder coming from asbestos contaminated mines can find its way into the finished product that can be inhaled during use and cause oi contribute to the development of mesothelioma Materials and Methods Laboratory A: product testing In Laboratory A, over 50 containers of this particular brand of talcum powder were acquired from a variety of sources for bulk testing Some of the containers were purchased online, while others were piovided directly from the manufacturer. All of the containers were verified to be the correct brand and product. Laboratoiy A tested talcum powder from each of the 50 samples using transmission election micro scope (TEM) methods The procedure for testing by Lab A was as follows' 0.01 g of talcum powder was removed from its vial and suspended in 1 ml of distilled water with one to two drops of ethanol by bnef sonication From this suspension. 10 pi aliquots were removed and placed on a senes of five formvarcoated nickel grids (100 grid openings each) In some cases, it was necessary to prepare additional sets of five grids from the same 0.01 g sample of powder The drops were allowed to dry in a covered Petri dish. The grids were then examined and analyzed with a Hitachi H-7000 STEM equipped with an Evex energy-dispersive spectrometer (EDS), for elemental composition and relative amounts of elements. The microscope was equipped with a tilt stage and a rotary specimen holder, which was employed with selected-area electron diffraction (SAED) analyses, as described below. Structures seen as fibers measuring at least five micrometers in length with aspect ratios of 5 : 1 oi greater were analyzed to determine if they were regulated asbestos mineral fibers. We used EDS to chemically establish the presence of asbestos fibers and the crystalline structure was assessed using SAED. All 100 grid openings were observed and analyzed on each of the five grids foi each pioduct sample (at least 500 grid openings per sample analyzed). Analyses were performed using a modification of the techniques described by Yamate et ai.. and similarly adopted techniques used by the Environ mental Protection Agency (EPA), American Society for Testing and Materials (ASTM). and Interna tional Organization for Standardization.2' 26 All techniques required the use of a TEM equipped with an EDS system. Only in Yamate level III is the tilt and rotary stage optional to perform advanced SAED zone axis analysis. Yamate et a! stated that zone axis diffraction analysis is useful m differentiat ing between otheiwisc unidentifiable fibers In the Laboratory A analysis, zone axis analyses were not necessary as the identified ampluboles dearly demon strated that they weie asbestiform tremolite and anthophyllite confirmed by morphology. EDS chem istry, and characteristic 5.3 A mter-row repeats on diffraction without tilting Both asbestiform and nonasbestiform particles and fibers were present However, m most cases this manuscript wall refer to asbestiform fibers and state when they are tremolite, anthophyhte, or chrysotile type asbestos. A non asbestos tremolite, anthophyhte will not be referred to as asbestos To calculate the fiber concentrations per gram of talcum powder, w'c first determined the number of asbestos fibers on average per grid opening This number was multiplied by 552 The product of that equation was multiplied by 100, and then divided by 0.01 to yield the fibers/gram talcum powder value The constant, 552, is the number of grid opening areas on the entire grid. One hundred is the numbei of 10 pi diops in 1 ml that the talcum powder was dispersed and the 0 01 was the weight of the talcum powder dispersed Quality control procedures, which included testing of blanks from water, working in a clean hood environment, and working with only one 2 International Journal of Occupational and Environmental Health ?0 14 / gl 0 0 0 mo 000 Gordon et al Asbestos m commercial cosmetic talcum powder Figure 1 Pouring of powder into hands in giovebox. sample at a time ensured that no laboratory contamination of samples Laboratory B: asbestos releasability testing To determine if the user could inhale asbestos during a talcum powder application. Laboratory B assessed asbestos releasability by air sample. Air samples were generated during simulation m a glove box, consis tent with normal product use in a controlled environment These three samples included the same samples tested by Laboratory A. Environmental and personal air samples were collected using standard airborne asbestos techniques, using high-volume air pumps for environmental (stationary) samples inside and outside of the controlled area, and low-volume air pumps for personal samples' taken at a distance comparable to the breathing zone of the person simulating application Standaid TEM 385 mm2 effective filter area 25 mm cassettes with 0.45 pm MCE filters were used on the flow-calibrated high (712 1/mm) and low- volume (1-4 1/min) air pumps (Figs 1 and 2) The resulting air samples were analyzed for airborne asbestos following the analytical procedures described in the U.S. Environmental Protection Agency Code of Federal Regulations 40 CFR part 763, subpart E, Appendix A -- AHERA for direct preparation of MCE filters.24 All final analyses by Laboratory B were conducted on a JEOL 2000FX TEM equipped with an energy-dispersive X-ray analyzer detector and SAED at magnifications up to x 50 000, using the fiber counting criteria specified by Yamate et a I's protocols 21 Laboratory C: product bulk testing and bathroom-sized chamber releasability Bulk methods Laboratory C examined nine samples under an Olympus.SZ-40 stereomicroscope at magnifications from x 7 to x 40. Portions of the paiticulate found in the sample were mounted in Cargille refractive index liquids for analysis by polarized light microscopy (PLM) using an Olympus BH-2 PLM with a magnification range from x 100 to x 1000 The PLM analysis followed the procedures for bulk analysis of building materials described by the US EPA m 1993 24 Characterization of the fibers was performed using a Philips EM420 100 kV TEM equipped with an Oxford INCA EDS x-ray analysis system and capable of SAED work involving tilting of amphibole fibers. Zone axis determinations weie also conducted. We used TEM asbestos fiber count ing criteria of fibers greater than 0.5 pm in length with at least a 5.1 aspect ratio as described in Asbestos Hazard Emergency Response Act (AHERA) and AS'lM methods: D6281, D5755. Figure 2 TEM cassettes in simulation area in giovebox. International journal of Occupational and Environmental Health ?014 voi 000 i u 000 j Cordon et al. Asbestos in commercial cosmetic talcum powder D5756, and D648.24~2R Data were recorded using the ASTM D6281 format XRD analysis was performed by an outside laboratory (DCM Science Laboratory, Inc , Wheat Ridge, CO, USA) scanning over a range of 3 45 2 0 using 40 kV, 25 mA Cu K, radiation Mineral phases were identified with the aid of computer-assisted programs accessing a CD-ROM powder diffraction database. Air testing Tests to determine airborne levels of asbestos fibers resulting from application of this bland of talcum powder were performed m a testing chamber The chamber was built to match the bathroom of the patient that used this brand of cosmetic talc. Her bathroom was measured at 7 feet, 9 inches high by 5 feet by 4 feet. 1 inch All talc products used in these chambei tests had previously been tested in Laboratories A. B, or both Air test -- shaker container Using Personal Protective Equipment, a volunteer applied one of the bulk tested cosmetic talcum powders to his body using a shaker container. This particular talcum powder contained approximately 0 1% by weight and approximately 18 million anthophyllite asbestos fibers per gram The container was weighed before and after the testing to determine the approximate weight of material applied The talcum user wore a respirator and a bathing suit The volunteer twisted the top of the container and shook material onto Ins hand. He applied the talc under his arm and around the shoulder and upper arm area. He then shook the talcum powder onto his other hand and applied it to the other undeiarm, shoulder and upper arm area. He shook out additional material and applied it to his neck and upper torso He shook out and applied material two more times for a total of five applications. The total talcum application time was approximately 1 mm and amounted to 0.37 g of the talcum powder. Two air samples were collected in the appher's breathing /.one at 0.5 1per minute (1pm) and two additional air samples were collected in the breathing one at 1 0 1pm with commercial open-face air cassettes. The five-minute sampling time included the application time and a waiting period. The bystander m the test chamber had two air cassettes in lus breathing zone foi the fivc-minutc period including application and the additional waiting time. The bystander wore a respirator and full protective clothing. These air samples were collected at rates of one and 2 1pm No activities were conducted during the waiting period other than checking the pumps and cassettes. The air filters and two additional blank filters were analyzed by phase contrast microscopy (PCM) using National Institute for Occupational Safety and Health (NIOSII) Method 7400.29 Two air samples and two blanks were also analyzed by NIOSH Method 7402 via transmission electron microscopy to determine the percentage of asbestos fibers among the fibers counted by PCM.29 An air sample collected from within the test chamber before the study was analyzed by a more sensitive TEM procedure following the EPA AHERA method 24 Air testing puff applicator In this test, a volunteer applied a different cosmetic talcum powder sample using a puff applicator Phis particular talcum powder contained approximately' 0.05% anthophyllite asbestos (approximately 70 mil lion asbestos fibers per gram). The container was weighed before and after the testing to determine the approximate weight of mateiial applied The talcum user wore a respirator and a bathing suit. I he talc user opened the puff container and applied the talcum powder as described above only this time with a powder puff He then repeated the process for a total of six applications. The talcum application time was approximately 1 minute. Two air samples were collected m the appher's breathing zone at 0.5 1pm for a sampling period of 4 minutes. One air sample was collected for a shorter period (3.3 min utes) that included the application period Another air sample was to be collected after the application period but this sample was voided because the volunteer hit the air cassette and the cassette fell off the vacuum hose. The bystander in this test followed the same protocol as described above. Both air samples were collected at a rate of 0 5 1pm. No activities were conducted during the waiting period other than checking the pumps and cassettes. The air filters and two additional blank filters weie analyzed by PCM using NIOSH Method 7400 as described above.29 One air sample and two blanks were also analyzed by NIOSH Method 7402 via TEM to determine the percentage of asbestos fibers among the fibers counted by PCM.30 An air sample collected from within was tested as described above by EPA AHERA method 24 Human Tissue Analysis TEM Tissue samples from a woman with no other known exposure to asbestos other than her use of the product tested was supplied to Laboiatory A Human tissue analysis was performed according to the techniques described in Wu et a l 29 Lung and lymph node tissue was received fixed in formalin Half of the. tissue was removed from the lung and the lym ph node tissue T w o grams of lung tissue were divided twice The two halves of the lymph node weighed 0 16 g together. The two specimen types weie separated throughout the study The tissue from each was fiist digested in a 5% solution of potassium 4 International journal of Occupational and Environmental Health 2 0 14 ya 000 no 000 Gordon et al Asbestos in commercial cosmetic talcum powder hydroxide (KOH) for approximately hour at 60 C. The dissolved lung and lymph node material was then centrifuged m a high-speed centrifuge to separate the inorganic material from the dissolved organic tissue. The solute material containing the dissolved organic material and KOH was removed and distilled water was added The inorganic material was re-suspended in the water by brief sonication The material was recentrifuged and the pi ocess of washing the inorganic material was performed five times After the fifth wash, the distilled water was removed and replaced with 10 ml of fresh distilled water and the inorganic material was re-suspended by brief sonication. Ten microhter samples were removed from the suspension and placed on formvar-coated nickel grids on a metal mesh m a covered glass Petri dish to dry. Five grids were initially prepared and an additional set of five grids was prepared for each tissue type for a second analysis The dried grids were observed with a transmission electron microscope. Four hundred grid openings on at least four grids were analyzed, and a fifth grid was used if grid openings were broken in the initial four examined grids The fiber concentrations per gram wet weight lung or lymph node tissues were calculated from the number of fibers observed, the area analyzed, the aliquot ratio, and the total weight of the tissue sample digested. Light microscopy Tissue sections Small lung tissue samples were put into 10% phosphate-buffered formalin and processed for embedding in paraffin. Five micrometer paraffin sections were cut, mounted on glass slides and stained with hemotoxylin, eosin, and an iion stain The tissue was evaluated for the presence of altered morphology and/or ferruginous bodies; two characteristics often seen in lung tissues that are a byproduct of iron-rich protein deposits on asbestos fibers resulting from macrophage frustrated phagocytosis. Digested lung and lymph node tissue Two hundred and fifty microliters of digested lung and lymph node material suspension used for TEM analyses was placed in a cytocentrifuge and the slides w'ere cover slipped and observed by phase contrast light microscopy. The entire area was counted for ferruginous bodies and calculated back to the weight of the tissue to determine the concentration of bodies per gram of wet weight tissue. Scanning electron microscopy (SEM) 5 EM samples were prepared by taking 250 pi of the suspended inorganic material used for the TEM and light microscopy analyses and placed on a 0.1 pm pore size Nucleopore filter mounted on a carbon planchcttc on an aluminum SEM stub. The material was allowed to dry m a covered Petri dish The stub was then coated with vaporized carbon and observed with a Hitachi S-4300 field emission scanning electron microscope equipped with an Evex EDS system. The entire filter sample surface was scanned for fibers and asbestos bodies Results All three laboratories confirmed in multiple tests the presence of asbestiform anthophyllite and asbestiform tremolite in the talcum powder pioducts, just as had been found and described by Rohl and Langer over three decades ago.6 Initial bulk analyses of 50 samples of this product m Laboratory A showed that all of the samples contained asbestos fibers Eighty percent contained only anthophyllite asbestos, 8% only tiemolite asbestos, 8% anthophyllite and tremolite asbestos and 4% anthophyllite, tremolite, and chrysotilc asbestos The lange in asbestos concentrations of fibers > 5 pm in length were calculated to be, at a minimum, between 1840 and 1 104 000 fibers per gram of talcum powder. More than 80% of the tested cans and plastic containers contained over 10 000 asbestos fibers/gram of talcum powder. Four of the containers had less than 5000 fibers per gram and six containers had moie than 250 000 fibers per gram However, it should be noted that there were many asbestos fibers that also had aspect ratios less than 8 1 These fibers were generally found to be shorter than 5 pm and were noted, but not counted m the original product testing oi m the lung and lymph node tissue testing by Laboratory A. There were also a number of fibrous tale particles that were easily distinguishable from asbestos by morphology If there was a question regarding their identity, both EDS and SAED were employed to recognize such fibers as talc. All the fibers that were actually counted in bulk and tissue preparations were 5 pm or greater in length, with aspect ratios for the most part greater than 10:1. The majority of asbestos structures counted demonstiated aspects iatios > 1 5 .1 , with many > 20:1. A minimum of four fibers was identified m each sample, making the concentration determinations of asbestos statistically significant and reproducible. Laboratory C. using PLM, TEM, and XRD. tested nine samples of the specific brand of talcum powder described above. Generally, the PLM analysis showed that the samples contained both platy and fibrous talc, less than 1% by volume of the PLM visible am p h ib o le fibers a n d som e q u a rtz Hie majority of the PLM amphibole particles had low aspect ratios (length to width) but some were >10 1 By XRD, one of the talcum powder samples was found to contain 4% anthophyllite No amphibole International Journal of Occupational and Environmental Health ?014 \Ot 000 kj 000 5 Gordon et al Asbestos n commercial cosmetic talcum powder Figure 3 Application ot powder from shaker in bathroomsized chamber. Figure 4 Application with powder puff in bathroom-sized chamber. minerals were detected in the other eight samples by XRD. The XRD detection limit was approximately 2% by weight. In TEM analysis, all nine samples weie positive for amphibolc asbestos (primarily anthophyllite), and were confirmed with zone-axis electron diffraction measurements. At least five asbestos fibers per sample were recorded in each sample, with concentrations ranging from 0.004 to 0 9% by weight and from 3 to 200 million asbestos fibers per gram of fibers greater than 0 5 pm m length with at least a 5 1 aspect ratio Air monitoring Releasability of asbestos into the air from the products was assessed by glove box simulation testing by Laboratory B, and by full chamber testing by Laboratoiy C In a manner consistent with methods used by the EPA, NiOSII or ASTM, study product body powders and dusting powders were applied hand to hand and hand to arm Consistent with bulk testing results, anthophyllite and tremolite asbestos was repeatedly found m the air tests resulting from these simulations (Figs. 6-8). Shaker container test The shaker application test used 0.37 g of talcum powder (Fig. 3). For the talc user, the average PCM fiber concentration in his breathing zone duiing application was 4 8 F/cc (3 1, 7.3, 3.9, and 4.9 F/cc). The asbestos to total fiber percentage as determined by TEM was 40%. Therefore, the asbestos concentra tion in the bieathing zone of the talc user duiing application was 1.9 F/cc. For the bystander the PCM fiber conccntiation w'as 1.35 F/cc (0 9 and 1 8 F/cc) and the TEM derived percentage of asbestos was 35%, which results in a bystander asbestos concen tration of 0.5F/cc. No asbestos fibeis were found m the sample collected in the chamber before the testing or m the blank filters. Puff application The puff application test used 6.25 g of talcum powder (Figs 4 and 5). For the talc user, the aveiage PCM fiber concentration m his breathing zone during the 5-inmute sampling period was 20 F/cc (23 6 and 16.5 F/cc) The asbestos to total fiber percentage as determined by TEM was 21%. Therefore, the asbestos concentrations in the bieathmg zone of the talcum powder user were 5 and 3.5 F/cc. The short term sample in the breathing zone of the applier had a PCM value of 60 F/cc. Using the TEM-denved percentage of asbestos of 10%, result for the short term sample was an asbestos concentration of 13 F/ ce. For the bystander, the PCM fiber concentration was 11,7 F/cc (13.7 and 9.7 F/cc). Using the mini mum TEM-derived percentage of asbestos of 36% results in a bystander asbestos concentration of 4 9 and 3.5 F/ce. No asbestos fibers were found in the sample collected m the chamber before the testing or in the blank filters. The tests performed independently by Laboratory C using a bathroom-sized room confirmed the findings for asbestos fibci release found by Laboratory B's glovebox testing. Samples showed that significant concentrations of anthophyllite. tremolite, and occasionally chrysotile asbestos were released in the simulated application of several iterations of the products. This confirmed not only Figure 5 Application with a powder puff in bathroom-sized chamber. 6 international journal of Occupational and Environmental Health ?014 /Cl 000 no 000 Gordon et al. Asbestos in commercial cosmetic talcum powder > ,<.iy//y'iy ^ 4 '%&' ' ' ' / '.......... >l* srf* C // r , !&> *'i- < M- 5 s ** ! ^ '%sv^ tv -... - rII2V >!*-% /J^ , ,r, ''44^^ t <' >'1Z1"1' ^ 'K***? *,. /> X # 7'lf" :3 !*>. -.y 1 '2H4&w'- ;" '* uss ,v'4 >>-"%<. ^ " ='" ^ y Ht tUM<V TLMhi^'4C-i!l'^ i-S/Jt/J* CB_A 4b Tffiit{3) 1 urn Figure 6 Tremolite asbestos from TEM analysis of releasability air testing of product (images, EDS, and SAED). the presence of asbestos in the talcum powders, but also that the asbestos contained m the friable powders was easily aerosolized m a manner consis tent with the products intended use, confirming the hypothesis that the cosmetic powders are capable agents of exposure to asbestos Human tissue analysis Electron microscopic analj sis of the lung tissue revealed amphibole type asbestos fibers m a calcu lated concentration of 1380 and 4150 fibeis per giant wet weight, respectively, with a limit of detection of 690 fibers per gram wet weight. All fibers counted International Journal of Occupational and Environmental Health ?014 voi 000 kc 000 7 Gordon ta l. Asbestos in commercial cosmetic talcum powder Figure 7 Anthophyllite asbestos from TEM analysis of releasability air testing of product (images, EDS, and SAED). were 5 pm or greater in length and had aspect ratios of 20:1 or greater. The amphiboles were identified by EDS and SAED analysis as anthophyllite (Fig 9) and tremolite (F'ig. 10) asbestos. The asbestos fibers were seen m a ratio of 1 -1 and 2 .1 , respectively (anthophyllite/tiemolite). There were many antho phyllite and tremolite asbestos fibers less than 5 pm in length that were not counted. The majority of these smaller asbestos fibers were of the anthophyllite type. Light microscopic analysis of the cytocentrifuge preparation revealed a calculated concentration of 140 asbestos bodies per gram wet weight of lung tissue by phase contrast light microscopy m both samples Electron microscopic analysis of the lymph node tissue revealed amphibole asbestos fibers in a calculated concentration of 12 738 fibers per giam wet weight, with a limit of detection of 2123 fibeis pel gram wet weight. AH counted fibers were at least 5 pm m length with aspect latios of 10 1 oi greater The amphiboles were identified by EDS and SAED analysis as anthophyllite and tremolite and they were seen in a ratio of 5.1 anthophyllite/tremolite. There were many anthophyllite and tremolite fibers less 8 International Journal of Occupational and Environmental Health ?014 /Ct 000 no 000  Gordon et al. Asbestos in commercial cosmetic talcum powder iS0M68 ChiyS*CluitM(MW 1S8eC(tiysil Figure 8 Chrysotiie asbestos from TEM analysis of releasability air testing of product (image and SAED). than 5 pm `n length that wcie not counted. We also observed blit did not count tiemolite cleavage fragments. Light microscopic analysis of the cytocentnfuge preparation levealed a calculated concentra tion of 92 asbestos bodies per gram wet weight of lymph node tissue by phase contrast light microscopy (Fig. 11). Histological sections of the tissue showed focal aieas of mild parenchymal fibrosis and a more generalized pleural fibrosis Although many ferrugi nous bodies were identified m the cvtocentrifuge preparation, most were relatively small and not seen in the H&E-stained paraffin sections These macro phages were clustered and contained a combination of fibrous and platy talc and small asbestos bodies. In addition to the fibrous and platy talc described above, other inorganic materials were seen. Aluminum silicates and magnesium aluminum sili cates m both fibrous and platy form were identified. We elected not to count these fragments. Their presence supports the hypothesis that the lung and lymph node samples match findings from the tested talcum powder. The two analyses performed on the lung tissue were from two separate tissue digestions The second was prepared with tissue not previously analyzed, but saved from the original half of the tissue retained by Laboratory A. The results proved to be completely reproducible with no finding of any additional fiber types other than those reported above. Confirmation of interlaboratory analyses After several years of independent testing m separate laboratories, the authors became aware of one another's work through litigation The finding that this historic brand of cosmetic talcum powder contained asbestos fibers with generally the same morphological and chemical assemblage was con firmed. A fourth laboratory (Laboratory D) tested many of the same samples, but did not report asbestos findings. Owing to the inconsistency with the other laboratories, re-exammation of results tiom Laboratory D was warranted. Two of the three authors of this study went to the Laboratory D and were supplied with the prepared filters on TEM grids or SEM stubs previously analyzed by Laboratory D They were also supplied with both TEM and SEM microscopes to re-analy/e the specimens, along with data and locator sheets, allowing For the same grid openings and areas to be observed as m the initial analyses Reanalysis of subject product samples One author re-analyzed the TEM preparations of 20 study products of talcum powder prepared by Laboratory D. Asbestos structures were found m the re-analysis, some of which were named in the original analysis as cleavage fragments, mtergrowths, or fibrous talc rather than as asbestos Although the author-reviewer agreed with many of the non asbestos fibers identified, he concluded the original analyses were incomplete. Additional analyses by the author-reviewers showed some of the incompletely analyzed fibers to be asbestos In other eases, asbestos found on re-analysis was located on aieas of the filter where no libers were recorded in the original bench sheets or reports In some instances, the overall distribution of particulates on the preparations was inhomogeneous, m contrast with the method of choosing grid openings for the original analysis by skipping -every other opening in a "checkerboard" fashion Furthermore, the methods named on the analytical count sheets were not the same as the methods cited in the reports from Laboratory D Laboratory D reported no asbestos fibers in the 20 samples analyzed. In contrast, asbestos fibers were identified in all 20 of the same products m Laboiatory A and m 16 of 20 products tested by Laboratory B In the re-analysis of those same 20 samples originally analyzed by Laboratory D via TEM, eight were found to contain asbestiform anthophylhte, six asbestilorm tremolite, and two were found to contain chrysotiie fibers These findings were significant because re-analysis was not a International Journal of Occupational and Environmental Health ?014 v<"u 000 i o 000 9 Cordon et al Asbestos in commercial cosmetic talcum powder 0000 3:39:12 P i 21-Jarv2014 kev 75W 40 Degree 10,230 Figure 9 This asbestos fiber is a representative sample removed from the lung tissue of the patient exposed to cosmetic talcum powder. Anthophyllite asbestos fiber is observed and its SAED pattern is demonstrated beside it with the EDS spectra. complete replication of the original analysis due to time constraints, damage, or unsuitable preparations. It was apparent that the technicians in Laboratory D missed fibers and misidentified asbestos fibers as non-asbestos Re-analysis of human tissue Laboratory D also performed fiber burden analysis on human tissue with differing results than the study o f the authors Similar to the re-evaluation o f bulk analyses, two author-reviewers analyzed the human tissue sample preparations of Laboratory D together and found significant differences m their analyses compared to the technicians who originally analyzed the grids and stubs. We determined that the technicians misidentified anthophyllite asbestos fibers that had been coated with iron and protein (anthophylhte asbestos bodies) as either cleavage fragments or as amosite fibers (Fig 12). Furthermore, it is the authors' consensus that there are no generally accepted criteria to classify individual fibers as cleavage fragments by TEM when the sample contains attributes o f an asbestos fiber or countable structure. When Laboratory D technicians initially looked foi asbestos bodies to determine the fiber core, they concluded that most were amosite However, when the two author-reviewers examined 10 International Journal of Occupational and Environmental Health ?014 ;u i 000 no 000 Gordon et al Asbestos in commercial cosmetic talcum powder Figure 10 This asbestos fiber is a representative sample removed from the lung tissue of the patient exposed to cosmetic talcum powder. Tremolite asbestos fiber with its corresponding EDS spectra. the same stiuctures, it was clear that the cores were either anthophyllite or could not be determined because there was exposed fiber core. In previous studies of human tissue having anthophyllite and anthophyllite bodies (Fig. 11), it was common to find that the entire anthophyllite core, even if quite long, was completely coated. Zone axis confirmation in bulk, tissue, and air Laboratories A, B, and C confirmed original amphibole asbestos stiuctures by zone axis diffraction Labora tories A, B. C, and D re-analyzed archived preparations with the intent of confirming amphiboles by zone axis diffraction. In all four sets of re-analyzed preparations, anthophyllite and tremolite asbestos were consistently Figure 11 These are asbestos bodies from the patients lung tissue taken by SEM. It is possible to see in the one to the left that the fiber is almost completely covered by the iron protein coating. This is compared to the one at the right which appears to have much more fiber exposed. However, upon EDS testing, it was determined that in both cases, these were anthophyllite fibers and they were both entirely coated, although much thicker is some areas as opposed to others. international Journal of Occupational and Environmental Health P014 voi 000 lo 000 11 Gordon et a i Asbestos in commercial cosmetic talcum powder Figure 12 Tremolite and anthophyllite asbestos from re-analyses of `Lab D' preparations (images, EDS, and SAED). confirmed by zone axis diffraction pattern measure ments This included confirmation of asbestiform am phiboles, including anthophyllite and tremolite asbestos from the original product testing, front the releasability air tests, and from TEM preparations of lung and lymph node tissues. 12 International Journal of Occupational and Environmental Health 2014 vci 000 mo 000 Discussion Historically, many mesotheliomas, particularly abdominal mesotheliomas in women, have been labeled idiopathic due to a lack of an identifiable source for asbestos exposure, Furthei, there has been an increase in the number of idiopathic pleural and abdominal mesotheliomas m women using this specific brand of talcum powder. There have been a few studies that have examined talcum powder and its potential to cause ovarian tum ors.! 5 The studies were inconclusive, but suggested that talc, asbestos, or both may cause these cancers through vaginal exposure.4 These studies attributed asbestos found within the women's lesions to result from contact with their partners. There was no consideration for the potential of the asbestos being a contaminant in the women's talcum powder 3-4 However, it has been leported that cosmetic talcum was contaminated with asbestos, and that asbestos was found in the mines from which the talc originated6,9 Our findings indicate that historic talcum powder exposure is a causative factor in the development of mesotheliomas and possibly lung cancers in women Talc has been identified as a causative for mesotheliomas m New' York talc miners.31 In recent years, more than 10 women developed mesothelioma and their only source of asbestos exposure was the use of one brand of talcum powder. This study demonstrates that the brand of talcum powder tested contained asbestos. Furthermore, we have traced the asbestos in the talc to the mines from which it originated, into the milled grades, into the product, and finally into the lung and lymph nodes of the users of those products, including one woman w'ho developed mesothelioma Based on the testing and re-testing conducted by the authors, it is evident that this product line has been consistently contaminated with asbestos tainted talc derivatives. The amount of asbestos was variable based on the time of manufacture and the talc source There have been numerous publications that have indicated that the talc m many talc deposits had asbestos contamination.32"35 The most common types of asbestos were tremolite and anthophyllite. These are the same asbestos fiber types found in the autopsied lungs and lymph nodes tested here for asbestos presence. In a few' containers tested in this study, chrysotile was also found, consistent with the source ore geology. Most, if not all, testing of cosmetic talc was perfoimed using techniques designed for light micro scopy, PLM , or by T L M criteria designed to test air and water samples. Testing determined if asbestos levels wei e above the EPA standards under AHER A or the Occupational Safety and Health Agency standards. These protocols ate based on the parameters described Gordon et a l Asbestos in commercial cosmetic talcum powder in the Yamate method.23 There are significant limita tions to these methods. PLM analysis misses small fine asbestos libers or fibrils because the limits of the resolution are approximately 0.2-0.5 pm for different forms of light microscopy. Based on our findings, approximately 90% of the fibers identified fall into this category. Determining the number of TEM grid openings to be counted during the analysis requires stopping factors, or limits on the quantity of analysis to be performed. The Draft Yamate method (1984) gives the guidelines of " 100 fibers or 10 grid openings, whichever is first."23 This counting rule was instituted foi cost limitation purposes. The Draft Yamate method describes that while this guideline of using 10 full-grid openings represents a judicious compromise between a reasonable experimental effort and a fairly low value of the detection limit, the analysis of additional TEM grid openings reduces the detection limit and improves the precision of the estimates In the talc study described here, a very low level of detection was desired and therefore, m some cases, as many as 500 plus grid openings were analyzed to reduce the detection limit and improve sensitivity of the test. TEM testing has been adequate for evaluating building material asbestos abatement projects, local air sam pling. and potential water contamination with asbestos.23 However, these criteria are not acceptable for assessing asbestos fiber burden analyses in human tissues and for low asbestos content products that are used intermittently in small quantities over long periods of time, such as cosmetic talcum powder 36 Talc related asbestos exposures can be heavy at times, above 4000 F/cc The inhaled asbestos fibers are extremely variable in the causation of asbestos related tumoi s and fiber burdens found m the deceased woman were within the reported ranges for amphiboles to be causative factors m the development of such a tumor '7 Therefore, it is imperative to analyze products such as talcum powder for small amounts of asbestos fibers This requires that the limits of detection be lower than levels required m a typical Yamate analysis. The author-reviewers observed that the Laboratory D analyses were done using Yamate methodology and no more than 10-25 grid openings on bulk TEM grid preparations were observed.24 Based on Laboratory D 's protocols lor testing, millions of fibers/gram of talc would have to present m order to find fibers Lower concentrations m the ranges found by Laboratones A, B. and C demon strated that fibers were detectable and present at levels sufficient to cause mesotheliomas A lthough long narrow asbestos fibers are highly carcinogenic, shorter, narrow fibers arc also dangerous.3<i 38 It is now more common to find shorter narrow fibei s in human tissue digestions than long narrow fibers, especially for chrysotile >9 This International journal of Occupational and Environmental Health ?014 000 ku 000 13 Gordon ta l Asbestos in commercial cosmetic talcum powder study provides evidence that low concentrations of asbestos in raw materials do not necessaiily correlate to low health risk 18,39 Examples of recent studies of low asbestos content producing significant airborne concentrations in simulated activity include activitybased momtoiing of asbestos as it naturally occurs in several sites, as conducted by the EPA and Agency for Toxic Substances and Disease Registry, and vermiculite-containing attic insulation studies40 These studies have repeatedly shown that substantial airborne concentrations could be derived from materials with only a fraction of a percent asbestos content,16 This has been especially true when a product was in a friable state, or where the obvious use of material intimates aerosolization of fibers. Significant airborne concentration can be easily generated from such conditions when asbestos is a constituent.411 41 The talc application studies were simulations of cxposuics to talc used by a deceased woman who had mesothelioma The air volume m the testing space was 158 cubic feet This is m the range of the chamber sizes used by talcum powder manufacturers m the 1970s in their studies of the quantity of talcum powder used m normal application The space used by Russell was 171 cubic feet and the space used by Aylott was between 152 and 163 cubic feet. The amount of material used in the shaker test w'as 0 37 g. The amount used lor the puff applicator test w'as 6.25 g.44,4:> The shaker test was a light application and the puff a heavy application However, the heavy application was within the ranges published by Russell of 8 84 + 8 32 g and Aylott of 2 5 + 12 5 g The "talcing times," or the duration of talcum powder application, were approximately 55 seconds for the shakei test and approximately 57 seconds for the puff applicator test 4445 These were within the ranges published by Russell of 83 + 33 seconds and Aylott of 28-78 seconds for adult dusting4441 Laboratories A and B determined that the contami nated talcum powder released inhalable asbestos into the air. Another issue in this study was the documentation and identification of cleavage fragments The scien tific community has not generally adopted cleavage fragment differentiation criteria46 It is unclear how to identify a cleavage fragment once the stone or material has been finely ground. Two criteria for distinguishing cleavage fiagments from asbestos fibers have been proposed. The first is that the ends of cleavage fragments have oblique angles and second is that the aspect ratios arc all less than 20:1. The ends criterion has not been validated with known asbestos/cleavage fragment standards and while an aspect ratio of 20.1 suggests that a fiber is likely to be an asbestos liber, some fibers with aspect ratios below 20. 1 are also asbestos. As the fiber aspect ratio increases, the percentage of asbestos fibers versus cleavage fragments also increases.4' However, this criteria falls short when the fiber is extremely thin and is the smallest unit of diameter of a fiber When these small fibers are removed and analyzed from human tissue, these criteria have to be discarded because enzymes with basic and acidic molecules withm cells can leach elements from the surface, causing a breakdown of the fibers, especially when thin in diameter van Orden et a/ propose criteria to identify cleavage fragments by SEM.46 The criteria are based on surface contours which identify a cleavage fragment.46 However, this method has not been verified and is not generally accepted. There were no photographs of TEM or high-resolution lughmagnification SEM provided by Laboratory D. which classified potential asbestos fibers as cleavage fragments In conclusion, we found that a specific brand of talcum powder contained identifiable asbestos fibers with the potential to be released into the air and inhaled during normal personal talcum powdei application We also found that asbestos fibers consistent with those found m the same cosmetic talc product were present in the lungs and lymph node tissues of a woman who used this brand of talc powder and developed and died from mesothelioma Disclaimer Statements Contributors All authors did studies relevant to the manuscript and all contributed and accepted all the writing Funding The work done was paid for by attorneys for litigation purposes No funds were for writing of this manuscript. Conflicts of interest Funding for all Labs was provided as part of litigation No funds were for writing this article. 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