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FILE NAME: Talc (TALC) DATE: 1975 Sept DOC#: TALC143 DOCUMENT DESCRIPTION: Published Conference Presentation Animal Experiments with Talc INHALED PARTICLES IV ' (in t w o p a r t s) Proceedings of an International Symposium organized by The British Occupational Hygiene , Society , Edinburgh 22-26 September 1975 Edited by W. H. WALTON Hon. Editor-in-Chief, Annals o f Occupational Hygiene Institute of Occupational Medicine, Roxburgh Place, Edinburgh assisted by BRENDA McGOVERN Editorial Assistant,' Annals o f Occupational Hygiene PART 2 PERGAMON PRESS OXFORD . NEW YORK . TORONTO . SYDNEY . PARIS . FRANKFURT U.K. U.S . A. CANADA AUSTRALIA FRANCE WEST G ERM ANY Pergamon Press Ltd., Headington Hill Hall, Oxford 0X3 OBW, England Pergamon Press Inc,, Maxwell House, Fairview Park, Elmsford, New York 10523, U.S.A. Pergamon of Canada Ltd., 75 The East Mall, Toronto, Ontario, Canada Pergamon Press (Aust.) Pty, Ltd., 19a Boundary Street, Rushcutters Bay, N.S.W . 2011, Australia Pergamon Press SARL, 24 rue des Ecoles, 75240 Paris, Cedex 05, France Pergamon Press GmbH, 6242 Rronberg-Taunus, Pferdstrasse 1, Frankfurt-am-Main, West Germany Copyright 1977uThc British Occupational Hygiene Society A ll Rights Reserved. No part o f this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic tape, mechanical, photocopying, recording or otherwise, without permission in writing from the Editor * First Edition 1977 Library of Congress Cataloging in Publication Data Main entry under title: Inhaled particles IV. 1. Lungs--Dust diseases-- Etiology--Congresses. 2. Dust--Physiological effect--Congresses. 3. Lungs--Dust diseases--Congresses. 4. Coal-miners--Diseases and hygiene--Congresses, I. Walton, W. H. _ II. British Occupational Hygiene Society. RC773.I55 1977 616.2'44 76-55009 ISBN 0-08-020560-7 Printed in Great Britain by Unwin Brothers Limited, The Gresham Press, Old Woking, Surrey, England CONTENTS Preface PART 1 SESSION 1. LUNG ANATOMY AND PARTICLE DEPOSITION Deposition of Inhaled Monodisperse Aerosols in Small Rodents O. G. R aabe, H.-C. Y eh, G. J. N ewton, R. F. Phalen and D. J. Velasquez Species Differences in Aerosol Deposition T. M cM ahon, J. D. Brain and S, Lemott A Theory of Predicting Respiratory Tract Deposition of Inhaled Particles in Man C. P. Y u and D. B. T aulbee Particle Deposition in Systems of Repeatedly Bifurcating Tubes W en-Ching L ee and Chiu-Sen W ang Inertial Deposition of Particles in Human Branching Airways John R. Johnston, K. D. Isles and D. C. F. Muir The Influence of Fibre Shape in Lung Deposition--Mathematical Estimates R. L. H arris and V. Timbrell SESSION 2. DEPOSITION OF INHALED PARTICLES The Human Head as a Dust Sampler 93 T. L. O gden and J. L. Birkett Deposition of Aerosol Particles in the Human Nose 107 J. H eyder and G. R udolf Influence of Respiratory Air Space Dimensions on Aerosol Deposition 127 E. D. Palmes and M. Lippmann Pulmonary Deposition of Inhaled Particles with Diameters in the Range 2.5 to 7.5 m 137 N. F oord, A. Black and M. W alsh Experimental Studies of the Deposition of Particles in the Human Lungs 151 C. N. D avies, M. J. Lever and S. J. R othenberg Lung Deposition in Freshly Excised Human Lungs 163. R. I. M itchell v Contents Deposition in the Lung and Uptake to Blood of Motor Exhaust Labelled with 203Pb A. C. Wells, J. B, Venn and M.'J. H eard SESSION 3. DEPOSITION AND CLEARANCE On the Deposition of Unipolarly Charged Particles in the Human Respiratory Tract C. M elandri , V. Prodi, G. Tarroni, M. F ormignani, T. De Zaiacomo, G. F. Bompane and G. M aestri Bronchial Deposition of Free Ions and Submicron Particles Studied in Excised Lung A. C. James Accumulation and Retention o f 137Cs-labelled Fused Aluminosilicate Particles by Beagle Dogs After Repeated Inhalation Exposures B. B. Boecker, R. G. Thomas and R. O. M cClellan Aerosol Deposition in the Dog Respiratory Tract D . L. Swift, Julia A. C. Cobb and J. C. Smith A Study of the Short-term Retention and Clearance of Inhaled Asbestos by Rats, Using U.I.C.C. Standard Reference Samples A. P. M iddleton, S. T. Beckett and J. M. G. D avis Deposition and Clearance of Inhaled Fibrous Minerals in the Rat. Studies Using Radioactive Tracer Techniques . A. M organ, J. C. Evans and A. H olmes The Distribution and Clearance of Inhaled Uranium Dioxide Particles in the Respiratory Tract of the Rat D onna J. G ore and M. C. T horne Comparative Measurements of the Short-term Lung Clearance and Translocation of PuOz and mixed N a20 + P u 0 2Aerosols in Mice J. Brightwell and R. F. Carter SESSION 4a. FACTORS AFFECTING CLEARANCE Factors Affecting Tracheobronchial Mucociliary Transport M. Lippmann, R. E. A lbert, D. B. Y eates, J. M. Berger, W. M. F oster and D . E. Bohning Effect of Sulphur Dioxide on Tracheobronchial Clearance at Rest and During Exercise R. K. W olff, M. D olovich, G, Obminski and M. T. N ewhouse The Effects of Selected Air Pollutants on Clearance of Titanic Oxide Particles from the Lungs of Rats J. Ferin and L. J. Leach Contents . SESSION 4b. BIOLOGICAL REACTIONS TO DUST (1) Investigations into the Determination of the Cytotoxicity of Quartz Dust by Physical Methods 345 W. K riegseis, R. Biederbick, J. Boese, K. R obock and A. Scharmann Short and Long-term Experimental Study of the Toxicity of Coal-mine Dust and of Some of its Constituents 361 J. C. M artin, H. D aniel and L. Le Bouffant Pathogenicity to Animals of Fine Dusts from Ruhr Mines 373 J. Bruch, W. Hilscher and U. K ramer . Long-term Test on Rhesus Monkeys for the PVNO Therapy of Anthracosilicosis 379 W. W eller Therapeutic Action of Aluminium Compounds on the Development of Experimental Lesions Produced by Pure Quartz or Mixed Dust 389 L. Le Bouffant, H. D aniel and J. C. M artin The Action of Quartz in the Presence of Iron Hydroxides in the Human Lung 403 G . Reichel, H.-D. Bauer and E. Bruckmann PART 2 SESSION 5. BIOLOGICAL REACTIONS TO DUST (2) The Effects of Inhaled Silica and Chrysotile on the Elastic Properties of Rat Lungs; Physiological, Physical and Biochemical Studies of Lung Surfactant 415 M argery M cD ermott, J. C. Wagner, T. T etley, J. H arwood and R. J. R ichards The Immunology of Asbestosis 429 E. K agan, I. Webster, J. C. Cochrane and K. M iller Topographic Distribution of Asbestos Fibres in Human Lung in Relation to Occupational and Non-occupational Exposure 435 P, Sebastien, A. F ondimare, J, Bignon, G. Monchaux, J. D esbordes and G. Bonnaud . The Biological Effect of Asbestos and Asbestos Cement Products 447 K. R obock and W. IClosterkotter The Influence of Varying Lengths of Glass and Asbestos Fibres on Tissue Response in Guinea Pigs 455 G. W. W right and M. K uschner ' SESSION 6. BIOLOGICAL REACTIONS TO DUST (3) Chrysotile Asbestos: Biological Reaction Potential 477 R. J. R ichards, P. M, H ext, R. D esai, T. Tetley, J. H unt, R. Presley and K. S. D odgson Vll Contents Predominance of Histocompatibility Antigens W18 and HL-A1 in Miners Resistant to Complicated Coalworkers' Pneumoconiosis E. R. H eise, P. C. M ajor, M. S. M entnech, E. J. Parrish, A. L. Jordon, and W. K, C. M organ A Cell Kinetic Study of the Alveolar Wall Following Dust Deposition J. Brightwell and A. G. H eppleston Immunological Studies of Experimental Coalworkers' Pneumoconiosis R. Burrell, D. K. Flaherty and J. E. Schreiber The Activation of Phospholipase A in Macrophages After the Phagocytosis of Silica and Other Cytotoxic Dusts P. G, M under and St . Lebert . Investigation of Alveolar Macrophages from Rats Exposed to Coal Dust E ula Bingham, W. Barkley, R. M urthy and C. V assallo The Effect of Increased Particles on the Endocytosis of Radiocolloids by Pulmonary Macrophages in vivo: Competitive and Toxic Effects ' J. D. Brain and G. C. C orkery SESSION 7. RADIOACTIVE PARTICLES Polonium-210 ; Lead-210 Ratios as an Index of Residence Times of Insoluble Particles from Cigarette Smoke in Bronchial Epithelium E. P. Radford and E. A. M artell 239Pu0 2Aerosol Inhalation with Emphasis on Pulmonary Connective Tissue Modifications H. M etivier, R. M asse, D. N olibe and J. Lafuma Therapeutic Effect of Pulmonary Lavage in vivo After Inhalation of Insoluble Radioactive Particles D. N olibe, H. M etivier, R. M asse and J. Lafuma Lung Response to Localized Irradiation from Plutonium Microspheres E. C. A nderson, L. M. H olland, J. R. Prine and R. G. Thomas Comparative Pulmonary Carcinogenicity of Inhaled Beta-emitting Radionuclides in Beagle Dogs F. F. H ah n, S. A. Benjamin, B. B. Boecker, C. H. H obbs, R. K. Jones, R. O. M cClellan and M. B. Snipes . SESSION 8. TALC Chemical and Physical Properties of British Talc Powders F. D. Pooley and N. R owlands Animal Experiments with Talc J. C. Wagner, G. Berry, T. J. Cooke, R. J. H ill, F. D . Pooley and J. W. Skidmore Talc-i-Recent Epidemiological Studies G . H ildick-Smith Contents SESSION 9. EPIDEMIOLOGICAL STUDIES (1) Effect of Quartz and Other Non-coal Dusts in Coalworkers' Pneumoconiosis. Part I. Epidemiological Studies W. H. Walton, L D odgson, G. G. H adden and M. Jacobsen Effect of Quartz and Other Non-coal Dusts in Coalworkers' Pneumoconiosis. Part II. Lung Autopsy Study J. M. G. D avis, J. Ottery and A. Le Roux Results of Epidemiological, Mineralogical and Cytotoxicological Studies on the Pathogenicity of Coal-mine Dusts M. T. R. R eisner and K. Robock Characteristics of Lung Dusts and Their Relation to Dust Exposure and Pathological Findings in the Lungs M. D obreva, T. Burilkov, K. Kolev and P. Lalova Chronic Obstructive Lung Disease in Gold Miners F. J. W iles and M. H. F aure . Factors Influencing Expiratory Flow Rates in Coal Miners J. L. H ankinson, R. B. Reger, R. P. F airman, N. L. Lapp and W. K. C. M organ SESSION 10. EPIDEMIOLOGICAL STUDIES (2) Smoking and Coalworkers' Simple Pneumoconiosis M. Jacobsen, J. Burns and M. D. A ttfield Possible Synergism of Exposure to Airborne Manganese and Smoking Habit in Occurrence of Respiratory Symptoms M. Sari and S. Lucic-Palai Physiological Changes in Asbestos Pleural Disease K. P. S. L umley Differences in Lung Effects Resulting from Chrysotile and Crocidolite Exposure H. Weill, C. E. R ossiter, C. Waggenspack, R. N. Jones and M. M. Ziskind Radiological Changes Over 20 Years in Relation to Chrysotile Exposure in Quebec D. Liddell, G. Eyssen, D. Thomas and C. M cD onald List of Delegates Author Index Subject Index ix 669 ' 691 703 717 727 737 759 773 781 789 799 815 825 829 ANIMAL EXPERIMENTS WITH TALC J. C. W agner,* G. Berry,* T. J. Cooke,! R. J. H ill,* F. D . P ooley$ and J. W . Skidmore* Abstract--Italian talc has been tested on rats using three routes, intra-pleural inoculation, inhalation and ingestion, Groups exposed to superfine chrysotile asbestos and untreated controls were included for comparison. In all the experiments animals were allowed to live out their lives. The intra-pleural inoculation o f talc produced no mesotheliomas in contrast to eighteen produced by the chrysotile asbestos. After ingestion, one leiomyosarcoma occurred with Italian talc and one with chrysotile asbes tos. Whether these tumours are a consequence o f the feeding is uncertain. The inhalation studies demonstrated that with equal dosage, talc can produce a similar amount of fibrosis as asbestos. However, the chrysotile exposed rats developed lung adenomas, adenomatosis and an adenocarcinoma, whereas the only lung tumour seen in animals, exposed to talc was a small adenoma, which may have been an incidental finding. IN T R O D U C T IO N In these studies we have investigated the biological properties of a single type of talc, using the same methods as we have applied to various types of asbestos. Talc was introduced into rats by three routes--intra-pleural, inhalation and ingestion. We had facilities for testing a single talc dust, and decided to use the Italian talc which is a major source of cosmetic talc used in Great Britain. The literature on the biological effects of cosmetic and consumable (used in confectionery) talc is confusing and some of the results unjustifiably alarming. The more sensational findings have naturally attracted'a lot of publicity. Therefore, we have been forced to devote a large amount of experimental resources in an attempt to put the possible hazards resulting from the inhalation of cosmetic and consumable talc into perspective. The inhalation study has not been completed and this is a preliminary communication, as far as this route is concerned. ' MATERIALS AND METHODS The main experimental material was Italian talc. The mineral sample used in this study was obtained from a shipment of talc material imported from a mine in Northern Italy where talc has been produced for over 70 years. Talc from this particular mine was chosen for two reasons. First, because it has been used in Great Britain for over 50 years and secondly, because over 40 % of the cosmetic grade talc used in Great Britain is obtained from this source. * Medical Research C ouncil Pneum oconiosis U n it, PenartH, Wales. t Department o f Paediatric Pathology, Welsh National School o f Medicine, Cardiff, Wales. t Department of Mineral Exploitation, University College, Cardiff, Wales. 647 648 * J. C. Wagner et al. This particular talc is referred to in the talc trade as Italian 00000 grade. It is imported in a ready-milled form, with an upper .particle size of 70 pm and a mean particle size of 25 pm. The talc sample was found to contain 92% talc mineral by weight together with 3% chlorite and 1% carbonate minerals; quartz was also found in the powder at approximately the 0.5-1 % level. Extensive investigation of this particular grade of talc had been performed for a 2-year period prior to the start of the animal experimentation, and samples which were over 30 years old had been examined as well as more recent imports. Virtually no change in the mineralogical composition of the material has been detected; No asbes tos minerals of either the tremolite or chrysotile varieties have been detected in the many samples of this powder examined. A mineralogical study of the talc mine itself has shown that tremolite can be found in isolated sections of the mine but this could not be traced into the final product. This is probably due to the selective mining procedures adopted in the mine where the talc is mined by hand. For comparison the sample of super-fine chrysotile asbestos (SFA chrysotile) which we have previously shown to give a high mesothelioma rate after intra-pleural inoculation (Wagner et al., 1973) was included and there were also controls exposed to neither material. The experimental animals were barrier-protected caesarian-derived rats of the Wistar strain bred at the Unit from a stock given to us by Imperial Chemical Industries, Pharmaceutical Division, Alderley Edge, Cheshire. In each group of animals there were equal numbers of males and females and allocation to the treatment groups was at random. The rats were caged in fours except when in the inhalation chambers when they were caged in sixes. The inhalation chambers were in a separate room. The rats were fed on a proprietary brand ot auto claved cubes and water ad libitum. The animal house was supplied with filtered air. Except for the scheduled killings, each rat was allowed to live until it died naturally or appeared to be distressed; full necropsy examinations were carried out. INTRA-PLEURAL INOCULATION EXPERIMENT The dose was 20 mg per rat made up as a suspension in physiological saline, with a concentration of 50 mg/ml. Injection was into the right pleural cavity using the method described by W agner and Berry (1969). There were 48 rats injected with the talc, 48 with SFA chrysotile and 48 controls injected with saline. Injection was in January 1973'when the rats were between 8 and 14 weeks old, and the last animal died in September 1975. ... The results are given in Table 1 which includes the mean survivals after injection and' the numbers with a mesothelioma. As expected, mesotheliomas occurred in a proportion of the rats injected with SFA chrysotile. In fact, fewer occurred than was expected since in our previous experiments (Wagner and Berry, 1969; W agner et aL, 1973) this material produced mesotheliomas in 65 % of the animals. However, in these previous experiments there was longer survival and after allowing for this the present mesothelioma rate was similar to that in our first experiment. The shorter survival in Animal Experiments with Talc 649 T able 1. I ntrapleural I noculation E xperiment Material injected No. injected Mean survival (days) Number with mesothelioma Italian talc 48 655 0 SFA chrysotile 48 598 18 Saline controls 48 691 0 the present experiment was probably due to the rats having lost their SPF status. Also, as expected, the mean survival of the SFA-injected rats was reduced in comparison with th controls. No mesotheliomas were observed in the talc-injected animals. Howevei, injection site granulomas were common and a small pulmonary adenoma was found in one rat which died 25 months after injection. There was no other relevant pathology of the lungs in these animals. The mean survival was about a month less than the controls, but this difference could have been due to chance (P > 0.25). INHALATION EXPERIMENT Rats were exposed in 1.4 m3 chambers which could hold up to 48 rats, caged in sixes. The dust clouds were generated for h a day and 5 days per week. The respi rable dust concentrations were measured daily using a Casella Type 113A size-selective gravimetric dust sampler, and variations were allowed for by adjusting the concentra tions on the following days, so that the required dosage, calculated as the product of concentration and time, was achieved uniformly in a specific time. The SFA cloud was generated using the generator designed for the U.I.C.C. standard reference samples of asbestos (T imbrell et al., 1968) and in the cabinets about 80% by weight of the cloud was respirable. The talc cloud was generated using a Wright dust feed mechanism and about 40% was respirable. Exposure started in February 1973, with 48 rats exposed to talc and 48 to SFA chrysotile. After 6 months' exposure half of the rats were removed and transferred to ordinary cages, and were replaced by another 24 per dust. These rats were in turn removed and replaced after 3 months' exposure, and all exposure ceased after another 3 months. Thus there were 96 rats exposed to each dust, 48 for 3 months, 24 for 6 months, and 24 for 12 months. There were also the same numbers of controls which were kept in ordinary cages in racks. At the start of each exposure period the majority of the rats were between 6 and 8 weeks old. The mean respirable dust concentration was 10.8 mg/m3 for each dust and the cumulative doses, i.e. the products of concentration and time, were approximately 4100, 8200 and 16 400 mg/m3 h for the 3-month, 6-month and 12-month exposures. Ten days after the end of each exposure period some rats were sacrificed and there were also sacri fices 1 year later. For the sacrificed rats an assessment was made of the severity of fibrosis in the lungs. Sections of both lungs were examined in random order without knowledge of the dust or length of exposure. The sections were observed on a viewing screen of a Projectina microscope 4013 BK at a magnification of. X85. The fibrosis in each lung 650- J. C. Wagner et al. was assessed on a seven-point scale: 1: nil, 2: minimal, 4: slight, 6: moderate and 8: severe. Illustrative examples can be seen in W agner et al. (1974). The mean fibrosis scores of the rats sacrificed at the end of exposure and one year later are shown in Table 2. The main features are that both Italian talc and SFA chrysotile produced fibrosis to a similar extent, and that there was some evidence of progression after exposure had discontinued in the longer exposed animals. T able 2. I nhalation E xperiment-- M ean F ibrosis at end of Exposure and 1 Y ear L ater (number o f rats) Material Time Length of exposure 3 months 6 months 12 months Italian talc SFA chrysotile Controls End of exposure 1 year later End of exposure 1 year later End of exposure 1 year later 2.2 (8) 2.4 (8) 2.8 (8) 2.2 (8) 1.8 (8) 1.6 (8) 2.7 (6) 3.4 (4) 3.0 (6) 3.2 (4) t.9 (6) 1.5 (3) 3.4 (6) 4.6 (4) 3.2 (6) 4.2 (4) 1.3 (6) 1.9 (3) Most of the animals in the 6- and 12-month exposure groups had died by 6th June 1975, but over half of those in the 3-month groups were still living. Therefore, the 3-month groups will not be considered further in this paper. The numbers of rats with lung tumours are shown in Table 3. None occurred in the control rats, there was one adenoma in the rats exposed to talc and there were seven Material Italian talc SFA chrysotile Controls T able 3. I nhalation E xperiment--L ung T umours Exposure Number exposed Sacri ficed Died Number of lung tumours Adenomas Adeno Adeno matosis carcinoma 6 months 24 10 12 0 0 0 12 months 24 10 12 1 0 0 6 months 24 10 8 0 12 months 24 10 11 3 1 0 2 1 48 18 27 0 0 0 lung tumours, including one adenocarcinoma ini the SFA groups. In addition, one rat in the SFA 1-year group had a widespread lymphosarcoma. However, as we showed previously (W agner et al., 1974) tumours of this type (lymphomas and leukaemias) are an occasional finding in our rats independent of treatment. Animal Experiments with Talc 651 INGESTION EXPERIMENT Rats were feci the test materials with a dose of 100 mg per day per rat, The food mixture was prepared in batches sufficient for 5 days. The basic mixture consisted of equal amounts by weight of coarsely powdered Spillers small-animal diet and Horlicks malted milk. This mixture was chosen because the rats liked it and could be easily trained to eat it quickly and completely. 244 g of the basic mixtures was added to 16 g of the test material in a 20 X 30 cm polythene bag. The bag was sealed and the con tents mixed by rubbing between the hands. The contents were then weighed into 5 equal parts and sealed in 5 x 13 cm polythene bags. On the day before a bag was required,5 cm3 of deionized water were injected with a hypodermic syringe and the contents kneaded into a uniform stiff"dough. This was rolled in the palms, still sealed in the bag, and then shaken out on a polythene rolling sheet. The cylinder of dough was the.n rolled out to the length of a dose-cutter on which it was then laid and divided into 32 portions by drawing a scalpel through the slots in the cutter. The doses were stored in the polythene bag until the next day. This improved the consistency, making them drier and firmer. The doses were administered by dividing the rectangular cages into four compartments. The mesh floor was covered with aluminium and the rats introduced. A daily record was kept for each rat of how much of the dose was con sumed. In fact after the first 2 weeks, it was rare for the whole pellet not to be con sumed and overall over 98% of the planned dose was consumed, the minimum consumption of one rat being 85 %. There were 32 rats fed talc, 32 SFA chrysotile and 16 controls fed with the basic mixture only. Feeding started in February 1973, when the rats were between 21 and 26 weeks old, and was carried out on 101 days in the next 5 months. Except when the doses Were being administered the rats had access to the normal diet. At post-mortem the abdominal organs were examined and the entire alimentary canal removed and fixed en bloc in 10% neutral formalin, together with the liver, spleen, kidneys, heart, lungs and any suspected pathological lesion. Tissue for histo logical examination was taken from the liver, spleen, stomach, ileum, caecum, rectum, omentum, lesser omentum, mesentery and parietal peritoneum and any other site of pathology. Tissues were processed on a tissue processor, embedded in paraffin wax, sectioned at 5 xm bulk stained in haematoxylin and eosin and mounted in DPX. Care was taken during the post-mortem and while handling wet tissues not to introduce possible contamination particularly from glove powder. No steps were taken to eliminate possible contamination via chemical reagents such as the fixatives. Further tissue blocks will be taken for electron microscopical and electron probe identification of minerals. Two animals from each treatment were sacrificed 3 months after the feeding had finished, and the last animal died in September 1975. The mean survivals from the start of feeding were 614 days for talc, 619 for SFA chrysotile and 641 days for the controls, ignoring the sacrificed animals. Abnormalities of the gut were found in only two rats. A rat fed talc had a leiomyo sarcoma of the stomach. A tumour of this type may have occurred in a rat fed SFA chrysotile although the diagnosis is not' certain (possibly it is a reticulum celled sarcoma). Other findings were an adrenal adenoma in a control rat, sarcomas of the 652 J. C. W agner et al. uterus in two rats fed talc, and in one fed SFA and a lymphosarcoma in a rat fed SFA. These latter findings are not considered as consequences of the feeding because of their location; also we have previously observed three sarcomas of the uterus out of 126 control rats (W ag ner et al, 1974). The two leiomyosarcomas of the stomach could possibly be a consequence of the feeding although malignant tumours of the digestive organs and peritoneum do occur in our rats in the absence of treatment, and we found three in the group of controls referred to above, although none was a leiomyosarcoma. REFERENCES T imbrell, V., H yett, A. W. and Skidmore, J. W . (1968) Ann. occnp. Hyg. 11, 273-281. W agner, J. C. and Berry, G. (1969) Br. J. Cancer 23, 567-581 * Wagner, J. C., Berry, G and T imbrell, V. (1973) Br. J. Cancer 28, 173-185. Wagner, J. C., Berry, G., Skidmore, J. W. and Timbrell, V. (1974) Br. J, Cancer 29, 252-269. Animal Experiments with Talc 653 DISCUSSION S. J. R othenbiTRg : Were the particle size distributions similar for the samples of talc and chrysotile? J. C. Martin: In the inhalation experiments, how much dust was retained in the lungs at the end of exposure? D o you think that retention and clearance are the same for each dust tested; if not, can one really say that the fibrogenic potential is the same? Mr Skidmore: Whilst continuous sampling was used to determine respirable mass concentrations only occasional samples were obtained for microscopical examination. These indicated that the respirable fraction of the talc cloud contained approximately 1300 particles/ml larger than 1 /mi. Between J and 2% o f these were fibrous with lengths up to about 20 /mi and diameters between 1 and 2 //m. The SFA cloud contained approximately 500 fibres/ml longer than 5 /<m plus a substan tial number o f shorter fibres and non-fibrous particles of similar chemical composition. Retentioh and clearance were certainly not the same for the two dusts. For talc, in those rats sacrificed at the end o f exposure, there were 2.5, 4.7 and 12.2 mg talc per rat in the lungs after 3, 6 and 12 months' exposure. With the chrysotilejnuch smaller amounts were found and even after 12 months, the mean was only 0.8 mg. One reason for using the inhalation route is to allow these differences to play their part; that is, the actual rather than the potential effect is determined. E. K. Cund y: You say that your mineral is free of fibrous particles of the tremolite type. Was any electron microscopy carried out on this sample to check the absence or otherwise of fibrous particles? W. Smither: You said that you found no tremolite or asbestos in this talc. Have you found any fibres at all? This conference has learned that fibre morphology is the important factor. Would you care to characterize the fibres you found in talc? Would you. say that they conform to the classical description of asbestos--that is a hydrated fibrous silicate? If so, must we change the classical description o f asbestos or must we accept that there are asbestos fibres in cosmetic talc? D r Pooley: The examinations performed on the talc included X-ray diffraction analysis, differential thermal and thermal gravimetric analysis on the bulk material while the transmission electron micro scope fitted with an energy-dispersive X-ray analyser was used to study single fibres in the samples in an effort to detect asbestos. The many fibrous particles analysed in the samples, i.e. those 'particles with a > 3 : I axial ratio, were found to be laths of talc or chlorite mineral, Dr Smither poses a very interesting question concerning the definition of asbestos. If we look very closely at industrial silicate materials imported into Great Britain for use by the various manufactur ing industries, we find that these materials contain numerous fibrous particles. One example is the mineral sepiolite which is a magnesium silicate which forms very fine fibre very similar to chrysotile but yet is not called asbestos. If you ask me whether or not the fibres found in talc look like commercial asbestos then I would say "n o". The diameters of the fibres found in the talc samples were generally in excess o f 1 /tm whereas the majority of commercial asbestos minerals have fibre diameters which are below 1 /tm. . W. Smither : Were there any ferruginous bodies in the pathological sections ? D r W agner: No. . . J. R . L y n ch: Since most of what we know about the biological effect of asbestos indicates that the fibre shape may be the most important factor, it seems that we should regard all respirable mineral fibres, regardless o f how they may be described industrially or mineralogically, as presenting a poten tial hazard similar to asbestos in the absence o f evidence to the contrary. Mr Skidmore: The talc sample we tested was mainly non-fibrous, i.e, less than 2% of the particles were fibres. A lso these fibres were coarser than typical asbestos fibres. Our experiments have surely provided evidence that this talc has an actual and potential carcinogenic hazard at least an order of magnitude less than asbestos, but provide no evidence on the possible effects of non-asbestos fibrous material. . . D. K. Craig: A t Battelle, we have exposed groups of 100 hamsters to talcum powder in various exposure regimens, the maximum cumulative exposure being 6000 mg b/m3, delivered over a year at a respirable concentration (as measured by the MRE horizontal elutriator) of 8 mg/m3. This talcum powder came from Vermont. We observed no significant difference in the lung pathology of the control and the exposed animals, we saw no significant fibrosis in animals, we saw no ferruginous bodies in their lungs and we saw no fibres in any of the samples we took of the talcum powder aerosols. G. W. Wright : Since fibrous as well as non-fibrous respirable particles are reported in the air to which these animals were exposed, one cannot be certain which, or perhaps both, may be the fibro genic agent. Which do the authors believe is the effective agent? A similar situation has recently come to my attention in humans exposed to flux-calcined diato- maceous earth. The chest roentgenogram of persons thus exposed often shows a diffuse non-nodular pattern similar to asbestosis. 654 J. C. Wagner et al. The dust examined by electron microscopy shows the presence of long thin fibres said to be quartz. This observation raises the possibility that it is these fibres that may account for the non-nodular component injthese roentgenograms.* R. J. R ichards: Mineral particles do adsorb organic materials on to their surface; perhaps this effect should receive some consideration in relation to the biological potential of the "mineral" . A. Morgan: Why did you select the SFA chrysotile for comparison ? Electron micrographs o f this material show that it is inhomogeneous and contains both very fine fibres and "chunks" of material which may consist of chrysotile compacted during milling. Tests such as protein adsorption and haemolysis show that it does not behave as a typical chrysotile. Would you hazard a guess as to whether it is the fibrous phase or the "chunks" which is responsible for the reported biological effects ? Dr Wagner: The SFA chrysotile has produced a higher mesothelioma rate after intrapleural injection than any of the asbestos samples we have used, and as we were looking for gastrointestinal tumours' after feeding for the first time we decided to include it for this reason. Actually in the injection and inhalation experiments other chrysotile samples, including one of the U.I.C.C. samples, were also used, but they are not reported in this paper which is primarily concerned with talc. J. C. M cD onald: Have you any information on the frequency of mesothelial tumours in animals' exposed to talc containing tremolite? G. W. Gibbs: Did you see pleural calcification in your animals? Dr Wagner: We have no experience o f talc or tremolite in our previous experiments but we have an injection experiment in progress involving tremolite. I saw no pleural calcification in the talc experiments, in contrast to one of our previous experiments with samples of Canadian chrysotile. M. Kxjschner: Was the pattern o f fibrosis in the lung produced by talc similar to that produced by asbestos ? Secondly, did the amount of fibrosis in the pleura on intra-pleural instillation differ with each of these materials ? Dr Wagner: In the lung the patterns were very similar but previously I have observed a slight difference between chrysotile and crocidolite. The pattern with chrysotile is similar to that described by Professor Heppleston with a focal alveolar lipoproteinosis around the respiratory bronchioles. The talc produced the same type o f reaction. As far as the pleura is concerned the fibrosis was much more marked with the talc than the chrysotile. V. Timbrell: Fibrous and flaky materials such as talc have in common the ability to produce large particles o f small aerodynamic size, the aerodynamic size o f a fibre being related to the diameter and that of a flake to its thickness. Thus thin flakes of talc can have diameters 25 pm or greater and yet be respirable. Like long fibres, such large flakes may not be cleared by macrophages, etc., and may thus remain in contact with the same cells indefinitely and produce fibrosis. It is necessary to differentiate between fibrogenicity and carcinogenicity which may not be directly related. Stanton and Wrench (/. natn. Cancer Inst., 1972, 48, 797) and ourselves have shown that the carcinogenic potential o f fibrous materials is related to the particle shape and not that fibrogenicity is dependent on the particles being fibrous. It is thus understandable how inhaled talc particles may be fibrogenic without being carcinogenic.