Document KzG0n0n95o3gzdp6Zo4X8aVrw

REFERENCES Vc />' 1. Taylor, J, 5. 1979. Environmental Chloracne: Update and Overview. Ann NY AcadSci 320: 295-307. 2. ,, Morse, O. L.; Baker, Jr., E. L., Kimbrough, R. D.;and Wlueman, til, C. L. 1979. Propanil-Chloracne and mtthomyl toxicity in workers of a pesticide manufacturing plant. Clin Toxicol IS: 13-21. 3. Sundstom, G.; Jansson, B.; and Renberg, L. 1 978. Determina tion of the toxic impurities 3,3 ,4,4 -tetrachloroaaoxybenaene in commercial diuron, linuron and 3,4-dichloroanillne samples. Chtmosphtrt 12: 973-79; Bunce. N. J.; Corke, C. T.; Merrick, R. L.S and Bright, J. H. 1979. 3,3 ',4,4-tttrachlo/oazobenzenc as a contaminant in commercial propanil. Chtmosphtrt 5: 283-84. 4. Bartha, R.: Unke, H. A. B.; and Primer, D. 1968. Pesticide transformations: production of chloroazobenzeqes from chloroanilines.Sc/fnce 161: 582-83. 5. Poland, A.; Glover, E.; Kende, A. S.i(DeCamp, M.;and Glandomenico, G. M. 1976. 3,4,3 ',4 -teuachloroazoxybenzene and azobenzene: potent Inducers of aryl hydrocarbon hydroxylase. Science 194: 627-30. 6. Hsla, M. T. & Sainton, F. V. Z.; Shih, L. C. T.; Pounds, J. C.; and Alien, J. R. 1977. 3,4,3,4-tetrachloroazobenaene: A potential environmental toxicant. Rts Commun Chtm Pathol Pharmacol 17: 225-36. 7. Hsla, MVT. S., and Burant, C. F. 1979. Preparation and spectral analysis of 3,3 *,4,4-tetrachloroazobeniene wd the correspond ing azaxy and hydrazo analogs. / Assoc Off Ana! Chtm 62: 746-50. 8. Taylor, J. S.; Wuthrlch, R. C.; Lloyd, K. M.; and Poland, A. 1977. Chloracne from manufacture of a new herbicide. Arch Dtrmatol 113: 616-19. 9. Kimbrough, R. D.; Carter, C. D.; Liddle, ). A.; Cline, R. E.; and Phillips, p. E. 1977. Epidemiology and pathology of a tetrachlorodlbenzodloxin poisoning episode. Arch Environ Health 77-86. * 10. Adams, E. M.; Irish, D. D.; Spencer, H. C.; and Rowe, V. K. 1941. The response of rabbit skin to compounds reported to have caused acneform dermatitis, tnd Med 10: {Ind Hvg Sec) 2: 1-4. 11. Schwetz, B. A.; Norris, J. M,; Sparschu, G. L.; Rowe, V. K.; Yehrlng, P. J.; Emerson, J. 1.; and Gerblg, C 1973. Toxi cology of chlorinated dibenzo-p-dioxins, Environ Health Perspect 5: 87-99. URL 05263 Effects in the Rat of Inhaling PVC Dust at the Nuisance Dust Level (10 mg/m ) R.j. RICHARDS, Ph D. F. A. ROSE, Ph.D. T. D. TETLEY, Ph.D. Department of Biochemistry University College Cardiff, Wales United Kingdom L. M. COBB, Ph.D. MRC Radiobiology Unit Harwell, Oxon United Kingdom C. \. HARDY Department of Inhalation Toxicology Huntingdon Research Centre Cambs., United Kingdom ABSTRACT. Rats inhaled a paste polymer polyvinyl chloride dust at an aerosol concentration of 10 mg/m3 for 6 hr/day, 5 days/wk for a 15-wk period. A small num ber of randomly scattered lung lesions were detected at IS wk; these lesions were also present 15 wk after exposure to polyvinyl chloride had ceased. Few if any biochemical changes were detected at the alveolar surface in lung tissues or other organs of polyvinyl chloride-exposed rats. It is therefore concluded that at "nuisance" dust level this form of polyvinyl chloride polymer exhibits a weak biological reactivity. DURING ONE STAGE in polyvinyl chloride (PVC) manufacture the material exists as a fine, easily respirable dust. Consequently, investigators have examined the effects of this dust on mammalian lung. 14 Archives of Environmental Health URL 05264 The few experimental studies that have been reported present conflicting data on PVC toxicity. This may be due to the different formulations of PVC examined and/or differences of experimental designs, in the only previ ously reported inhalation study,' the authors suggest that continuous (24 hr) exposure of guinea pigs for 2-7 months in a PVC bagging plant resulted in the formation of granulomatous foci in the lung. The nature of the PVC used in that study-possibly a mixture of homopolymer and paste polymer-is not described, and the level of exposure is poorly defined. Using a simple in vitro technique of hemolysis it has been shown that different formulations of PVC exhibit differential biological potential, as assessed by their ability to react with red blood cell (RBC) membranes.3,3 Thus, homopolymers are not hemolytic, whereas paste polymers containing surfactants (detergents) on their particle sur face produce extensive RBC damage in vitro. The chemi cal formulation of the detergent used in the preparation of the PVC polymer was,' therefore, considered important in its hemolytic reaction.3 Similar findings were reported by other investigators4,3 who found that certain emulsion (paste) polymers were cytotoxic to rat peritoneal macro phages maintained in vitro, which is a test system consid ered by some investigators to be indicative of particle fibrogenicity in vivo. These investigators,5 however, subse quently concluded that some paste polymers containing detergents produce a "false-positive" result in the in vitro system because the intratracheal 2-mg injection of the identical polymers into rat lungs produces no progressive fibrogenic response (unlike a quartz). All the polymers tested in experimental animals produce only a mild inflam matory response, and it was therefore concluded that PVC per se is an inert .material.5 Intratracheal instillations of high doses (25 mg) of PVC * have been sh<?wn to induce biochemical and histopatho' logical changes in rat lung tissue;6 other studies9 have indi cated that the cellular and biochemical effects at the ^alveolar surface and in lung tissue of instilled PVC particles are dose-dependent However, aggregates of PVC dust given intratracheally, like many other materials given in this way, can produce a different, usually more vigorous, response from that produced by inhaled dust Additionally, It is not possible to convert a single intratracheal instilla tion of a known volume of dust to a period of exposure at \ a multiple or fraction of the dust level, which means that . for assessing safe occupational exposure levels, the results from intratracheal instillation studies are open to critical interpretation. The aim of the present study was to determine whether . the inhalation of a paste polymer, PVC-7, containing the detergent sodium dodecyl sulphate,3'7 at "nuisance" dust level (10 mg/m3) would produce any alteration in the iungs of experimental animals. The parameters chosen for Investigation included a study of the biochemical integrity v of the alveolar surface by estimation of free cell number and enzyme activities, and determination of pulmonary : surfactant levels and soluble protein. In addition to histo. pathological examination of the lung, DN A, and protein */ 'synthesis'and hydrolytic enzyme activity of alveolar tisV sue were monitored. MATERIALS AND METHODS Animals. Eighty 10-wk-oid female albino rats of the Sprague Dawiey C.D. strain (Charles River, U.K. Ltd., Margate, Kent) were used in this study. These were ran domly allocated to either the control or PVC-exposed group so that each group consisted of 40 rats. The rats were housed in groups of four, in polypropylene cages with stainless steel mesh floors. Pood and water were sup plied ad libitum while the rats were in the cages. The room temperature was maintained at 20 2C, and light ing was controlled to provide a 12-hr light period each day. Aerosol exposure. A paste polymer, PVC-7, which con tains sodium lauryl (dodccyli sulphate surfactant was used throughout the study. The PVC-treatcd animals were exposed to a mean aerosol concentration of 10.mg/m3, i.e., "nuisance" dust level (Guidance Note EH 15/777* Health and Safety Executive), on 5 days for 6 hr/day for 5 days/wk up to 15 wk. Groups of 10 exposed rats were examined with control animals at 3, 9, and 15 wk after the experiment was initiated. One group of animals was maintained without further contact with PVC for 15 wk after being previously exposed to the dust for 15 wk. A nonexposed control group was also maintained during this time period. Exposures were carried out in a perspex, cubic, 240-L chamber which was operated under dynamic conditions at a slight positive pressure. The aerosol of PVC panicles was generated using a Wright dust feed mechanism (L. Adams Ltd.; Minerva Road, Chase Estate, London NW10). Following preliminary experiments to determine optimum operating conditions, a dust packing force of 0.2 tons was used. The mechanism was operated with standard canister liner at a gear ratio of 4 : 1 reduced. Total chamber air was supplied through the dust feed which was run at 1.05 kg/cm2 (25 L/min). The aerosol was introduced into the exposure chamber at the base center and then ascended through an area clear of holding cages to descend through the cages in which the animals were individually housed, and left the chamber through a series of holes at the base. The chamber was held in a fume cupboard which was at negative pressure with respect to the laboratory, and the exhausted dust was removed by an absolute filtration unit Control animals were placed in an identical chamber, but were exposed only to the same compressed air as that used to operate the dust feed mechanism. Aerosol concentration was determined twice daily by gravimetric analysis of samples collected with an openface fitter holder operated in the vertical position. Due to the extremely low aerosol concentration, a sampling rate of 10 L/min was required to ensure accurate assess ment of total chamber concentration. The particle size distribution in the chamber was measured using an Ander son mini-sampler. Treatment of animals prior to biochemical analyses. In the biochemical studies six exposed and six control rats were examined at each chosen lime interval and treated identically. Each animal was given an intravenous tail vein injection of 75 pQ of [methy!-3H)-thymidinej52Ci/mmol) and 2.5 siCi of c-fu-l4Cl*proline (282.5 mC/mmoJ) and left > Jamiary/Ftbruary 1981 (VoJ. 36, No. 1) * *% m Table 1.-*Cellular and Biochemical Studies on Rats Expoaed to 10 rngta* PVC Dust for Different Time Periods Exposure Period/ Animal Group Lung Weight/ Body Weight Ratio x 102 Number Free Cells/ Animal x 10~ Puimonaryf Surfactant mg/Animal Puimonaryf Lavage Protein mg/Animal Acid RNAase unlts/ioe Cells Acid RNAase unlts/g Lung V* f 3 Weeks Control PVC-Ex posed 0.53 (.086) 0.55 (.082) 6.71 (0.93) 8.86 ( 1.52) 0.50 0.52 2.25 2.62 0.68 (0.12) 0.69 (0.29) 74 (10) 75 (19) 3 Weeks * Control .. .j* PVC-Exposed ; IS Weeks Control PVC-Ex posed -0,47 (.0J6j -0.48 (.037) 19.35 (4.64) 13.79 (4.63) 0.60 1.S0* 0.43 (.023) 0.40 (.045) 11.00 ( 3.61) 11.00(1 1.08) 0.66 0.62 3.19 3.96 2.86 3.11 t.17 (0.27) 1.39 (0.31) 106 (12) 104 (12) 0.90 (0.31) 1.10 ( 0.21) 83 ( 5) 104 (16)* 15+ Wk 15 Wk Clearance \ *T Control PVC-Exposed 0.39 (.02B) 0.41 (.063) 9.26 ( 2.93) 11.59 (1.97) 1.28 1.20 3.S0 4.13 0.96 (1 0.22) 0.72 (0.23) 45( 8) SO ( 9) Mr. v-2% NOTE- Number* within parentheses refer to standard deviation, Significantly different from control. Values represent mean of pooled samples from six rats. DNA mg/g Lung 4.54 ( 0.76) 4.06 (0.43) 4.93 ( 1.071 S.30 ( 0.871 4.01 (0.87) 4.78 ( 0 32) 4.23 (0.25) 4.27(0.29) 1 | mH; for exactly 1 hr. They were then sacrificed by intraperitoneaJ pentobarbitone injection, a blood sample was then removed from the hepatic portal vein, and the lungs per* fused via the heart with 0.1 5 M NaCI to remove blood from the vascular bed. The lungs were removed from the pleural cavity and lavagedsix times with 0.1 S.M NaCI, after which they were dried between paper towels and weighed. The lavaged lung tissue and pooled washes were kept on ice prior to further treatment (see below). Other tissues taken for examination were the liver, spleen, kid* ney and sections of the gastrointestinal tract which were stored frozen until required. Fractionation of lavage fluid and biochemical analysis. The free ceil population from each animal lavage was obtained by centrifugation at 300 ? for 20 min at 4C The supernatant fraction from six rats in each group was pooled and centrifuged at 1000 g for 1 hr at 4*C. The supernatant fraction from this centrifugation contained the majority of soluble alveolar lavage protein.1 The pellet was resuspended in AM NaCI, mixed well, and centrifuged at 1 SOQg for 25 min at 4C, and the resulting separation gave a pellicle of lipoprotein-rich material, designated pul* monary surfactant, which floated to the top of the tube. This was collected, dialyzed against distilled water, freeze* dried, and weighed.* The free cell population was counted and the levels of acid RNAase and acid protease were. 16 determined as described previously.* The incorporation of [3Hj-thymdine into tissue DNAand [l4C] -proline into tissue protein was determined as follows. Body tissues were homogenized in 0.15 ,W NaCJ and samples suspended in a final concentration of 0.2 M perchloric acid (PCA) for 1 hr. The mixture was centrifuged at 1000? for 20 min. and the supernatant, containing free label, discarded. This procedure was repeated once more and the resulting pellet was then resuspended in 0.5 M PCA at 70*C for 20 min. to solubilize the DNA. The supernatant derived from centrifuging this mixture at 1000? for 20 min. was stored and the process repeated. The supernatant fractions were pooled and samples taken for chemical analysis of DNA and direct counting of (3H] label. The remaining pellet was suspended in distilled water and samples digested in 1 M NaOH to assay for protein content or taken up in Sotuene for determination of {**C} radiolabel. The efficiency of counting was determined by using internal standards and results were expressed as disintegra tion per minute of incorporated radiolabel per mg DNA or protein for each tissue examined Hlstopathology. The trachea and lungs from four con trol and four PVC-exposed rats were removed after the rats had been lulled by exsanguination under deep pento barbitone anaesthesia. The lungs were infused to constant pressure (10 cm water) with buffered 10% formalin, and C rX--J nVooI o> VI Archives of Environments! Health iffc . . Y.V vi-.- 3!Hl; Thymidine -'in Lung DNA dpm x 10"*/mg DNA Protein mg/g Lung " 7?5 ( 4.5) 10.4 (1 3.6) 40,0 (148.0) 25.6(414.2) 14.3 ( 6.9) 10.3 (i 2.3) 50.6(1 8.52) 52.0 (4 7.46) 77.3 (19.72) 87.7(410.68) 70.9 (10.50) 77.5 (4 4.14) 1*10Proline in Lung Protein dpm/mg Protein 164 (4 $5) 165 (47) 160 (23) 120 (23)* 156 (420) 165 (21) 7.7 ( 2.4) 7.5(4 1.9] * 80.5 ( 6.84) 91.2 (411.58) 94 (133) 74(1 6) Fl|. 1. Lung from rat expoted to PVC dust for 3 wk, Transmission electron microscopy of alveolar macrophage containing numerous smooth-surfaced particles (arrows) identical to the inhaled PVC dust (scale unit 2 ten). ` transverse sections prepared from embedded material. t Sections were stained with hematoxylin/eosin, Masson's r tfichrome stain for collagen,10 silver stain for reticular fibers,11 %and with modified Sudan IV to identify PVC particles.*3 Some material m is also processed for electron microscopy. RESULTS Body .i,. win . There was no effect on body v. any clinical signs that c' during the study. Ch: ,,mber aerosol was maintainrd ai. a.*, 3"crag>.- . non of 10.6 mg/m3 [standard deviation ($>; - 1- 146]. The highest and lowest cnn;cn;ratsnn rcco-H * .vas 15 mg/m3 and 7 mg/m3, respectively. The mass mod1:' !?rodynamic diameter of the aerosol was 1 7 pm (og 4/in). Biochemical studies. Very few significant changes in lung weight/body weight ratio, pulmonary surfactant and alveolar surface protein, frea cell number and enzyme activities or lung enzyme ar i . ics, protein and DNA 'syn thesis' were detected in animals exposed to PVC dust throughout this study (Table l). In addition, DNA and protein `synthesis' in the liver, spleen, and kidney, together with changes in acid protease levels in the free cells and lavagrd lung tissue, were not detected in PVC-exposed rats (data not shown). Three sipiificant changes in rats exposed to PVC were found: (1) elevated pulmonary sur factant, (2) depressed lung protein `synthesis' at 9 wk, (3) elevated lung acid RNAase activity at 15 wk. Histopathology. At 3 wk the lungs of both.conirol and PVC-exposed rats appeared normal, except that in the exposed group the alveolar macrophages were enlarged and contained numerous, 1-4 pm diameter, smooth surfaced particles similar in appearance to PVC dust {Fig. 1). At 9 wk there was evidence of a mild respiratory tract infection in both control and PVC-exposed groups characterized by increased numbers of mononuclear cells in the perivascular spaces, and in most animals, an increase in the volume and extent of bronchus-associated lymphatic tissue. The foamy macrophages seen at 3 wk in PVCexposed animals were less evident, possibly because of an increased turnover of cells in the 9-wk group. At 15 wk there was no sign of the respiratory tract infection seen in the rats at 9 wk. In the PVC-exposed animals there were aggregates of foamy macrophages occupying groups of up to 9 alveoli; also associated with these aggregates was hypercellularity due to an increase in mononuclear cells and fibroblasts, in the interstitium of the alveolar wall (Fig. 2). This interstitial reaction was also associated with minimal increase in collagen and reticular Fibers. The smooth- January/February 1981 (Vol. 36, No. 1) 17 99ZS0 "WO Is3$ .? .3 *- r- URL 05267 Fig. 2. Lung from rat exposed to PVC dust for IS wk. Not* aggregation of foamy macrophages and hyprclluiirity of ad|ac*nt alveolar wall {arrows). Hematoxylin and cosin stain (X 250), surfaced particles that filled foamy macrophages stained red with modified Sudan IV, indicating that they were PVC dust.1* Fifteen weeks after the final exposure (30 wk from commencement of the experiment), the lungs of control animals appeared normal, while those previ ously exposed to PVC showed no significant change in the lesions detected at 15 wk (termination of exposure). DISCUSSION The results show that a paste polymer preparation of v;PVC dust inhaled by rats at "nuisance" dust level (10 mg/m*) M^ for 6 hr/day, 5 days/wk, produces small, randomly scat* ' ' tered, lung lesions after 15 wk of exposure. These lesions - are characterized by hypercelfularity of the interstitium of J the alveolar walls in areas adjacent to macrophage aggre gates containing PVC In addition, the lesions persist 15 wk after the cessation of animal exposure to the particulate. The PVC-induced lesions, however, show only minimal increase in collagen and reticular fiber formation, with no evidence of extensive fibrotlc reaction. Similar conclusions are reported by other investiptors' who studied the effects of intratracheal instillations of different PVC formulations. The results of the current histopathological study there fore suggest that at "nuisance" dust level PVC has a rela tively weak biological reactivity, and this conclusion receives further support from the biochemical investiga tion. While PVC-exposed animals have elevated levels of pulmonary surfactant and show a decrease in lung protein 'synthesis' 9 wk after exposure, the presence of a mild respiratory tract infection in all the animals during this time period prevents the establish men* of any definite con clusions. Thus relatively few, if any, biochemical changes in PVC-exposed rats are detected at any exposure periodeither at the alveolar surface, the lung tissue, or other body 18 Archives of Environmental Health organs. Such results are in contrast to previous inhalation studies where rats exposed to 12 mg/m3 chrysotile asbestos - for 5 days/wk for 15 wk have elevated free cell numbers (2-3 X control animals) free cell and lung enzyme activity {3-10 and 1-8 X control, respectively), and pulmonary sur V .factant (12X control).* Elevations in pulmonary surfactant ,^' and free cell numbers are also detected in rats inhaling ~amosite asbestos and fiber glass (12 mg/m3,5 days/wk, for ' ;,:;8wk).13 With dusts of "high" biological reactivity these :' 2 biochemical changes at the lung surface persist with the V:. progressive pathological development of lung disease. In summary, the present short-term study indicates that at "nuisance" dust level one form of paste polymer PVC has a weak biological reactivity, only detectable by histo* pathological examination, which reveals a small number of lung lesions in experimental animals. It is not possible at this time to interpret this finding in terms of the lesion likely to arise in man under similar conditions of exposure. *. Drs. Richards and Tetley would like to thank the Medical Research Council for financial support. We are also grateful to the - ' Huntingdon) Research Centre for the provision of Inhalation expo*' ' sure facilities. Submitted for publication November 14, 1980;reviscd; - accepted for publication December 8,1980. ; Requests for reprints should be sent to: Dr. R. ). Richards, . .Department of Biochemistry, University College Cardiff, P. O. Box ,"78, Cardiff CFtlXL, U.K. v? .* REFERENCES . -,;1. Frongia, N.; Spinazolla, A.; and Burarelli, A. 1974. Lesionl ' polmonari sperlmentali da Inalazione prolungata dl polveri dl PVC in ambiente di lavoro. Med Lav 65: 321*41. 2. Richards, R. J.; Desai, R.; Hext, P. M.; and Rose, f, A. 1975. Bioioglcai reactivity of PVC dust. Nature 256: 664-65. 3. Richards, R.).; Desai, R.; and Rose, F. A. 1976. A surface- active agent Involved in PVC-induced haemolysis. Nature 260: 53*54. 4. Pigott, G. H. 1976. In vitro studies of a range of formulations of PVC. Polymer Preprints 19: 29-34. 5. Pigott, G. H., and Ishmael, J. 1979. A comparison between in vitro toxicity of PVC powders and their tissue reaction in vivo. Ann Occup Hyg 22:111 *26. 6. Agarwal, D. K.; Kaw, /. L; Srivastava, S. P.; and Seth, P. K. 1978, Some biochemical and histopathoiogical changes induced by polyvinylchloride dust in rat lung. Environ Res 16: 333*341. 7. Tetley, T. D.; Rose, F. A.; and Richards, R. j. 1980. Biochemi cal and cellular reaction of PVC paste polymers and latg^ following intratracheal instillation into rats. Inflammation (in press). g. George, G., and Richards, R. J. 1979. Preliminary studies on the isolation, separation and identification of pulmonarylavage proteins from the rabbit. Bioehem 5oc Trans 7: 1 285-87. 9. Tetley, T. D.; Hext, P, M.; Richards, R.and McDermott, M. 1976. Chrysotilt-induced asbcstosis: Changes in the free cell population, pulmonary surfactant and whole lung tissue of rats. Br J Exp Pathol 57: 505-14. 10i Bradbury, P., and Gordon, K. 1977. Connective tissue and stains. In Theory and Practice of Histological Techniques, J. D. Bancroft and A. Stevens, eds., p. 104. Edinburgh, Lon don, and New York: ChurchOI-Uvingstone. 11. Gordon, H., and Sweets, H. H. 1936. A simple method for the silver impregnation of retlculln. Am I Pathol 12: 545*51. 12. Wilson, N. 1979. A method for staining polyvinyl chlorine In sections using Sudan IV. Stain Techno154:101-02. 13. Richards, R. J.; George, G.; Hunt, J.;and Tetley. T. D. 1980. Relationship between the hemolytic potential of certain par ticulates and their reactivity at the lung surface In vivo. In The In vitro Effects of Mineral Dusts, R. C Brown, M. Chamberlain, R. Davies, and I. P. Gormley, eds,, p. 323-332. London: Academic Press. * INDUSTRIAL HYGIENE SAMPLING STRATEGIES (NIOSH 553), sponsored by the Midwest Center for Occu pational Health and Safety, will be held on April 22-24, 1981, at St. Louis, Missouri. The course content includes introduction to statistical sampling strategies, legal aspects of sampling strategies, fundamentals of statistics, estima tion and decision I and II,exposure measurement sampling strategies, compliance vs. non-compliance, full period sam pling, grab sampling decisions, ceiling limit sampling, com pliance officer sampler strategies, and statistical workshops. Industrial hygienists and supervisors who are responsible for sampling industrial atmospheres, making decisions on such sample results and taking appropriate action in com pliance with OSHA regulations should plan to attend. Three points will be awarded toward maintenance of certification from ABIH. CEUs will also be awarded. There is a fee of $375.00. For further information, write or call Ruth K. McIntyre, Director, Continuing Education, Midwest Center for Occupational Health and Safety, 640 Jackson Street, St. Paul, Minnesota, (612) 221-3771. URL 05268 January/February 1981 (Vol. 36, No. 1) 19