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IN-VITRO STUDIES OF A RANGE OF FORMULATIONS OF ?VC /i 2- I U by C. H. I'igott Imperial Chemieni Industries Limited jjprAV,^ . Central Toxicology Laboratory Alderley Par!*, Nr.' Macclesfield Cheshire, SKIO 4TJ Acs v+j INTRODUCTION s:t y w u The polymer PVC is generally considered -as biologically inert, although there are isolated reports in the liecrature'of lung changes in PVC workers (SZENDE at al 1970, .VESTl-CIN MAMONTOV 1970, LILIS ec al 1975). Earlier work in this laboratory (STYLES & WILSON 1973) demonstrated neither in-vitro cytotoxicity of PVC or fibrosis in rats given an intrapericoncal injection of polymer. However FRONCIA, SPINAZZCLA BUCARELLI (1974) reported pulmonary changes in guinea pigs and rats continuously exposed to a-high concentration of PVC dust. More recently RICHARDS et al (1975) reported high hacnolycic activity in a sample of PVC dust, though a second, sample proved essentially inert. This latter result suggests the possibility of differential effects attributable to the varying formulations of PVC required to produce materials for different end usages. It was therefore decided to subject a range of formulations of PVC to an in-vitro assessment of cytotoxicity, ' and to test representative samples of the various grades in whole animal studies. The results df the in-vitro tests are summarised in this paper. .* . * METHODS AND MATERIALS _ ' .. ' ' ' ' The method used to assess cytotoxic potential of dusts was essentially that of Styles & Wilson (1973) using peritoneal macrophages. 2C0-4O0 gm SPF Vistar rats (Alderley Park Strain) of both sexes were injected with 2 mis II glycogen (from shellfish)' as a chemocactic agent for stimulation of peritoneal exudates (CHAMBERS GRAND 1936). The peritoneal macrophages-were harvested by n wash-out technique (CONNING FIRTH 1969) and resuspended in medium 199 (Biocult Labs) containing 20 1U heparin/ml to yield a concentration of approximately 10 cell$/nL. Conical centrifuge tubes (115 x 25 era); treated with a silicone water repcllant to retard macrophage adhesion, were used as culture vessels. Cultures were stirred with a 12 cm FIFE coated bar magnet rotated at 180-200 rpa on .a magnetic stirrer and maintained at 37- in a constant temperature room. Particulate suspensions ir. physiological saline ware added- Co give a final dust concentration of 0.5 mg/10 cells.. After two hours, L ml of culture was mixed with an equal volume of 0.51 trypan blue in phosphate buffered S2liacp':! 7,-4 (FAPPENKEIMER 1917) and a sample of the mixture examined with a x40 phase contrast objective. Random fields were examined for live and dead cells containing dust visible by optical microscopyanc in addition for live and dead cells without visible dust. Where soluble materials were examined an aliquot was ndecd from a stock solution co give the desired final concentration in the medium.' Subsequent 'examination for live and dead cells vns as previously described. SP/-02686 o a non-cy toto::i c PVC powder (D) and of another non-cyto toxic polymer (polyme^'-yl catha cry late) were stirred * *it h 5 els of 10% alkyl bcnac . sulphon.ite i:i alcoholic stisp. sion for 30 minutes, recovered by centrifugation and washed four times with 25 ml aliquots of saline before resuspension as a stack solution. Results of these tests are shown in Table 5 from which it is obvious that surfactant creacr.vn t resnlicc! in a signi fjeant increase in cytotoxicity of thu part ir (of:. Thus it way ho tentatively concluded that surfactant residues are responsible for the observed cytotoxicity of emulsion type I'VC powders. The original test of Styles S Wilson suspended the macrophages in .5% calf scrum. The serum has been omitted from this series of tests as it has been observed (Styles & Pigott, unpublished observations) that such omission expands the observed range of cell mortality vich no . apparent effect on the relative toxicity of a varietyof dust samples.' Although the test in either form has been found unreliable where a soluble component is present in the particles, a series of tests vich scrum present was performed, ns this may be more relevant to human exposure. Results are given in Table 6. It may be seen that non only > was there a marked reduction in cell mortality but also a small change in relative toxicity with respect to a quartz. A parallel L. reduction in cell death with surfactant alone was also observed, reinforcing the belief that this agent is responsible for observed cytotoxicity. . . CONCLUSIONS ^. . .. The test of Styles S Wilson lias previously shuwn'a good correlation vich fibrogenicity in-vivo for a vide range of powders, both organic and inorganic, provided r.o soluble component is present in the particles. On the-basis of this test the PVC samples examined would not be expected to produce lung fibrosis. The cytotoxicity associated vich the emulsion type polymers seems to be associated with the presence of surfactant residues ar.d is reduced, but not abolished by the presence of serum in the culture medium. The relevance of this to an in-vivo situation is doubtful as it is well established that the in-vicro cytotoxic effects of surfactants generally are not necessarily indicative of high toxicity in-vivo .In any event the use of ir.-vicro testing for polymers with diffusible additives uay not ba relevant to longterm tissue reactions. (STYLES 1974). _ REFERENCES SPI-02688 CHAMBERS, R. and CRAND , C. C. (1936) J. cell comp. Physiol. 8 1. CONNING, D. M. ar.d FIRTH, J. (1959) Fd. and Cosr.ee. Toxicol. T 461. FRONCTA, N., SPI.VAZZCLA, A. and 3UCAISI.LI, A. (1974) Med. Lav. 65 321. LISIS, R., AliLZ'MO::, NIC.'iELSSN, V. J., DAW. 5., FZSCKRal.V, A.A. and sr.l.lKOFF, I. J. (1973) Anna Is of the New York Acad, of Sciences 266 22. PAITEtmiltMEK, A. :t. (1917) J. C.\p. Med. j!5 633. RICILAKUS, R. Ji, D2SAI, R., HZN.T, ?. M. and ROSE, F. A. (1975) Nature 256 634. . STYLES, J. A. (1974) In 'Cur.'vtiC Approaches Co Toxicology1 (Symposium Forton Down, Wilts. Sept. 1974) in press. STYLES, J. A. and WILSON, J. (1973) Ann. Occup. Hyg. 16 241. SZENDE, B., LAPIS, K., NEMES, A. and PINTER, A. (1970) Mad. Lav. 61^433. VERTKIW, JU.I. and MAMONTOV, JL'.R. (1970) Gigicna Truda Pro'f. Zabolevar.iya 14 29. I Sanplc F G 2 colls vith dose 74 61 2 dust bearing cells, dead 10.52 14.9Z TABLE 4 - Surviv.il of Macrophages in Surfactant Solution Cone11 of Surfactant (w/v). 2 cell dcach Test Control . *-- l O .o I 5^5 . 0.01Z 0.005% .. .0.0022 0.001% 0.0005% 0.00022 100% 882 76% 63% 122 5Z sz 42 8Z 82 107. ' SPI-02690 flammation g .1' endothelial cells '< disrupted at the rocytes from the iesc two evenu. .me and pericytes i.|jonant role as a o the subendothe- Purification and I TnmrresurusJlavovm;Mi9-399, Gordon and . V.. 197-4, Cinemato: induced by the main ", limerrsurus Jlavoviridig, j g TRAUMA AND INFLAMMATION &5S 235 6.6. In Vivo Skin Capillary Abnormalities in Vinyl Chloride Workers H. R. MARICQ, M. N. JOHNSON, C L. WHETSTONE, and E. C UROY Some workers exposed to vinyl chloride (VC) have been found to have clinically detectable proliferative and fihroiic lesions of the periphery , (acroosteolysis, Raynaud's phenomenon, and sclerodermalike skin pla ques) and the viscera (angiosarcoma and hepatic fibrosis. Band type) (Harris and Adams, 1967; Wilson et al., 1967; Dinman ei al., 1971; Juhe and Lange, 1972, Falk el aL, 1974; Lange et al., 1974; Marstellar et aL. 1975; Thomas et aL, 1975). Techniques of wide-field capillary microscopy have shown distinc tively abnormal patterns in patients with spontaneous Raynaud's syn drome and scleroderma, as well as a strong positive correlation between severity of capillary pattern abnormality and visceral involvement (Maricq and LcRoy, 1973; Maricq et al., 1974). Therefore, these techniques were used to examine 152 VC workers (44 selected, 108 unselected) and 50 control manual workers not exposed to VC. In 44 VC workers selected for known disease (angiosarcoma, hepatic fibrosis, acroosteolysis, Raynaud's phenomenon, and skin lesions), 39% had capillary abnormalities, of which 16% were sclerodermalike patterns and-23% were isolated, discrete capillary lesions. The possibility that trauma and not VC was the cause of capillary ' abnormalities in VC workers, many of whom had been reactor cleaners, was studied by comparing 108 VC workers and 50 manual workers outside the VC industry. The results showed (Table I) that capillary abnormalities were significantly more common' in the VC-exposed group than in non-VC manual workers (x* = 15.50, df = 2.p < 0.001). More of the selected VC workers with VC-specific lesions (angiosarcoma, hepatic fibrosis, severe acroosteolysis, and fibrotic skin changes) had capillary abnormalities than did VC- workers with inactive, stable, or nonspecific lesions (healed acroosteolysis, nonspecific skin rashes, and Raynaud's phenomenon alone) (X* * 13.85, df = 1. p < 0.001). Among the 108 VC employees, no correlation was found between capillary abnormalities and length of employment or length of reactor-cleaning assignment. H. R. MARICQ - Medical University of South Carolina. Charleston, South Carolina {9404. M. N. JOHNSON - B. F. Goodrich. Akron. Ohio 44509. C. L. U'HETSTONE and E. C. LeROY * Conoco, Inc., Ponca City. Oklahoma 74601. I 6...WMATION g TRAUMA AND INFLAMMATION ! 131 i.mplovees of VC VC I and lyjicinic disease in vlrnxlcnua. I'nasliii|:s ul die Ki"litli Kaimpcim (iinlcr- cncc for ihe Microcirculation. le Tom|m-t. ! Marslcllar. H. J.. Lclliach, \V. K.. Moll. r. R.. ami (Icdigk. I'.. I UTS. Umuual iplcnnmc I galic liver disease as evidenced hy jH ni.Mio.vnp. ami guided liver biopss anmng ^ I polyvinyl chloride production uiukctv..<. V >. Ann! SVi. 246:03. I Thomas. L. B.. Popper. H.. Herh. IV l),, .Vlilnfl. I., ami Falk. II . 1975. Vinyl chloride v /' i < 0.1 KM ,,> induced liver disease: From idn>|iallui jkhi.iI liy|tenenvinn (Hand's ssndrrnney lo ' angiosaicomxs, V. Engl. J. Mol. 192:17. Wilson, R. II.. Midonniik, \y. FI.. I alum. II. F.. ami (ariali. |. I., I'.NiT, (>"!iji.iihiiijI ; .-ttrixisicolysis.y. Im. Mrtl d,.,. 201:577. r, 4 1 workers (unscnr not found in . present in VC uiiv (Wilson etal., in compare these k! i-tidotheHa in Capillary micros~i\e technique to !'c:i>es in "suscepee I ..ml Diiclieck, T.. hri. J.nviron. Health .! V . 197-1, Hepatic v / -Im. Mtd. Assoc. : . ;i. iiirit engaged in lit-.ingcn, Raynaud* ici. Industrie.Dtsch. i ,.r.;in- Yinvlchlorid32:1, :,vl i .it 'rule: Current .<> .iliitornuilities in . ! .i< iill.ir\ patterns ar 3 BFGoodrich Th BFGoodrich Company 500 South Main Slreat Akron. Ohio 44318 RECEIVED SEP 2 1 BS1 Addrau Rop/y To: Dept. 0020 BWg. 5-H September 16, 1981 Fran Lichtenberg Society of the Plastics Industry 355 Lexington Ave. New York, N.Y. 10017 Dear Fran: Enclosed is some material that I spoke to you about on residual VC in PVC and ambient exposure levels. I hope it will be of some use to you and if you have any questions or need additional information, please contact me. There is no need to return this material. Cordially M. N. Johnson, M.D. Director of Toxicology v SPI-02696 A Model for the Diffusion of Vinyl Chloride Monomer rom PVC Under Venous Conditions of Storage LINWOOD B. CRIDER, MICHAEL M. O'MARa, ond ROBERT L. BOWLES BFGoodrich Chemical Division Avon.Lake Technical Center Avon Lake, Ohio 44012 This example of employee safety activity in the vinyl resin industry is illustrative of the efforts put forth by many to achieve acceptable . VCM levels in work areas during all conditions of handJ ing. Since use conditions differ greatly at temperatures above T, temperatures and are nonstatic, it was deemed necessary to develop a model embracing many variables to allow prediction of atmospheric vinyl chloride monomer levels for any combination of the variables. INTRODUCTION rrithin the past several years significant advance- V V mentshave been made within the PVC industry to minimize employee exposure to vinyl chloride monomer (VCM); Improvements in PVC manufacturing processes to reduce the residual vinyl chloride monomer (RVCM) content of finished resins has been a key factor in maintaining acceptable VCM levels in work areas during packaging, processing. shipment and stor age. The diffusion of vinyl chloride monomer out of finished PVC resins has been adequately studied (I) at temperatures above the glass transition temperature (T,). The results of these studies have been particularly useful in reducing RVCM during manufacturing and to predicting RVCM escape to the environment during processing. This new knowledge has also led to a rapid, simple gas chromatographic method (2) for the determi nation of RVCM in PVC from the analysis of the vapor phase (head space) over PVC powders in a closed con tainer. This somewhat ideal behavior does not exist at tem peratures below T,, however, and consequendy one cannot use existing equilibrium data to predict the con centration ofVCM in the air space above PVC resins and in particular under non-static conditions such as exists during the storage and transportation of bagged or bulk resin. In addition to the relative'slow diffusion rate at or near ambient temperatures, other factors that affect VCM release include variable ventilation rates, massto-volume ratios, RVCM content of the resin and resi dence time in the storage or shipment compartment. The primary objective of the research reported in this paper was to develop a physical model for the diffusion of VCM from PVC resin under various conditions of expo sure during storage and shipment. The developed model is based on a statistically significant number of large scale diffusion experiments to show the interaction of all the above variables and to allow the prediction of atmospheric vinyl chloride monomer (AVCM) levels for any combination of the variables. EXPERIMENTAL It can be postulated that some reasonable understand ing of these storage variables and their interactions can be developed from a physical modeling of a storage or shipment compartment. It can also be rationalized that. the accuracy of such a model will be much improved if the experimental design is on a large scale that can physicily simulate all of the storage variables but under highly controlled conditions. Any experimental design of this type, however, must be thoroughly tested to establish the validity of the test measurements and to assure the absence of any errors as may occur through poor test controls, adsorption of VCM by the system, and possible interfering components that could lead to spurious results. Using a Computer Optimized Experimental Design (COED), 24 large scale experiments were selected to study the variables listed below to describe their effects and interaction on AVCM in a storage compartment or warehouse. The variables and ranges included: 1. Temperature--74-ll5F 2. Ventilation rate--0.3-3.0 tumovers/h 3. Loading (PVC volume/storage volume)--11 -34 percent 4. Resin RVCM--0.01-123 ppm 5. Storage time--1-170 h In order to meet the demands of the statistically de signed (COED) diffusion study, the experimental phase of this program had to be capable of controlling the following experimental parameters: temperature, venti lation rate, and mass/volume ratio. The basic physical model that was chosen to meet these requirements was one based on sealed metal storage bins (53-gal drums housed in an environmental chamber where the tem perature could be varied and controlled. A schematic of the entire experiment is shown in Fig. I. The schematic <1 168 JOURNALOF VINYL TECHNOLOGY, SEPTEMBER 1979, VOL. 1. SO. 3 SPI-02697 I