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Acute Inhalation Toxicity of Vinyl Chloride ' to Laboratory Animals E. MASTROMATTEO, M.D., A. M. FISHER, Ph.D., H. CHRISTIE, B.Sc., and H. DANZIGER, M.D. Division of Industrial Hygiene, Ontario Department of Health, Toronto, Ontario; Department of Physiological Hygiene, School of Hygiene, University of Toronto, Toronto, Ontario; and Welland County General Hospital, Welland, Ontario Introduction scribed as very slight. Elimination from the body was very rapid. These authors quoted the work FIRST prepared in 1833, vinyl chloride has come into increased use in the last decade in of Schaumann* with experimental animals. Mice, rats and dogs were able to endure repeated nar hdkotfnbhianreuladeeNveosrcentwtaoLhAronirneipatmemidi.ncrlgpeeAdieaHtrrvupsxroiyicdorpVsdgaaoelutauonirpndesilsceuanliiatyneorCliistosdniofebtnoaousneshfft.sfeoaaetm5norflzri0ietnaoapi0rnerdleodcsaemppelcyroopaaauvfrmsottttieeeetsnfhodrytweiGco.ealaixxalmocIs,ptitvhcehooeeldionswotsryuworntawia.rmdmesebAievtelethieshranTrrherea,ticharsni.ersldtidenfwouhssIbiens.arhteyselslTheiatowccw1nieoon2recossVenroupiessckioelantdeiordhrwnyndbenbcdltiistnreeetiaehccanycnrthoattivaoealureogwdndrdtdrecasoiiadonmamuicnettnol.aiducyfgOaahsebtlrascpsole.resutayscvlhdtitAarxebhhuarcaenmonmdcenalgooionadtaecgogrnsrstdasiosicsvittuunuaoesiissntgdlofnye.icedeaaitvpaodoisitesnfdeheraestoStrshn*hicroccen*teauhe*otnisaeritpmtoeuxeonmdftiexsnarpelsattiahiuvenmbanaerrnliideeir,tr of special interest when two fatalities occurred in a plant handling vinyl chloride. This plan ;j en gaged in the polymerization of the gaseous n onomer with the aid of catalysts. One worker died while cleaning out a polymerization vessel and the other while working in a pit. Details of these two fatalities have been reported by one of the au ment. Carr and others7 found that vinyl chloride produced sensitization of the myocardium in three of seven dogs in which it was administered as an anesthetic agent. Two cases of vinyl chloride gassing occurred in a factory in Great Britain where polyvinyl chlor ide resin was being made. These were reported by thors.1 Published information on the acute toxicity of vinyl chloride to experimental animals proved scanty. Patty, Yant and \Yaite: in 1930 reported on the acute response of guinea pigs to vinyl chloride. They found that exposure to 20 to 40 per cent in air killed guinea pigs in a short time; exposure to ten per cent was endured for several hours. Congestion and edema of the lungs with hyperemia of the liver and kidneys were noted. Peoples and Leake3 found the minimal anes thetic range of vinyl chloride in mice exposed for ten minutes was 8 to 12 per cent in air; 25 to 30 per cent was fatal in ten minutes. Dogs and rab bits were anesthetized within one minute when the Chief Inspector of Factories.* In one ease a process worker was standing outside a polymeri zation vessel unci washing it with a water stream. After ten minutes of this he suddenly collapsed across the open manhole. Artificial resuscitation was successfully applied. Subsequent symptoms experienced by this worker were tightness of the chest, nausea, abdominal pain and headache. The second ease occurred in a maintenance worker who was overcome while repairing a vinyl chloride leak. Both men wore hospitalized. Another worker whose hands were accidentally sprayed with vinyl chloride liquid under pressure developed a burn. This latter case w-as reported by Harris.' exposed to a concentration of alxmt IS |>er cent .Such burns may occur with other highly volatile in air. Recovery was very rapid with no apparent materials when sprayed onto the hands us a liquid untoward effect even after prolonged anesthesia. under pressure. Lehmann and Flurv* noted that vinyl chloride Filatova and GronslK-rg1* iti 1957 reported on wns highly narcotic hut had a wide margin l>e- hygienic conditions in a (K)lyvinvl processing tween its narcotic and lethal concentrations. Its plant in the U.K.S.lt. The plant was engaged in local irritating effort and its toxicity were de the iKjlymerizatiim of vinyl chloride by means of m. BOR 009672 Industrial Hygiene Journal 395 :i 'catalyst. Air sampling was done and tire con centration of vinyl chloride was found to vary from about 20 ppm to alsmt 315 ppm. (U-S.S.R. Threshold Limit Value: 1 ing/litcr or about 400 ppm). A spastic ty|>c blood vessel disorder was ileseriU'd in workers from this plant. Because of the recent fatalities mentioned above it was tlecidcd to undertake acute inhala tion studies in laboratory animals with commer cially available vinyl chloride. This was done both for comparison with earlier work and to provide information on the acute response and pathologi cal changes in more than one species of laboratory animal. Experimental Materials and Procedures Vinyl Chloride: The vinyl chloride was supplied in a metal cylinder under pressure. A commercial grade with the following specifications was used: Specific ftsvlty it S0*C Water cootcft* Boilin* tiofa *C Hydrogen chloride (Sorted Acetylene Acetaldehyde Iron Purity (per cent) Impuntie* (per eent) O.Sffl-O.MS None -11 to -9.5 None 10 ppm maximum 90 ppm maximum EaaectiaLiy iron frea 99.6 maximum 0.5 maximum Pure vinyl chloride is & colorless gas at room temperature; its boiling point is -- 13.9eC. It has a sweetish odor. Its flash point is given at --78C and its explosive limits in air from 4 to 22 per cent. It has the molecular formula CHjiCHCl. Animals: Mice, rats and guinea pigs were used in the study. Test and control animals were taken from the same laboratory stock. All were fed a standard commercial diet and housed in the same way. All were of the same stage of development. Equipment: The inhalation chamber capacity was 56.6 liters. It was equipped with a viewing window and an inlet tube. Vinyl chloride was re leased in gaseous form through an adjustable valve on the top of the containing cylinder, then through connecting rubber tubing and a recali brated Fisher flow-meter. Fresh air was pumped by motor at an adjustable controlled rate through a meter. The streams of air and vinyl chloride were combined at appropriate rotes of flow by a glass Y-tube leading through further rubber tub ing to the nnimal chamber inlet, to deliver a continuing stream in the desired proportions. Calculations and adjustments were made to pro duce the following flow concentrations of vinyl chloride in air for delivery: 10, 20, 30 and 40 per cent. All concentrations are expressed in this re port us per cent by volume in air. Xo determina tions of vinyl chloride concentrations were done in the test chandler during the experiments. Experimental Procedures: Different groups of five mice, five nits and five guinea pigs were placed in the ehamlxT and exposed for thirty minutes to concentrations of 10, 20 and 30 per cent vinyl chloride in air. Similar groups of con trol animals were maintained but not exposed to vinyl chloride. At the end of thirty minutes exInsure the test animals were immediately re moved to fresh air. An additional group of five guinea pigs was exposed to a concentration of 40 pci cent vinyl chloride in air. In all 65 experimen tal animals were involved. Observations were reeoided on the animals during and after exposure. The animals which died either during the ex posure or after a delay [icriod were autopsied soon after death. Two weeks after the exposure sur iving test animals and controls were sacrificed uy a blow to the head with the exception of four control rats which were killed by exsanguination. All animals were examined for gross pathologii al changes. Tissues were removed from all ani mals for microscopic examination. The lungs, liver, kidney and heart were removed in all eases. The brain, adrenal, spleen, trachea, lymph nodes, and the eye were removed from represen tative animals in each group for study. The tissues were preserved in formalin and sections made for staining with hematoxylin-eosin. Special stains were made where indicated. Observations Control Animals: Xo symptoms were exhibited by the 15 control animals and no deaths occurred. Ten Per Cent Vinyl Chloride: The response of animals exposed to this concentration is recorded below in summary form; Exposure Time (minutes) X 6 10 15 30 36 30 JUtpcni Slight irritation in mice and reta. Increased motor activity tat in `miss, then rsts end guinea pigs. Increased motor activity in all apsciaa; twitching of extremities in mice. Pronounced tremor, unsteady gait and muscular incoordination in all gpsore, Mice and reU in aide position; muscular incoordination, tremors and twitching of eitremitiea in cuius* pig* Mice and reU unconscious. guinea pigs very unsteady but still standing. Mien and mu in deep narcosis; guinea piga in aid* position with tnmnn nwa Exposure was stopjKil and the animals removed to fresh air. All recovered within five minutes. Twenty Per Cent Vinyl Chloride: The response BOR 009673 396 October, I960 of animals exposed to this concentration is re corded below in summary form: Exposure Tim* (mtautu) 1 I I 10 IK 10 u 10 RtipOM* Immediate imutioo in mice and rtUMuacular incoordination m mion and nti, Mion and mu on their iden with marked tremor* and twitching of the extremi ties; uantatdineM and muscular inco ordination in guinea pigs. Mice and rau unconscious with rapid irregular breathing, guinea pigs un< eoneciou* but showing marked twitchin. AJl animals in deep aarcoris; respirations irregular and rapid. Deep narco*!*; breathing alow and sUallow. Breathing ceased in one mouee; frothing at mouth and nostrils in mioeand rau. AU animal* in deep narcosis- Exposure was stopped and the animals removed to fresh air. Mice and rats recovered faster than the guinea pigs and appeared normal within five minuted except .for one mouse which was dead. The guinea pigs continued to show muscular in coordination, unsteadiness on their feet until 20 minutes after removal from exposure.. Thirty Per Cent Vinyl Chloride: The response of animals exposed to this concentration is recorded below in summary form: Etporate TiAie (mitiute*) Response Mite showed irritation immediately and th* hit* showed imtatioa quickly thereafter. 1 Muscular incoordination in mice and reU, 2 Mice mod rat* in aide position with tnatked tremor* and twitching of the axtremttiea; muoeular tnroordi nation io guinea piga with developing paraly* si* of the extremities. I All animals unconscious; rapid irregular respiration* in mice and rat* with frothing about nose and mouth; twitching of extremities still occurred Occasionally in guinea pigs. 10 Respirations Stopped in mire; breathing low and h*Uow in rate; guinea pigs stilt exhibited occasional twitching movements of the extrenn tics 10 Breathing Stopped in r*U; respiration* slow and shallow in guinea pigs; twitching of extremities still preaent in guinea pigs. 30 Mice and rats atill: guinea pigs in deep narcosis with slow iliaNow breathing E\]>osure wns stopixxl and the animals removed to fresh air. The mice and rats were dead. The guinea pigs took 25 minutes to return to their normal apix-aranee and activity. One guinea pig from this group died within 21 hours following exposure. ITable Number of Deaths in Different Croups of Five Mice, Rats and Guinea Pigs Exposed for Thirty Minutes to Varying Concentrations of Vinyl Chloride in Air Vinyl chloride concentration fper cent by volume in air) Laboratory animal Mice Rati Guinn pigi Tout 10 20 30 40 Toul 0/5 1/5 .s/s -- a/15 0/5 0/5 5/S -- 5/15 C/5 0/5 1/5* J/5* 3/20 0/15 1/15 11/11 I/S 14/50 * A delayed death occurred withm24 hours following aspocurs. Forty Per Cent Vinyl Chloride: Only five guinea pigs were exposed to this concentration. Sigcs of irritation were immediately apparent. Muscular incoordination ap|>cared within seconds. After five minutes all the guinea pigs were uneonse ous with slow shallow* breathing. At the end of the exposure period one guinea pig was dead and the remaining four were in deep narcosis. These, sur vivors took 30 minutes to return to their normal appearance and activity, but one died within the following 24 hours. The number of deaths occurring in different groups of five laboratory animals exposed for thirty minutes to varying concentrations of vinyl chloride in air is shown in Table I. Pathological Findings Gross pathological and histological studies were carried out. The findings are summarised below. Control Animals: Those animals showed no gross or microscopic evidence of damage. Ten Per Cent Vinyl Chloride: All test auimnls survived this exposure. They were sacrificed two weeks later. On gross examination there was evi dence in mice of slight hyperemia of the lungs. This was less marked in rats. Guinea pigs showed no difference from control animals. On histologic examination the mice showed very slight engorge ment of the pulmonary vessels. One mouse shower! degenerative changes in the tubular epi thelium of the kidney with hydropic swelling. The mts showed slight congestion of the capillar ies in the lung. Lungs in the guinea pig were also slightly more hyiwremie than those of the control animals. Twenty Per Cent Vinyl Chloride: One mouse died us a result of 30 minutes exposure to this concentration. Pulmonary congestion was evident on gross examination. Microscopically, there was BOR 009674 Ind^trial Hygiene Journal 397 1`iipiW`iin`itt "(tin* blood Ye.-scI* in flu; lung with imtcliy areas nf atelectasis anti minimal edema. Thn kidneys showed minimal degenerative change* in the epithelium of tlu* convoluted tu bule*. Tin- ti-'t animals surviving this exposure were sacrificial in two weeks. On gross examination, congestion of the lungs was present in all species, hut it was more marked in the mice and rats than in the guinea pigs- Histologically, there was evi dence of pulmonary congestion in exposed ani mals. Sane fatty infiltration was present in the liver of one rut. All other tissues studied appeared normal. Thirty Per Cent Vinyl Chloride: Gross examina tion of the animals which died as a result of this ex|K>sure revealed congestion of the lungs with hemorrhagic areas. The mice and rats in addition showed congestion of the liver and kidney. The one guinea pig d'-ath was delayed. In this animal there was marked congestion of the lungs with hemorrhages and the liver was distended and very friable. The microscopic changes in the animals which died included marked engorgement of the pulmonary blood vessels with edema and hemor rhages in tlie lungs. The trachea of one rat showed superficial desquamation of the epithelium. Con gestion was also evident in the liver and kidney of the mice and rats. The liver of the guinea pig which had the delayed death showed severe fatty degeneration of the liver confirmed with frozen sections stained with Sudan III. The four surviving guinea pigs were sacrificed two weeks later. Marked pulmonary congestion was present with hemorrhagic areas and edema. In one case there was evidence of secondary bac terial infiltration. The liver in these guinea pigs gave the appearance of fatty infiltration but no fat was demonstrated on frozen section. Forty Per Cent Vinyl Chloride: One of five guinea pigs exposed to this concentration died during the exposure and another died within 24 hours. The two which died showed marked con gestion of the lungs with hemorrhages on gross examination. This was also evident on micro scopic examination. The liver of one of these gave the appearance of fatty infiltration, but no fat could be demonstrated on frozen section. Tho three surviving guinea-pigs were sacrificed two weeks later. Marked congestion of the lungs with hemorrhage was evident on both the gross ami microscopic examination. In one guinea pig, the trachea! epithelium wns complctdyuliacnt. Pathological studies which were made of the brain, heart, spleen, adrenals, lymph nodes and the eves showed no difference iietwccn controls and animals dying as a result of exposure or be tween controls and the surviving animals sacri ficed two weeks after cxt>ostiro. There was a tendency for the blood to remain unrlottcd in the animals dying during exposure. This feature was noted in the two human fatalities mentioned ear lier but it is not a s[ccific characteristic of vinyl chloride. Discussion The response of guinea pigs to inhalation of vinyl chloride was similar to that reported by Patty, Yant and Waite. The guinea pigs in our study, however, tolerated a greater cxjxisurc dur ing the experimental period than either the mice or rats. The results in mice agreed closely with those reported by Peoples and Leake. Rata were similar to mice in their response. The pathological changes in animals which died as a result of exposure were mainly those of vascular engorgement of the lungs with hemor rhages and edema. The severity of these changes varied with the severity of exposure. In the higher concentrations, pulmonary change was marked with severe damage to the tracheal epithelium. Congestion of the liver and kidneys also occurred in test animals. These changes are similar to `hose reported by Patty and associates. Evidence of pulmonary congestion was still present in surviving animals sacrificed two weeks after exposure. Patty and associates reported that such changes had disappeared in about eight days Degenerative changes in the tubular epithelium of the kidney were noted in one mouse dying as a result of exposure and in one mouse sacrificed two weeks after being exposed. Such changes, however, were minimal and not shown by other animals in the group. One of the guinea pigs with delayed death fol lowing exposure showed severe fatty infiltration of the liver. Changes suggestive of fatty infiltra tion were observed in other exposed animals but not confirmed by frozen sections stained with Sudan III. Summary Separate groups of laboratory animals com prising five mice, five rats and five guinea pigs were exposed in an inhalation chamber to concen trations of 10, 20 and 30 per cent vinyl chloride in air for 30 minutesi An additional five guinea pigs were exposed to u 40 per cent concentration for a similar period of time. The rcs|x>n8C by each of the three s|K-eies to these concentrations is noted. Inhalation of these relatively high concentra tions produced narcosis und death. Mice were tho most susceptible with guinea pigs considerably more resistant. Rats were similar to mice in their BOR 009675 -4 388 October, i960 response. Exposure to ten per cent vinyl chloride ia sir produced deep narcosis in mice and rats but no deaths; 30 per cent concentrations killed mice and rats. Exposure of guinea pigs to 20 per cent produced deep narcosis; but three of five guinea pigs survived exposure to 40 per cent con centration. Animals dying as a result of exposure were autopsied shortly after death. Survivors and con trol animals were sacrificed two weeks later. Groin pathological and microscopic studies were done and these findings are described. Tne principal pathological changes in animals d'-iug from exposure were congestion of the lungs wiin pulmonary edema and hemorrhages in some, and congestion of the liver and kidneys. Failure of the blood to clot was also observed. One of the three guinea pigs which died following exposure showed severe fatty infiltration of the liver. Survivors sacrificed two weeks after exposure shewed little difference from the control animals. Pulmonary congestion was still evident but live: and kidney congestion was not. References 1. Damioib, It.: Accidental Folioalnc by Vinyl Chloride: Re port of Two Caen. Canadian Wed. Auac. J. U: 80S CApril 1930). j 2. Pattt, F. A., W. P. Yawt, akS C. P. Wait*: Acute Reepone* of Guinea Pice to Vepote of Some Now Commercial Ore*cue Compound# V. Vinyl Chloride. /**. HtalU Ktft. it: 1983 fAutuet 1030). | S. Fiono, A S., ako C. D. Loaki: The Aneatbatie Aetioa of Vinyl Chloride, J. Piarmaeoltn it: 289 (1933). 4. Lehwame, K. B., A F. Fiver: Teiicdotf and Ayyine / Industrial Sdwnd, Tranalated by E. Kins and H. F. 8mith, Jr., Williame tc Wilkin, Baltimore (1993). 9. Scmacuanw, O.: Cited by Lehmans. K. B. and J Fluty. 9. OtTut, R. H., C. J. Cask, J. C. Kbaht*, add M. J. Saves- wald: Anertheeia XXVII. Narooeid with Vinyl Chkride. AtuUkmotm t: 339 (1997). 7. Cabs, J., R. M. Bneonujn, J. F. Vtiona. aim J. C. Khaim J*.: Aseotheaia XXIV, Chemical Constitution of Hydtocarbon and Cardiac Automatieity, J. Ptarm. and Freer. Tkmop. >7: 1 (1999). t. Annual Report of the Chief Inepocter of Faawiee for Ike Fear . Ml. London : H.M.S.O. Cmd. 3772 (Kerch 1933). 9. Haeats, D. K.: Health Problem* in tba SUnufaetiuo and Um of Pintin, Brti. J. lad. Ned. 10:233 (1933). 10. Filatova, U. 8 , AND E. 8. Ghotnnno: Hytionie Worlrin* Condition in Polyvinyl Chloride Tar Plants Gifuno pp. 38-12 (January 1937). Abstracted in AM. f World Hod. tt: m (July 1937). / Back Isbues of AIHA Quarterly FOR 18 YEARS THE AIHA QUARTERLY was the publication of AIHA, then in 1958 it was changed to be the AIHA Journal with six issues per year instead of four. Now limitation of storage space is forcing us to dispose of all back issues of the Quarterly. For individual issues quantities on hand vary from a low' of 8 upward. All issues except March 1946 (Vol. 7, No. 1) and June 1956 (Vol. 17, No. 2) are available as we go to press. Some issues are reprinted without cover. The cost is $1.50 per issue {31.00 each when more than three issues are ordered) from Volumes 7 through 18. From 1940 to 1946 AIHA Quarterly appeared as a section in Industrial Medicine and Surgery, however these issues of these sections have been reprinted and are available as a unit. The complete reprint, Vols. 1-7, costs $10.00 to members, and $50j00 to companies or departments of government. Handsomely bound, complete seta of Vols. 1-18 with cumulative index are available for $250.00 per set. Address all orders to: George D. Clayton, Executive Secretary, AIHA, 14125 Prevost, Detroit 27, Michigan. (Back issues of AIHA Journal, 1958 to present, may be obtained directly from our publisher, The Williams Si Wilkins Company, 428 E. Preston, Baltimore 2, Maryland, at $1.50 each issue.) BOR 009676