Document nmL72RQ34o8G8okNKDmQ1qoR2

*J URL 18022 Reprinted from American Indupttuai, Hygiene Association Jocrnal Volume 21, No. 8, October, 1960 Printed in V.S.A. 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 oj Industrial Hygiene, Ontario Department oj 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 FtfnkbohienrladeeeorcnIctwahRLAoonnerimtmSemdi.cgepTeAeiartripsxorincdpVrpdatoaerloauoenpndlcspeuiiaitnanoreCisotcrdnieoernoouednhsffsafoaetsm5iofzrrne0ieanoapd0nrdlod1cuaempl8esycrop3aeauvfm3otttiei,etnefnhrytwveiGoealtiahnxxmlocs,epiyhvchooelelilonostasrywcuorntsia.hrmdemteblAevodeleeireasrnrTircdhrea,tehaaeesnirditlddnewehhssIbieaa.nhitnyssslTl heoaworccw1aon2ofraotsVenSsspuiskcsicelnvtdaiehhdrweynnaenbcrldtiutereyteiahmccyndntohritoaaaoalugongwdpnrtsrnceaisoidod*nmauw.tetnwol.iaedcyTfgOrihethebthasspoee.esataseevhbdeAtrbxehulaecnpeaoamnceenlgutoonatrdoetoigrnhmrdasiseoocistetunnruaeisnsnsdgdlont.uiacqeeariltvpuaedoasiotedehnrtraeseeoiSsnm8pdcrccnteeaaheotatnlahisttpmtoeuee.odmidieMsnwlsatniaihvoicnbaanaerenlridker,rt, dustrial Hygienists. No industrial fatalities associated with its use have previously been reported. It was therefore incoordinated muscular activity of the extremi ties and cardiac arrythmias of a serious nature were observed in all six dogs used in their experi of special interest when two fatalities occurred in a plant handling vinyl chloride. This plant is en gaged in the polymerization of the gaseous mono mer 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 thors.1 Published information on the acute toxicity of ment. Carr and others* 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 the Chief Inspector of Factories.8 In one case a process worker was standing outside a polymeri vinyl chloride to experimental animals proved scanty. Patty, Yant and Waite* 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 Leake* 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 exposed to a concentration of about 18 per cent zation vessel and 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 case occurred in a maintenance worker who was overcome while repairing a vinyl chloride leak. Both men were hospitalized. Another worker whose hands were accidentally sprayed with vinyl chloride liquid under pressure developed a burn. This latter case was reported by Harris.* Such burns may occur with other highly volatile in air. Recovery was very rapid with no apparent materials when sprayed onto the hands as a liquid untoward effect even after prolonged anesthesia. under pressure. Lehmann and Flury* noted that vinyl chloride Filatova and Gronsberg** in 1957 reported on was highly narcotic but had a wide margin be hygienic conditions in a polyvinyl processing tween its narcotic and lethal concentrations. Its plant in the LLS.S.R. The plant was engaged in local irritating effect and its toxicity were de the polymerization of vinyl chloride by means of S94 ) y URL 13023 r k o s i lan Industrial Hygiene Journal S95 a catalyst. Air sampling was done and the con centration of vinyl chloride was found to vary from about 20 ppm to about 315 ppra. (U.S.S.R. Threshold Limit Value: 1 mg/liter or about 400 ppm). A spastic type blood vessel disorder was described in workers from this plant. Because of the recent fatalities mentioned above it was decided 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 gravity at 20'C Water content Boiling range *C Hydrogen chloride content Acetylene Acetaldehyde Iron Purity (per cent) Impurities (per cent) 0.983-0.984 None -11 to --9.4 None 10 ppm maximum 20 ppm maximum Essentially iron free 99.4 maximum 0.4 maximum Pure vinyl chloride is a colorless gas at room temperature; its boiling point is -- 13.9C. 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 CHitCHCl. 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 some 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 rates of flow by a glass Y-tube leading through further rubber tub ing to the animal 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 as per cent by volume in air. No determina tions of vinyl chloride concentrations were done in the test chamber during the experiments. Experimental Procedures/ Different groups of five mice, five rats and five guinea pigs were placed in the chamber 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 ex posure the test animals were immediately re moved to fresh air. An additional group of five guinea pigs was exposed to a concentration of 40 per cent vinyl chloride in air. In all 65 experimen tal animals were involved. Observations were re corded on the animals during and after exposure. The animals which died either during the ex posure or after a delay period were autopsied soon after death. Two weeks after the exposure sur viving test animals and controls were sacrificed by a blow to the head with the exception of four control rats which were killed by exsanguination. All animals were examined for gross pathologi cal changes. Tissues were removed from all ani mals for microscopic examination. The lungs, liver, kidney and heart were removed in all cases. 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 I'inyl Chloride: The response of animals exposed to this concentration is recorded below in summary form: Exposure Time (minutes) 1 4 10 14 20 25 30 Response Slight irritation in mice and rats. Increased motor activity first in mice. then rats and guinea pigs. Increased motor activity in alt species; twitching of extremities in mice. Pronounced tremor, unsteady gait and muscular incoordination in all species. Mice and rats in side position; muscular incoordination, tremors and twitching of extremities in guinea pigs. Mice and rata unconscious, guinea pigs very unsteady but still standing. Mice and rats in deep narcosis; guinea pigs in side position with tremors--one unconscious. Exposure was stopped and the animals removed to fresh air. AH recovered within five minutes. Twenty Per Cent Vinyl Chloride: The response 396 October, 1960 URL 18025 of animals exposed to this concentration is re corded below in summarv form: Exposure Time (minutes) 1 2 5 10 15 20 25 30 Response Immediate irritation in mice and rata. Muscular incoordination in mice and rata. Mice and rats on their aides with marked tremors and twitching of the extremi. ties; unsteadiness and muscular incoordinHtioi) in guinea piss. Mice and rats unconscious with rapid irregular breathing; guinea pigs un conscious but showing marked twitch ing. All animals in deep narcosis; respirations irregular and rapid. Deep narcosis; breathing slow- and shal low. Breathing ceased in one mouse; frothing at mouth and nostrils in mice and rats. All animals in deep narcosis. Exposure was stopped and the animals removed to fresh air. Mioo and rats recovered faster than the guinea pigs and appeared normal within five minutes 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: Exposure Time {minutes) Response Mice showed irritation immediately and the rats showed irritation quickly thereafter. Muscular incoordination in mice and 2 Mice and rata in aide position with murked tremors and twitching of the exttemitiea; muscular incoordination in guinea pigs w ith developing paraly sis of the extremities. 3 All animals unconscious; rapid irregular respirations in mice and rata with frothing about nose and mouth; twitching of extremities atill occurred occasionally in guinea pigs. 10 Respirations stopi>ed in mice; breathing slow and shallow in rata; guinea pigs still exhibited occasional twitching movements of the extremities. 15 Breathing stopped in rats; respirations slow and shallow in guinea pigs; twitching of extremities still present in guinea pigs. 30 Mice and rats still; guinea pigs in deep narcosis with slow shallow breathing Exposure was stopped and the animals removed to fresh air. The mice and rats were dead. The guinea pigs took 25 minutes to return to their normal appearance and activity. One guinea pig from this group died within 24 hours following exposure. Table I Number of Deaths in Different Groups of Five Mice, Rats and Guinea Pigs Exposed for Thirty Minutes to Varying Concentrations of Vinv] Chloride in Air Vinyl chloride concentration 1 (per cent by 1 volume in air) I Laboratory animal Mice Rais {Guinea pigs Toul 10 0/5 0/5 C/5 0/15 20 1/5 0/5 1 0/5 1/15 30 5'5 5/5 i i -S* 11/15 40 -- -- 2/5' 2/5 Total 6/15 5/15 320 14/50 A delayed de*thoecurmdwithin24 hours following exposure. Forty Per Cent Vinyl Chloride: Only five guinea pigs were exposed to this concentration. Signs of irritation were immediately apparent. Muscular incoordination appeared within seconds. After five minutes all the guinea pigs were unconscious 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 summarized below. Control Animals: These animals showed no gross or microscopic evidence of damage. Ten Per Cent Vinyl Chloride: All test animals 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 showed degenerative changes in the tubular epi thelium of the kidney with hydropic swelling. The rats showed slight congestion of the capillar ies in the lung. Lungs in the guinea pig were also slightly more hyperemie than those of the control animals. Twenty Per Cent Vinyl Chloride: One mouse dic'd as a result of 30 minutes exposure to this concentration. Pulmonary congestion was evident on gross examination. Microscopically, there was Industrial Hygiene Journal 397 URL 18026 engorgement of the blood vessels in the lung with patchy areas of atelectasis and minimal edema. The kidneys showed minimal degenerative changes in the epithelium of the convoluted tu bules. The test animals surviving this exposure were sacrificed in two weeks. On gross examination, congestion of the lungs was present in all species, but it was more marked in the mite and rats than in the guinea pigs. Histologically, there was evi dence of pulmonary congestion in exposed ani mals, Some fatty infiltration was present in the liver of one rat. All other tissues studied appeared normal. Thirty Per Cent Vinyl Chloride: Gross examina tion of the animals which died as a result of this exposure 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 death 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 the 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. The three surviving guinea pigs were sacrificed two weeks later. Marked congestion of the lungs with hemorrhage was evident on l>oth the gross and microscopic examination. In one guinea pig, the tracheal epithelium was completely absent. Pathological studies which were made of the brain, heart, spleen, adrenals, lymph nodes and the eyes showed no difference between controls and animals dying as a result of exposure or be tween controls and the surviving animals sacri ficed two weeks after exposure. There was a tendency for the blood to remain unelotted in the animals dying during exposure. This feature was noted in the two human fatalities mentioned ear lier but it is not a specific 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 exposure dur ing the experimental period than either the mice or rats. The results in mice agreed closely with those reported by Peoples and Leake. Rats 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 those 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 minutes, An additional five guinea pigs were exposed to a 40 per cent concentration for a similar period of time. The response by each of the three species to these concentrations is noted. Inhalation of these relatively high concentra tions produced narcosis and death. Mice were the most susceptible with guinea pigs considerably more resistant. Rats were similar to mice in their 898 October, 1960 response. Exposure to ten per cent vinyl chloride in air produced deep narcosis in mice and rats but no deaths; 30 per cent concentrations killed mice aDd 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. Gross pathological and microscopic studies were done and these findings are described. The principal pathological changes in animals dying from exposure were congestion of the lungs with 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 showed little difference from the control animals. Pulmonary congestion was still evident but liver and kidney congestion was not. References 1. Dan*K3eh, H.: Accidental Poiaoning by Vioyl Chloride: Re port of Two Caeet, Canadian Uti. Alsoc. J. SS: 828 (April I960!. 2. Patty, F. A., W. P. Yant, and C. P. Waite: Acute Response of Guinea Pisa to Vapors of Some New Commercial Organic Compound* V. Vinyl Chloride, Pub. Health Rept. IS: 1963 (August 1930). 3. Peoplcb, A S., and C. D. Leake: The Anesthetic Action of Vinyl Chloride. J. Pharmacology 18: 284 (1933). 4. Lehmann, K. B., it F. Flubt: Torieology and Hygiene o! lrtiuetriol Solvents, Translated by E. King and H. F. Smith, Jr., Williams a Wilkins, Baltimore (1943). 5. Schaomann, O.: Cited by Lehmann, K. B. and F. Flury 0. Osteb, R. H-, C. J. Cabb, J. C. Khanty, and M J. Saceb- wald: Anesthesia XXVII. Narcosis with Vinyl Chloride. Anesthesiology 8:359 (1947). 7. Cabb. J., R. M. Btjboisqk. J. F. Vitcha, and J. C. Krantz Jb.: Anesthesia XXIV. Chemical Constitution of Hydro carbons and Cardiac Automaticity, J. Pham, and Ezpcr. Therop. 97:1 (1949). B. Annual Report o} the Chief Inspector of Factories for Me Year mi, London : H.M.S.O. Cmd. 8772 (March 1953). 9. Harris, D. K.: Health Problems in the Manufacture and Use of Plastics, Bril. J. Ini.Sfed. >0:255 (1653). 10. Filatova, U. S , and E. 8. Gronbbebo: Hygienic Working Conditions in Polyvinyl Chloride Tsr Plsot*. Gigiena pp. 38-42 (January 1957). Abstracted in Abst. of World Med. tt: 89 (July 19571. URL 18027