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* ._ X /V43 (: ? o Effects of Single and Repeated Exposures of Humans and Rats to Vinyl Chloride -f I^LESTER, Ph.D,,* L. A. GREENBERG, Fh.D.,* and W. ROBERT ADAM^ M.D.t Laboratory of Applied Biodynamics, Yale University and Department of Pathology, School of Medicine, Yale Unioeriity, New Haven, Connecticut 0 Rats exposed eight hours daily to vinyl chloride at concentrations of 2% for three months and 5r/r for 19 days exhibited changes in liver and spleen weight and in red and white cell counts. Except in the case of liver, tissue alterations did not accompany these changes. The alterations in liver morphology were within the normal range and were not pathologic in character. Because other facets of the animals* response, such as rate of growth, serum transaminase and hemoglobin, were unchanged, it is suggested that the present threshold limit value of 0.05% need not be lowered. Production THE TOXICITY of vinyl chloride has ^ been reviewed recently.12-* Because the mhold limit value of 500 ppm is based m limited data4 using vinyl chloride less pure Ian that now obtainable* the effects of scute exposure in man and rats and long fttm exposure in rats was investigated by us a 1959 and is described here. Our data &> not support the conclusion of Torkelson, it at* that the threshold limit value should hr reduced ten-fold; indeed their data Indi an- a need for further studies prior to any vision of the threshold limit value. together with a trace of air and carbon dioxide. Table I Analysis of Vinyl Chloride Monomer Add Acetylene Aldehyde Iran Sulfur Phenol Non. Vol. Water Color Aisey 2*2 ppm 0.0 1.9 ppm 0.0 0.0 * 60 pin 11 ppm 170 ppm w,w +% Methods Experimental ^VUftrial The vinyl chloride monomer was supplied ^ the Perkins Plant of Solway Process Di- twm. Allied Chemical Corporation, in four A lb. cylinders with the characteristics touu in Table I, presumably differing ma rtially from the vinyl chloride used by fctkrlson, ft at.* only in the presence of ppm of the inhibitor, phenol. Cas chroutography of the liquid phase indicated the awence of more than 99% vinyl chloride, it the TwfiiT-tkin) Aioml Mfffini of the Imlmuiil Hvjtfite Awvutwn, ViuhitiiM, D- ' M2. wu supported in pert by a mrirrh inn front Chnicil Corporation and in port b> L' S. Public H'i brrvice Grant C?73f. addins: Biurhmmtry and Phvipotoci, Nelson l.iboratorr* RuitnvTbt Suir L'annuiy, New New Jenry. tripirM oi U. S. Public Health Smirr Senior Rnnrdi No. SF*37. Desired concentrations were obtained by metering air and vinyl chloride through flow meters calibrated for these gases and pass ing the appropriate flows through a 2-liter mixing chamber. The concentration was also continuously monitored by a thermal conductivity meter calibrated for vinyl .chlo ride versus air. The desired concentration of vinyl chloride was maintained with less than 5% deviation. The total gas flow was about 50 liters per minute (1pm) in all but one of the experiments. Five experiments were conducted: (1)- a five-minute exposure of human beings to concentrations of vinyl chloride ranging from 0.0 to 2.0%; (2) an exposure of rats (Sher man strain rats from Rockland Farm, New City, N. Y.) for as long as two hours with concentrations up to 15%; and (3, 4, and 5) 265 725187 o 266 O May-June, 1963 exposures of rats to concentrations of 8 to 10% for 15 days, 5% for 19 consecutive days and 2% for 92 days. Pathological The experimental and control animals were killed by ether inhalation after expos ures to 2, 5 and 8 to 10% vinyl chloride were completed. Each animal was autopsied according to standard practice. In addition to the gross examination of all animals, all the livers, kidneys and spleens were examined microscopically and some animals received a complete microscopic examination. Heart, lung, liver, spleen, urinary bladder, testes, prostate, ovary, brain, spinal cord, pituitary, tibia, pancreas, stomach, small and large intestine, adrenals, kidneys, uterus, fallopian tubes, thymus, thyroid, parathyroid, eye, knee joint, bone marrow, skeletal muscle, salivary glands and skin were removed from each animal and preserved in 10% buffered for malin. All tissues were examined grossly both at autopsy and after fonnalin fixation. Following fixation, representative samples of all tissues were processed according to stand ard histological procedure and stained with hematoxylin and cosin; separate specimens of liver were stained for fat with the Flam ing Red technique. The animals receiving such a complete examination of the tissues listed included two males and one female rat that had been exposed 15 times and one female rat exposed ten times to the 8 to 10% level; eight experimental and nine control animals surviving the 5% exposure; and 20 randomly selected rats, equally di vided as to experimental and control groups and to sex, exposed to 2% vinyl chloride. The marked increases in liver weight ac companying the exposures to 2% and 5% of the gas, the decrease in spleen weight in the 2% expsoure and the reported kidney changes in exposure to 500 ppm1 led us to examine these particular tissues with a blind technique, thus excluding the operation of any bias or prejudice from the judgments. All slides, including duplicates, were ran domly numbered so that it was impossible to distinguish, without the code, which had been experimental and which control. The slides of the liver, spleen, and kidney wwr examined and classified, then recoded and re-examined; in this manner a measure d the consistence and reliability of the pathol ogist's technique was obtained. This con sistency was nearly perfect in the case of liver slides, and only slightly less so for spins and kidney. The results of these examina tions are reported in the appropriate follow ing sections. Results Experiment l ` Because the main objective was the deu* mination of the effect of long term exposure to vinyl chloride, the maximum concentra tion of the gas to which humans might conceivably be exposed without any imme diate acute effects was determined; this con centration then became the basis for deter mining the concentration used during jthr 92-day exposure of rats. Three men (26, 35, 50 years; 86, 78,.71 Kg.) and 'three women (25, 40, 55 yew;. 64, 52, 61 Kg.) were exposed twice each, day', separated by a 6-hour interval,' fecj three successive days to six different coo-' - ccntrations of vinyl chloride: 0.0, 0.4, 1.2, 1.6 and 2.0%. The concentrations wot j presented in a different order to each ject to make it possible to factor out aa4 possible adaptation to either the gas or the experimental situation; the 0.0% concent/*-: tion was included so that some asses***, of suggestibility could be made. : Until its conclusion, the subjects were tell neither the effects to expect from the exjw ure nor the purpose of the experiment; wr' was information vouchsafed as to the w-j centrations that were used at any time- *Tlfj subjects each sat in a chair separated f*, the gas mixing equipment by a screen, *, simple plastic breathing mask affixed $ the face, covering the mouth and nose. Tta ; rate of air or air-gas mixture passed thra^ the mask was sufficient (50 1pm) to prevent.1, any dilution effects from the atmosphere^ After five minutes of breathing the nuxttnv. the exposure was terminated and the jects were asked to compare their feeling at this time to the time immediately 725188 *lniu<trial Hygiene Journal 267 ptinr to putting on the mask; no suggestions fi any kind were made. The responses of tv subjects are summarized in Table II. I: u apparent that the maximum conccntratom causing no effect in any subject lies beJ^crn 0.8 and 1.2%. From the responses it evident that vinyl chloride causes clearcut intoxicating symptoms which can serve at adequate warning signs of its presence. Table II Responses of Human Subjects to Varying Concentrations of Vinyl Chloride C*afCatefnjttioa Ropoinc ** No differesoo reported by Subverts 1, 2. 4, 2 and Subject 3; "diflhtly di*iy*`- 9i>4B .No differences reported by tU subircu. No difference* reported by Subjects 1, 2, 4, S aed 6. Subject 3: **Iiht)y heady". No dffuiiKO reported bv Subjects 1. 3, 4 and 5. Subject 2 unsure, somewhat diaay ia middle of exposure. Subject 6, reeling. swimming hcadt "ju*t !.< like getting pi". No effect reported by Subject 3. All others report various degree! of intoxication with dininru. lighi- htededneiSt some tuuua. dulling ol visual and audi* lory cues; those symptoms disappeared rapidly upon ermiftatioo of the exposure. All subject* reported imoxicaiinc effects. Subject 1 reporting * headache that persisted for 30 minutes. Them symptom* appeared earlier in the exposure than at 1.6% and the symptoms were more intense than at 1.6%. Etfh rimcnt 2 To gain further insight into the intoxicat ing effects of vinyl chloride rats were ex?oicd to varying concentrations of vinyl ikloridc for periods up to two hours. The (fluent gas from the mixing chamber, at tbe desired concentration, was passed through i 10-liter all-glass exjjosure chamber con taining two rats. At a 5% concentration in dication is moderate but the righting re vs is lost: intoxication is more intense qt *Tr but the righting reflex is still present. This reflex is lost at a concentration of 7%; &r corneal reflex disappears at a conccntra* of 10%. On removal from the chamber, he animals return to the pre-exposure state rapidly. One animal was sacrificed after ex posure to the 10% concentration and showed no visible gross pathology. Exposure to a concentration of 15% resulted in deep ancsJxsa within five minutes. Effusion of fluid lawn the mouth preceded respiratory failure a one rat after 42 minutes; autopsy revealed (dona and congestion of the lungs. The sccd rat was maintained under this deep anes thesia for two hours; on removal to air, there was an uneventful and prompt recov ery. Experiment 3 Some notion of the distinctive and specific pathology that might be caused by vinyl chloride was our object in exposing rats to a concentration of 10%. Thirty-six rats, equally divided as to sex, were divided randomly into an experimental and a control group; the 18 experimental rats were exposed to the gas in a 1100-liter steel chamber. The concentration was initial ly raised rapidly to the desired level by ad mitting vinyl chloride without' admixture with air until the effluent from the chamber attained the desired level as noted. on the thermal conductivity meter. A' fan' within the chamber, connected by a flexible cable to an electric motor outside of the chamber (thus avoiding the hazard of an explosion), mixed the vinyl chloride with the air with in the chamber. Thereafter, the effluent^ from the 2-liter mixing vessel was' admitted to the chamber; to conserve gas, the through put of this highest concentration was 20 lpm. The experimental rats were exposed daily from 0830 to 1630 hours while the control animals were exposed from 0000 to 0800 hours. At this concentration/ as already noted, rats lose consciousness, regaining it five to ten minutes after removal to air-. After two consecutive 8-hour exposures, how ever, the appearance of the animals sug gested that there would be no survivors if this concentration were maintained for the con templated 15-day period; consequently, bpginning with the third exposure, the con centration was reduced to 8|%. The test was interrupted for one day after the sev enth daily exposure because of a mechanical breakdown. Of the group exposed in this fashion, three female rats died, after the sec ond, fifth and fourteenth exposures; the two animals that died earliest were replaced with substitutes for the remaining exposure pe riod; eight female rats were thus alive at the end of the fifteenth exposure. Female rats exposed 10 and 15 times were autopsied 725189 I 1II **1I 1 irii jil f f\ \ I1 268 May*]unf,`t90tk Table III Cliuificition of Morphologic Changes in Liitn Class DoeriiMioM 1 No swelling, no varuolrt, sinusoid* visible. 2 Slight swelling of cHU. only a few faint vacuoles or none, sinusoids visible but compressed. t' 3 Moderate twrlliAR of cells, most reUs with definite fine to medium vacuoles, sinusoids compressed. 4 Marked swelling of cells, large irregular "vacuoles" or clear spaces, compression of sinusoids. Changes focal in distribution. 5 Similar to 4 but rhauga more widespread and diffuse. Table IV :; Classification of Morphologic Changes in Kttorp *' Clast Description 1 Cells well preserved, no vacuoles, glomeruli mnd r 2 \ UMliutioA and "pyknmk" of ioni rnEneng tubules. Otherwise a* 1. 3 As 2 but with more extensive vaewditaiiaa mi ' "pyknosis" including proximal collecting ta^aksJpi,' Wtrx. ^ 4 As 3 but with vacuolization and "pykooo".ettm4;`: ing to convoluted tubules. ^ L yVtiik at the termination of the exposure; the re maining six rats were autopsied 15 days later, five of these rats having been cxjx>scd 15 times and the sixth rat 13 times. The mortality was greater among the male rats; only two males survived 15 exposures, the remaining males, and their replacements, surviving only an average of eight exposures. The two male rats exposed 15 times were autopsied at the termination of the exposure. There was no weight gain in the initial days of the exposure to this concentration, al though after the ninth day some resumption of growth seemed to occur, possibly an in dication of the development of tolerance to the effects of the gas as the exposure con tinued. Upon termination of the exposure, growth resumed promptly at the same rate as the control animals. Both at the 16th and at the 30th dap, there appeared to be no differences in liver: body weight ratios between experimental and control rats, al though too few livers were weighed at the termination of the experiment to make mean ingful comparisons of this ratio. The external appearance, coat and tail, of all the animals was within normal limits. About a third of the animals had parasitic liver cysts. No differences in appearance, color, consistency or degree of congestion were observed between the livers of experi mental and control animals. The lungs of three experimental animals sacrificed im mediately upon cessation of the exposure had numerous focal fibrinous pleural exu dates overlaying nodular yellow-brown pa renchymal lesions which had the appearance of regions of acute necrotizing focal pneu monia. Of the six experimental animals sacrificed two weeks after the exposure, the lungs of two revealed a few adherent fibrous pleural adhesions which were interprets! representing regions of healed pneumonia' All other organs and tissues were wiling normal limits, with no differences bed^w* experimental and control animals. _ :?/*$ The experimental animals sacrificed at ife termination of the exposure received a tw^i plete histologic examination. The lesions*, the lungs of the three animals in tills gnxf showing gross pleural pathology were;*' to be due to acute focal necrotizing pm* monia in varying stages of organizatwiC Some pulmonary edema was present asw|; as diffuse infiltrates of mononuclear c|' in the alveolar septae. Some areas of: plasia were present in and near the-regia*i of pneumonia and were interpreted as stgQK dary to the pneumonia. , ,-i The parasitic liver cysts seen grosdjr confirmed microscopically. The liver sect** stained for fat revealed individual variafi*1 of some degree but no significant deviaoa from normal. The coded slides of the tfu were classed according to the descripua* in Table III while kidney and spleen judged according to the criteria in Td IV and V; Figures 1, 2, 3, 4, and 5 Table V Classification of Morphologic Changes in Spin* Description Avenge site follicles, little or no congmtioa, MMdj between lymphocytes, germinal epithelium me isttiirtfianl tnisauitef. . . .1 J. Obvious congestion, no change in Iqumms cmf uoi] volume of germinal centers less than em peripheral lymphocytes. . Germinal centers approximately same w pbeml lymphocytes, moderate congestion incnawd number of lymphocytes, hyr Krminal centers (greater tana volume of nphocytes). . . ,. * Active hyperplasia: 2 diner of lymphocytes, tone immediately around germinal centers young lymphocytes surrounded by (2) a tone of small dark lymphocytes. . Lymphocytic hyperplasia extending to mdadr a of the interstitium. __ _ 725190 illustrative of the class descriptions in Table Ul. There were no significant differences in the scoring of the groups, although there ws a class "5" and no class "3" in the rats killed at the termination of the exposure, whereas there were no livers in class "3" among the control animals. There were no (lass "5" livers among the experimental rats killed two weeks after the exposure: the slides were evenly divided between scores of 3 and 4. Kidney slides from the experimental ani mals were not graded differently than from the controls but all spleen slides from cxprrituencal animals received the highest xore, differing significantly from the con trol spleens, although some controls also received such high scores. i Experiment 4 These preliminary tests seemed to indicate that vinyl chloride was an anesthetic gas which might also act as a lung irritant. In order to assess this feature of its action, to highlight significant pathological features and to avoid the potentiality of damage arising (com anesthesia alone, five female and five male rats, matched with controls, were ex posed for eight hours daily to five per cent vinyl chloride in air for 19 consecutive days. The experimental animals were placed with in the chamber at 0830 and removed at-1630 and the control animals from 0000 to 0800' hours. To prevent contamination of food or water, both groups of animals were placed within the chamber in empty'cages; hence experimental and control animals were de prived of food and water for eight hours daily. The requisite concentration of the vinyl chloride was attained initially as in Ex periment 3, but the chamber was ventilated at 50 1pm. Although the body weight of the experi mental rats decreased initially, this trend was reversed by the fourth exposure, the rate of grow th thereafter being no different than the controls. The drop in weight at the start and the subsequent resumption of growth was paralleled by an apparent increasing tol erance to the gas as the exposures continued. At the start, the intoxication of the rats was marked, instability of the hind legs being a prominent feature of the exposure. With" each exposure, however, there was an obvious diminution of these symptoms, so that by* the fifth or sixth exposure, it was not possible to distinguish any evident symptoms of neu rological deficit. Hemoglobin determinations during the ex- 725191 J270 May-]unr, 190 - 4J Table VI Formed Elements of the Blood At Termination of Exposure to 5 and 2% Vinyl Chloride | Control--5% (9) ExpU.--5ft (8) **p" lcat than Control--2ft (26) EiptL--2% (23) "pP* 1h than RBC X lOVmm4 Mm S.D, .13 0.49 .87 0,75 0. 05 6.50 0.83 6.82 0.83 NS WBC XI O'/mm4 Mean S.D. 12.67 2.75 7.96 1.85 0. 01 11*34 2.63 9.12 3.08 0. 01 Lymphocyte! ft Mma S.D. 89.4 3.7 92.3 3.5 NS 84.8 4.80 89.7 3.84 0.01 Neutr<o-r phil* I Mcu S.D. - 9.4 *.S " 6.3 M ' '# NS 12.62 4.71 7 8.02 4*4 0.01 posure period revealed no difference between experimental and control groups. On the twentieth day experimental and control animals were anesthetized with di ethyl ether, blood was drawn by cardiac puncture and 1/9 volume of 0.1M sodium oxalate was added to the blood. The ani mals were then killed with an overdose of the anesthetic and autopsied. Measurements of hemoglobin, prothrombin time, hemato crit, red cells, white cells, differential white cells and scrum transaminase were performed on the blood drawn. Serum transaminase, hematocrit values and prothrombin times were normal for both groups. Table VI lists the values for some of the formed ele ments of the blood. Monocytes and eosino phils formed only a small proportion of tie; white cells; no differences between conoti; and experimental animals occurred. The ltd' ceils were somewhat elevated and the *&*" cells lower in the experimental group. Tahir' VII shows that the liver:body weight rati# of the experimental animals was significant)!, elevated. All five male experimental animals- *. posed to 5% vinyl chloride had coats vdudt; were somewhat thinner than normal; the uk' of these animals were scaly. The three fr% male experimental animals and all the a*-' trol animals had normal coats and ta3k One male experimental animal had fibw*', pleural adhesions on the left side; the fibiw* nature of these adhesions suggested that da Table VII Liver and Spleen Weights of Rats Exposed to Vinyl Chloride Group Vapar Cant. t7> Casual Expd. Cootrol eupu. Casual EiptL Csaual ExptL Casual* EiplL- CootrolEiptL* 0 2 0 2 0 5 0 & 0 8-10 0 8-10 *Putttrd fr t*o wwhi *v<0 001 fc"p"<0.0| Day* on F.ipt. 89 92 89 92 l* t* i 19 !wt Tnl Sot Tnt Number of Antmila Sax 14 P 14 F 12 M 15 M 5r 3 -r 4 x< 5M 4P 6P 5M 4M % of Body Weifht Liver Splotn Mcu 3.65 4.76* 3.73 4.74* S.D. 0.36 0.30 0.19 0.30 Mcu 0.49 0.38* 0.38 0.33- ia 0 19 * 0 .MA . o oa ' 0 ** 5.10 6.95* 0.23 0.51 6.17 6.71* 0.46 0.40 . 4.98 5.13- 5.45 6.09* 0.20 0.35 0.57 1.03 * **p"<0.0S **V<0.02 not liinifieMt 725192 `liduuriat Hygiene Journal 271 process was several weeks old and probably * related to the exposure. Both experi mental and control animals had parasitic c,Tr O'*11- No differences in appearance, `rior, consistency or degree of congestion vere noted between the livers of the two pwips. The other organs and tissues were "'thin normal limits in their gross appear ance, with no differences between experi mental and control groups. With the exception of the pleural adhevens in one animal, the microscopic appear ed of all the organs and tissues was norul. The gross observation of parasitic liver owes in all animals was confirmed microvwpically. Liver sections from all animals we stained for fat, but none revealed evi dence of increased fat nor were there any deferences in intracellular fat between the groups. Classification of the liver slides for the *rphological designations of Table III flowed differences between the control and rtpcnmental groups. The mean score of the tmurol group was 2.93 and that of the npcrimental group was 4.56, with only one tmtrol animal being graded "4" and no "spcrimental animal being graded less than The differences between the means "u thus highly significant, yielding a "p" d less than 0.001. Xo differences in kidney or spleen slides between experimental and control animals we noted. Uperiment 5 Because human exposure to vinyl chloride i-ms unlikely at concentrations of the gas trjch greater than the concentration causing ns of intoxication, that is, at 1.2 to 1.6%, he long term exposure of rats was conducted it a concentration of 2.0%. Sixty rats, each weighing about 75 grams, we separated randomly into two groups of ; 5 males and 15 females and placed in eight rparate cages. In the week before the ex posure was started, the rats were observed, righed twice and blood withdrawn for sonogiobin determination. The experimen ts! animals were exposed in the 1100-liter chamber to 2.0% vinyl chloride for eight hours per day (0830 to 1630) on Monday through Friday for a period of three months. Tite control animals were exposed to 0.0% of the test gas, that is, to a flow of 50 1pm of air, in the same chamber as the experi mental group for eight hours per day on the same days of the week. No food or water was present in the cages during either ex posure. All rats were weighed at approxi mately weekly intervals; hemoglobin deter minations, from tail blood, were made at monthly intervals. In neither body weight nor in hemoglobin values were there any sig nificant differences between the control and* experimental groups. During the course of the exposure, there were five deaths; of these, four occurred in the control group. No data from these ani mals are included in any of the tables. On the 89th day blood was withdrawn from the control animals under anesthesia as previously described (Experiment 4); these animals were then killed with ether and autopsied. A similar procedure was followed on the 92nd day for the experimental ani mals. The livers and spleens of all animals were weighed prior to fixation in formalin. The mean values of the tissue: body weight ratios are shown in Table VII. The dif ferences in the means were in all instances significant, the livers larger and the spleens smaller in the experimental as compared with the control animals. No significant differ ences between the groups appeared in the values for hematocrit and prothrombin. The scrum transaminase was not determined. Monocytes and eosinophils showed no dif ferences between the groups; values for the other blood elements are shown in Table VI. The external appearance of all animals was normal. Parasitic liver cysts were pres ent in all animals. There were no differ ences in appearance, color, consistency or degree of congestion between the livers of tiic two groups. All other organs and tis sues were similarly normal, no differences between the groups being apparent. All the organs and tissues examined histo logically were within normal limits, no path- 725193 272 May-June, 196 ology being evident in either cx|>crimental to produce some singular or charactcrie. or control animals. pathology. It cannot be said that this px ; / The parasitic liver cysts seen grossly were was achieved, the results being qiicuioiuK confirmed microscopically. Liver sections and uncertain. Lung lesions were crruicl stained for fat revealed normal variation, but present but these could not be ascribed wa no animals had increased intracellular fat nor certainty to any irritant properties of the gs were there differences between the experi since they might well have arisen from th \\ mental and control animals. Graded in long-continued anesthesia; there was an s; accord with the morphology in Tabic III, parent regression of these lesions in rat* *. the liver slides revealed differences between lowed a 14-day recovery period. 'Hie the groups. The mean score of the control cncc of pneumonia certainly raises the pu group was 1.58, that of the experimental sibility of an acute toxic effect on hint; to group 3.63. No liver in the control group sue at these concentrations; however, h J! scored more than 2 and none in the ex pneumonia could just as well lie caused t perimental group scored less than 3; one liv secondary infection during the severe V!1J er scored 5. Because there was no overlap tral nervous system and respiratory dqno the differences between the means arc high sion, an interpretation favored somewhat b ly significant the diffuse lesions and by the irregular tr, There were no differences in score for the eurrence of the pneumonia. , '* i spleens in the two groups, but the kidney The kidney and liver changes drsrribn slides of the experimental animals scored by Mastromatteo, et at.* in rats exposed Jo significantly less than their controls. 30 minutes to 20, 30 and 40% of vim / `i chloride, were not observed here, aithour ! 15 repeated 8-hour exposures to an anewbr Discussion tic concentration is also a relatively sew {'U^;i The data from the present investigation stimulus. The findings in the lungs agir j -> confirm the acute effects to be cx|ected from with the relative lack of effect found b various concentrations of vinyl chloride: Mastromatteo et at. in rats exposed for 5 f.* concentrations below 1%, when exposure minutes to 10% vinyl chloride, except tL. i ' is limited to five minutes, cause no ob continued exposure, for days, docs result * servable intoxicating effects; five minutes, mortality'. The relative lack of pathology* however, is shorter than the time necessary' the result of 30 minute exposure at`UK* to reach an equilibrium level in the circula found by Mastromatteo et at. would tree tion; from behavioral observations in the to support the view that lung lesions fotm rat, it may be estimated that in five minutes after repeated exposures could well are some two-thirds of the equilibrium level is from the anesthesia and not from sonic * reached; consequently a concentration of 0.6 tion peculiar to vinyl chloride. There . to 0.?%, if long continued would not pro no indication at either the 2% or 5% In- duce intoxication. As the concentration rises of any untoward or other effects upon it above this level the intensity of the intoxicat lung tissue. ing signs increases until at a concentration of As an overall measure of general hcaki 7% the righting reflex is lost: at 10% the body weight and rate of growth are scnnC" corneal reflex disappears and at concentra indicants. Even in the exposure to a no tions of 15% and above respiratory failure centration of 5%, which at first products takes place. Vinyl chloride thus acts as an marked and severe intoxication, the shan anethetic gas, its depressant action increasing drop in the weight of male experiment, with increasing concentration of the gas in animals seen at the start of the exposure the air breathed, the corres|>onding increas soon reversed so that by the end of the IN ing neurological deficits ending in death at day exposure there was no difference b concentrations greater than 15%. The pur tween experimental and control rats pose in exposing rats to concentrations caus either sex. Body weight and rate of gnret ing anesthesia (8 to 10%) was an attempt similarly showed no differences between tt> 725194 ,l*dutirial Hygiene ]ou,nal 273 ;juu| in the 2% exposure. It must be emT/ju/cd again that the growth of the rats `hiring the 5% c.\|x>surc support the observatam of the rat's behavior which indicated `Sat there was a rapid development of toler ance to the intoxicating effects of this con centration. Neither the 2% nor the 5% concentrations caused changes in the prothrombin time, hrmatocrit or hemoglobin values. At both Ac 2c/c and the 5% concentrations, the white cell count was lowered significantly, although still well within the normal range. The increase in red cells, although signifi cantly elevated in the 5% exposure, was not correlated with cliangcs in the hemoglobin content. The increase in concentration of the vinyl chloride is associated with a greater iall in the white cells and a greater (and significant) increase in the red cells at the J'r level. Although lymphocytes and neu trophils are increased, only the change at the 2% concentration unlike the previous cell changes, reaches the statistically signifi cant level. It is difficult to knosv if these changes have any toxic significance, since no tissue changes were seen upon microscopic examination that would account or be aswiated with a drop in the white cells or an increase in red cells. The decrease in spleen: hodv weight ratio is in a direction opposite to that usually associated with a severe drop in white cells; although the white cell count iiid not suffer a severe drop, the decrease was wbstantial at the termination of the ex posure. The only finding suggesting a specific *sic action of vinyi chloride is the increase in liver weight on exposure to 5% for 19 <lays and to 2% over 92 days. The increase in liver weight is not only highly significant statistically but is also substantial, amounting to a 30% increase over the controls. It is unfortunate that no information is available as to whether this increase is reversed on dis continuing the exposure. The increase in liver weight may be interpreted as indicating alterations in water, electrolytes and protein content of the liver parenchyma, but that inch changes presage the development of actual histologic lesions is not certain in view of the wide range that the liver: body weight ratio may encompass (Table VII). Kntlicr this increase may signify a non-spe cific response to metabolic derangements oc casioned by mild and moderate intoxication for daily 8-hour periods. From the data ob tained here, it is not certain that histopathological change would have occurred had ex posures been carried out for longer tiroes. In the paper by Torkelson el al., histopathological changes in the liver and in creased liver:body weight ratios are ,re[>ortcd in male rats exposed to 500 ppm vinyl chloride for 4.5 months. That there is no causal relation between the reported hislopnthology and the increases in liver:body weight ratio is evident from ,the extensive data gathered by these investigators. Female rats exposed to 500 ppm vinyl chloride for 4.5 months showed no statistically significant increase in liver weight but are reported to have histopathological changes in the liver. Female rats exposed to lower concentrations (100 and 200 ppm) for six months had sig nificantly increased liver:body weight ratios hut no pathology. On the other hand, rab bits of both sexes exhibited liver pathology without showing any increase in liver weight after exposure for six months to a concen tration of 200 ppm. The authors correctly point out that organ`.body weight ratios may well be artcfactual, illustrating the point by the significant decrease in kidney weight' found in female rats exposed to 50 ppm for six months; such rats exhibited no changes when cx)xed to higher concentrations. By the same token, but overlooked by the authors, the increased liver: body weight ratio in female rats at 100 and 200 ppm is with equal reason artefact because no stat'stically significant increase occurred at 500 ppm. Apparently, also, species differences are of importance in the reactivity to vinyl chloride, male guinea pigs suffering a signi ficant decrease in their liver: body weight ratio when exposed to 100 ppm for six months. There are six possible combinations of the presence or absence of pathology and in creases, decreases and no change in liver weight. The one combination not observed 725195 r 274 Slay-June. Ml by Torkelson, el al. is histopathological change associated with a decrease in liver weight. Obviously, if five of the six possible combinations have been observed in a rela tively small sample, no causal connection can be said to exist between these two measures. Changes in the liver:body weight ratio may well have some toxic significance, but if they are unaccompanied by histopatho logical alterations, increased fat content or serum transaminase changes it is impossible to conclude that taken alone they signify much. Reference to Table VII will show that the liver:body weight ratios of the rats used as control animals in the 5% exposure were significantly higher than the experi mental animals in the 2% exposure. This illustrates well the fact that though control animals are used, unknown and non-specific changes in the environment, time of the year, temperature, diet, etc., may be responsible for changes in organ: body weight ratio with out at the same time producing pathological alterations in the organ. Similar considerations apply to the de crease in spleen weight; here, not only was pathology not observed, but there was no difference in the morphological character at either the 2% or 5% level between the experimental and control groups. Unlike the interstitial and tubular changes in kidneys of rats exposed to 500 ppm of vinyl chloride rqjortcd by Torkclson, rt al., was the lack of any pathology in our animals exposed to 2% and 5c/o and the fact that the only morphological alteration in which a significant difference between control and experimental animals occurred (kidney at 2%) indicated that the control animals were further from "nonnal". Because it is un reasonable to attach toxic significance to changes associated with a control air ex posure, it is our belief that the morphologi cal alterations we have observed should not be interpreted as manifestations of pathol ogy- The concentrations to which the rats were subjected in these experiments were at least 40 times those used by Torkclson, rt al. U any reliance is to lie placed in a dov-rtlm relationship, path logs' of some considcral.# degree should have been found in out n. periments. Yet only morphological altera tions of the character already dcsrriltrd and j pictured were seen, which, in our knowledge and experience, are of no pathological signi ficance. Whether the explanation resides k ~ a difference between the rat strains used U\.! us and by Torkclson rt al. or elsewhere b not known, but without additional data- a " is impossible to resolve the contradiction. ;. On the basis of the present data, and tbc. seeming unimportance of the liver frigid changes seen by Torkelson et al., and with. out further evidence, a change of the ptV' ent threshold limit value of 500 pptn sna unwarranted. `t `> Summary From 5-minute exposures of human sub-/ jeets to concentrations of vinyl chloride rang ing from 0.0 to 2.0c/c, it is estimated data" prolonged exposure to a level of more ihaar, 0.6e/o is necessary to produce tnininmk' symptoms of intoxication. Rats exposed foi up to two hours to higher concentrations et-.. hibited moderate intoxication at 5f, Iwt * their righting reflex at 7% and the contra! reflex at 10%. Respiratory- failure oeeuned at 15%. If the exposure to 10% was ks continued (two 8-hour daily exposures(, mortality increased; death was apparcath caused by a pneumonic process, but it' wj impossible to decide whether this was the result of a primary action or secondary to tie ancthesia. Rats exposed eight hours daily to 5# 19 days or to 2% over 92 days did not sh* any lung involvement. These levels had effects upon growth rate, hemoglobin, hema tocrit or prothrombin time. At both co' centrations there were increases in the liver: body weight ratio and decreases in the white cells; at 2% the spleen:body weight rat* decreased and at 5% there was an incna* in red cells. No gross or microscopic changes wtn found in any tissue that was correlated with 725196 . .p 'Industrial Hygiene Journal n 275 the changes in the formed elements of the blood. The increases in liver:body weight ratios were associated with, morphological alterations in the'liver which'appeared to Have no pathological significance, while no meaningful morphological alterations were evident in either kidneys or spleens. Because, in the long term exposures, it was impossible to attach any toxic significance to the changes noted or to observe any histopathology, and because other aspects of the animals' reactions, such as growth rate, were unimpaired, the presently accepted threshold limit value of 500 ppm for vinyl chloride, one-fortieth of the concentration tested here, seems to offer an adequate margin of safety for human exposure. Acknowledgment It is a pleasure to acknowledge the tech nical assistance of Malcolm Nicholson, Frederick A. Putt and Miss Lillcmor Wallmark. References 1. you Oettikou.-, W. F.: Tk. Hml.ttm.ttd Hydrmttt. tons; TexiYiiy <ni Dunttn. U, S- ftafclic Health Service* Publication 414. Government Prifltiae Office, Washington, D- C, (1955). 2. Masthomattto. E., A, M. Fimm, H, Ctcftisrn, and II, DAMZtom; Acute Inhalation Toxicity of Vinyl Chloride to Laboratory Animals. Amir, Imd. Hrt. An*. /. 21: 39* (Oct. I960). " 3. ToaKEiio.N, T. R-, F. Ovlm, and V. K. Rowe: The Toxicity of Vinyl Chloride as Determined by Repeated Exposure of Laboratory Antmah. Am*r, l*d, Hyw. Att*. /. 22: 354 (Oct. 1961). 4. Smyth. H. F-, Ja.: Improved Commu*k*ioa--Hy giene Standards for Daily Inhalation. Am*. Ind. Hy, Au*. J. 17: 129 (1956). " 5. Patty, F. A., W. P. Yamy, and C. P. Warn: Aeute Response o< Guinea Pift to Vaooca o( Some New Com. mereial Ornaic Compounds, V. Vinyl Chloride. Fm4- lie Httitk Report! (US.) 4$: 1963 (AuS, 1930). Sources and Use of Toxicological Information The New York University Medical Center in cooperation with the American Industrial Hygiene Association will present a course on Sources and Use of * Toxicological Information. The course will be given October 14-18, 1963, at the Onchiota Conference Center, Sterling Forest, Tuxedo, New York. The course is designed to acquaint those concerned with advanced methods of securing and interpreting pertinent toxicological information. Application and principles will be stressed rather than details of experimental toxicology. The design of the course eliminates the need for specialized scientific back ground so that all administrative and technical persons having responsibilities for use of toxicological information can benefit. Guest lecturers who are spe cialists in selected fields will supplement the staff of N.Y.U. Institute of Indus trial Medicine for presentation of the subject material. Areas to be covered are: biological principles involved, sources of toxicological information, appli cations to in-plant problems. Federal regulations, and requirements of various agencies. Inquiries and registrations should be directed to New York University Medical Center, Institute of Industrial Medicine, 550 First Avenue, New York 16, New York. The registration fee is $150 with a deposit of $25 required. Ac commodations (double occupancy 1 at the Onchiota Conference Center for the evening (dinner) October 13 through lunch October 18 are available at $21 per day including meals (payable to the Center on departure). .t ? . -.-DfA'y , }: ' ?; 725197