Document v55rjKm5Jngobamy07wrx036

R&S 136106 Effects of Single and Repeated Exposures of Humans and Rats to Vinyl Chloride D. LESTER, Ph.D.,+ L. A. GREENBERG, PhJV and W. ROBERT ADAMS, M.D.f Laboratory of Applied Biodynamics, Yale University and Department of Pathology, School of Medicine, Yale University, New Haven, Connecticut )g Rata exposed eight hours daily to vinyl chloride at concentrations of 2% for three months and 5% lor 19 days exhibited changes in liver and spken weight and m 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.. Introduction '"THE TOXICITY of vinyl chloride has -** been reviewed recently.1'3' * Because the threshold limit value of 500 ppm is based on limited data* using vinyl chloride less pure thai^^nt now obtainable3 the effects of icut^^Kposure in man and rats and long term exposure in rats was investigated by us n 1959 and is described here. Our data Jo not support the conclusion of Torkelson, :t ai* that the threshold limit value should )e reduced ten-fold; indeed their data inditate a need for further studies prior to any evision of the threshold limit value. together with a trace of air and carbon dioxide. Table I Analysis'of Vinyl Chloride Monomer Acid Acetylene Aldehyde Iron Sulfur Phenol Non, Vol, Water Color Aa*ay ' 2.2 ppm 0.0 1.9 ppm 0.0 0.0 60 ppm 11 ppm 170 ppm w,w 99+% Methods Experimental dateria! The vinyl chloride monomer was supplied >y the Perkins Plant of Solway Process Di-' ision. Allied Chemical Corporation, in four 0Q lb. cylinders with the characteristics hown in Table I, presumably differing ma nually from the vinyl chloride used by 'orkelson, ct aL3 only in the presence of 0 ppm of the inhibitor, phenol. Gas chro matography of the liquid phase indicated the resence of more than 99% vinyl chloride. Presented at the Twenty-tblrd AbiruJ o( the' aaericaj* Industrial Hyxtctrt Aaaociauoa. Waahiaftna, D, . May. 1962. Thk work wu tupported in part by a research grant from lied Chemical Corporation end in part by U S- Public ralth Service Grain C-2738. * Present addrtu; Biochemistry and PhytioJofy, Nekoa ouflouw^yioj^hLfaebaocrwatoJreyr,aayR, uCfen-The State Uftrvantfy, New U. S. Public Health Service Senior Rsnwdi SP'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 Gty, N. Y.) for as long as two hours with concentrations up to 15%; and (3, 4, and 5) 265 R&S 136107 266 May-June, 1963 exposures of rats to concentrations of 8 to slides of the liver, spleen, and kidney were 10% for 15 days, 5% for 19 consecutive examined and classified, then recode^ days and 2% for 92 days. re-examined; in this manner a mea the consistence and reliability of the Pathological ogist's technique was obtained. This con 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 sistency was nearly perfect in the case of liver slides, and only slighdy less so fpr spleen and kidney. The results of these examina tions arc reported in the appropriate follow ing sections. to the gross examination of all animals, all the livers, kidneys and spleens were examined microscopically and some animals received Results Experiment 1 a complete microscopic examination. Heart, Because the main objective was the deter . lung, liver, spleen, urinary bladder, testes, mination of the effect of long term exposure ' .j- prostate, ovary, brain, spinal cord, pituitary, to vinyl chloride, the maximum concentra tibia, pancreas, stomach, small and large tion of the gas to which humans might intestine, adrenals, kidneys, uterus, fallopian conceivably be exposed without any imme tubes, thymus, thyroid, parathyroid, eye, knee diate acute effects was determined; this con joint, bone marrow, skeletal muscle, salivary centration then became the basis for deter 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 formalin fixation. Following fixation, representative samples of all tissues were processed according to stand mining the concentration used during the 92-day exposure of rats. Three men (26, 35, 50 years; 86, 78, 73 Kg.) and three women (25, 40, 55 years; 64, 52, 61 Kg.) were exposed twice each day, separated by a 6-hour interval, for ard histological procedure and stained with three successive days to six different con hematoxylin and eosin; separate specimens centrations of vinyl chloride: 0.0, 0.4, 0.8, of liver were stained for fat with the Flam 1.2, 1.6 and 2.0%. The concentrations were ing Red technique. The animals receiving presented in a different order to cach^^B- such a complete examination of the tissues ject to make it possible to factor out^Hy listed included two males and one female rat possible adaptation to either the gas or the that had been exposed 15 times and one experimental situation; the 0.0% concentra female rat exposed ten times to the 8 to tion .was included so that some assessment 10% level; eight experimental and nine of suggestibility could be made. control animals surviving the 5% exposure; Until its conclusion, the subjects were told and 20 randomly selected rats, equally di vided as to experimental and control groups and to sex, exposed to 2% vinyl chloride. neither the effects to expect from the expos ure nor the purpose of the experiment; nor was information vouchsafed as to the con The marked increases in liver weight ac centrations that were used at any time. The companying the exposures to 2% and 5% subjects each sat in a chair separated from of the gas, the decrease in spleen weight in the gas mixing equipment by a screen, a the 2% expsoure and the reported kidney simple plastic breathing mask affixed over changes in exposure to 500 ppm1 led us to the face, covering the mouth and nose. The examine these particular tissues with a blind rate of air or air-gas mixture passed through technique, thus excluding die operation of the mask was sufficient (50 1pm) to prevent any bias or prejudice from the judgments. any dilution effects from the atmosphere. All slides, including duplicates, were ran After five'minutes of breathing the mixture, domly numbered so that it was impossible the exposure was terminated and the sub to distinguish, without the code, which had jects were asked to compare their feelings been experimental and which control. The at this time to the time immediately vV; Industrial Hygiene Journal 267 prior to putting on the mask; no suggestions of any kind, were made. The responses of the subjects are summarized in Table II. It is apparent that the maximum concentra tion causing no effect in any subject lies be tween 0.8 and 1.2%. From the responses it is evident that vinyl chloride causes clearcut intoxicating symptoms which can serve as adequate warning signs of its presence. thesia for two hours; on removal to air, there was an uneventful and prompt recovexy. Experiment 3 Some notion of the distinctive and specific pathology that might be caused by vinyl chloride was our object in exposing rats ter a concentration of 10%. Taali II Response] of Human Subjects to Varying Concentration] of Vinyl Chloride Per Ceot CoocentTitioa Refpmia 0-0 No differences reported by Subjects 1, 2, 4, 5 nd 6. Subject 3: "slightly diny". 0.4 No differences reported by subjects. 0:8 No differences reported by Subjects 1, 2, 4, 3 and 6. Subject 3: "slightly heady1'. {.2 No differences reported by Subjects 1. 3, 4 sad 5. Subject 2 unsure, somewhat dizzy in middle of exposure. Subject 6, reeling, swimming head, "just Hie getting 1.6 No effect reported by Subject 5. Afl other* report various degrees of (otaxicsiion with dixrineu, light* headedness, some uusCai, dulltog of visual end audi tory cues; these tymptoms disappeared rapidly upon termination of the exposure. 2.0^Mhd subjects reported intoxicating effects, Subject 1 ^^^Borttng a heedsrhc that persisted for 30 minutes. ^^HHese tymptoms appeared earlier in the exposure ^^mthaenn xatt 11-6.6%%. sod the symptoms were more intense Experiment 2 To gain further insight into the intoxicat ing effects of vinyl chloride rats were ex posed to varying .concentrations of vinyl chloride for periods up to two hours. The effluent gas from the mixing chamber, at the desired concentration, was passed through a 10-liter all-glass exposure chamber con taining two rats. At a 5% concentration in toxication is moderate but the righting re flex is lost; intoxication is more intense at 5% but the righting reflex is still present. This reflex is lost at a concentration of 7 %; the comeal reflex disappears at a concentra tion of 10%. On removal from the chamber, the animals return to the pre-exposure state 'apidly. One animal was sacrificed after ex posure to the 10% concentration and showed io visible gross pathology. Exposure to a toncentration of 15% resulted in deep anesheria within five minutes. Effusion of fluid rom the mouth preceded respiratory failure n one rat ei'.rr 42 minutes; autopsv revealed tdcnu^^id (vogestion of the lungs. The sectnd ^^vas maintained under this deep anes 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 1pm. 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; consequendy, be ginning 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 feniale rats were thus alive at the end of the fifteenth exposure. Female rats exposed 10 and 15 times were autopsied r &S 136109 jt ' i7 j d.1 t'-V. -*..fe .t. ' `f*5 '. . 265 May-J une^^63 Tabls III Clarification of Morphologic Changes in Liven CLu* Description 1 No fwtllinff, no vacuoles, linuaoidi visible. 2 Slight swelling of cells, only & few (lint vacuoles or none, sinusoids visible hut compressed. 3 Moderate swelling of celts, most cells with definite fine to medium vacuoles, sinusoids compressed, 4 Marked swelling of cells, Large irregular "vacuoles" or clear spaces, compression of sinusoids* Changes focal Ln distribution. 5 Sdiifmfuislaer. to 4 but changes more widespread and Table IV Classification of Morphologic Change* in Kidneys CLlu Description 1 CetU well pftxinxd, no vacuoles, glomeruli norm*!. 2 Vacuolization and "pykoosli" oi tome collecting tubule*. Otherwise u 1, 3 A* 2 but with more extensive vacuolization v>d "pvknoeis" including proximal collecting tubuJcs in cortex. 4 As 3 but with vacuolixatsoa end "pytaos'u" extend ing to convoluted tubules- at the termination of the exposure; the re maining six rats were autopsied 15 days later, five of these rats having been exposed 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 tennination of the exposure, growth resumed promptly at the same rate as the control animals. Both at the 16th and at the 30th days, 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 tennination 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- v mental and control animals. , The lungs of three experimental animals sacrificed im mediately upon cessation ` of the exposure had numerous focal fibrinous pleural exu\ v 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 interpreted as representing regions of healed pneumonia. All ocher organs and tissues were within normal limits, with no differences between experimental and control animals. The experimental animals sacrificed at the termination of the exposure received a com plete histologic examination. The lesions in the lungs of the three animals in this group showing gross pleural pathology were seen to be due to acute focal necrotizing pneu monia in varying stages of organizauon. Some pulmonary edema was present as well as diffuse infiltrates of mononuclear cells in the alveolar septae. Some areas of tnctaplasia were present in and near the r^^fts of pneumonia and were interpreted as s^^mdary to the pneumonia. The parasidc liver cysts seen grossly were confirmed microscopically. The liver sccdons stained for fat revealed individual variation of some degree but no significant deviation from normal. The coded slides of the livers were classed according to the descriptions in Table III while kidney and spleen were judged according to the criteria in Tables IV and V; Figures 1, 2, 3, 4. and 5 are Table V Classification of Morphologic Changes in Spleen CUu Description1 2 3 * * 6 1 Average loffklei, liule or no congeitioa, b*Un between Irtapbocytei, germinal epithelium and imer- ftitsxl trnuc. 2 Obvious congestion. do change in loUklo or epiiheli- ' tun. volume of germinal center* leu thin volume of peripheral Lymphocyte*. 3 G-erraiaal center* approximately time volume u peri- fherJ lymphocyte*, moderate cooteitioo, acre number of Lymphocyte*, hyperplasia oi ger-mlaa! centen (greater than volume of peripheral brmphocrtetl. 3 Active hyperplasia: 2 flam-* of Nmphoeytet, (!) a xone immediately around terrain*! Center* containing young lymphocytes rurrouoded by (2) * peripheral tone of small dark Lymphocyte*. 6 Lymphocytic hyperplasia extending to ioeludc moat of the iotentitium. Industrial Hygiene Journal 269 Fioukes 1-5. Rat liven, illustrating the morphologi cal classes described in Table lilt (1) Clan 1; (2) Clan 2; (3) Clan 3: (4) Clan 4; (5) Clan 5, H & E itain. a 200. illustrative of the class descriptions in Table III. There were no significant differences in (flooring of the groups, although there was^HRass "5" and no class "3" in the rats killed at the termination of the exposure, whereas there .were no livers in class "5" among the control animals. There were no class "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 ex perimental animals received the highest score, differing significantly from the con trol spleens, although some controls also' received such high scores. Experiment 4 These preliminary tests seemed to indicate that vinyl chloride was an anesthetic gas which might also act as a lung irritant. In jrder to assess this feature of its action, to lighlight significant pathological features and o avoid .the potentiality of damage arising ;rora anesthesia alone, five female and five nale rats, matched with controls, were cx5o*-d 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 growth 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- R&S 136111 270 May-Jun Table VI Formed Elements of the Blood at Termination of Exposure to 5 and 2% Vinyl Chloride ,*; ri*-. ' ' - -) 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 0AM sodium oxalate was added to the blood. The ani mals were then killed with an overdose of the anesthetic and autopsird. Measurements of hemoglobin, prothrombin time, hemato crit, red cells, white cells, differential white cells and serum 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 the white cells; no differences between control and experimental animals occurred. The red cells were somewhat elevated and the white cells lower in the experimental group. Table VII shows that the Hver: body weight ratio of the experimental animals was significantly elevated. All five male experimental animals ec.x\- posed to 5% vinyl chloride had coats were somewhat thinner than normal; th^ of these animals were scaly. The thrreeee"temale experimental animals and all the con trol animals had normal coats and tails. One male experimental animal had fibrous pleural adhesions on the left side; the fibrous nature of these adhesions suggested that the Table VII Liver and Spleen Weights of Rats Exposed to Vinyl Chloride Croup Control ExptL Control ExptL Control ExptL Control Expel, Control* ExptL* Control* ExptL* Vpof Cone. (%) 1----------------------- Dy Number of AnimtU Expt. - * 0 2 ' 0 . '2 0 -v *0 '* ft 0' 8-10 0 ft~10 89 ` 92 , 4 14 . u- 89 * 12 92 - 15 19 - ^ i ' 19 . s 19 ' 19 , : See Text 4 '. * `4 4 See Text S 4 Sts r F U M F T M M '? F M M % of Body Weight Liver SfAwa U*ftn S.D. Mm a d. 9.4ft 4,79- 0.34 0.90 0.49 Q.t4 O.lflO 0.068 8.73 4.74* 0.19 0.30 0.38 0.384 0.0&2 0.060 a, io 8 96- 0,23 0.61 5.17 4 71* 9.44 0.40 4.98 5.13* 0.20 0 35 5.45 5.09* 0.57 1 03 pastured for t*o "'V'CO.QS *V<0.02 a**p" sol tifniftcAfit p--.;. ' . -;.r: - Vs- : 'V. j-1- ' - .v.r - . , V. : - Industrial Hygiene Journal 271 process was several weeks old and probably chamber to 2.0% vinyl chloride for eight not related to the exposure. Both experi- hours per day (0830 to 1630) on Monday f mental and control animals had parasitic through Friday for a period of three months. I liver cysts. No differences in appearance, The control animals were exposed to 0.0% [ color, consistency or degree of congestion of the test gas, that is, to a flow of 50 1pm | were noted between the livers of the two of air, in the same chamber as the experi 'groups. The other organs and tissues were mental group for eight hours per day on - within normal limits in their gross appear- the same days of the week. No food or water . ance, with no differences between experi was present in the cages during either ex mental and control groups. posure. All rats were weighed at approxi With the exception of the pleural adhe mately weekly intervals; hemoglobin deter sions in one animal, the microscopic appear minations, from tail blood, were made at ance of all the organs and tissues was nor monthly intervals. In neither body weight mal. The gross observation of parasitic liver nor in hemoglobin values were there any sig cystes in all animals was confirmed micro nificant differences between the control and scopically. Liver sections from all animals experimental groups. were stained for fat, but none revealed evi dence of increased fat nor were there any differences in'intracellular fat between the two 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. Classification of the liver slides for the morphological designations of Table III showed differences between the control and exp^^ntaJ groups. The mean score of the cont^^group was 2.93 and that of the experimental group was 4.56, with only one control animal being graded "4" and no experimental animal being graded less than `4". The differences between the means was thus highly significant, yielding a "p" if less than 0.001. No differences in kidney or spleen slides Mttwecn experimental and control animals vere noted. 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 Zxpcrrmrnt 5 values for hematocrit and prothrombin. The Because human exposure to vinyl chloride eems unlikely at concentrations of the gas auch greater than the concentration causing igns of intoxication, that is, at 1.2 to 1.6%, serum 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. he long term exposure of rats was conducted The external appearance of all animals t a concentration of 2.0%. was normal. Parasitic liver cysts were pres Sixty rats, each weighing about 75 grams, ere separated randomly into two groups of 5 males and 15 females and placed in eight parate cages. In the week before the exisure was- started, the rati were observed, eighed twice and blood withdrawn for ent in all animals. There were no differ ences in appearance, color,, consistency or degTee of congestion between the livers of the two groups. All other organs and tis sues were similarly normal, no differences between the groups being apparent. rmoglobin determination. The experimen- All the organs and tissues examined histo were exposed in the 1100-liter logically were within normal limits, no path- 1 r . _' < J. - T , , : '` -* * .` v * .-f* 'r '' Da w CO a> --1. to 'f V :.y - . *: - * .pr-v-p- - , -lilJ'nv-'- y.: . iMMi-fr - , , * -i w . . V f'l - .. : *. . - -Jvr .'; it-- \ >/* f: y ; - ' L.t ys v ; ' \' . V& i'ir i 272 May-June, ology being evident in either experimental to produce some singular or characteristic or control animals. pathology. It cannot be said that this goal The parasitic liver cysts seen grossly were was achieved, the results being questionable confirmed microscopically. Liver sections and uncertain. Lung lesions were certainly stained for fat revealed normal variation, but present but these could not be ascribed with no animals had increased intracellular fat nor certainty to any irritant properties of the gas were there differences between the experi since they might well have arisen from the mental and control animals. Graded in long-continued anesthesia; there was an ap accord with the morphology in Table III, parent regression of these lesions in rats al the liver slides revealed differences between lowed a 14-day recovery period. The pres the groups. The mean score of the control ence of pneumonia certainly raises the pos group was 1.58, that of die experimental sibility of an acute toxic effect on lung tis group 3.63. No liver in the control group sue at these concentrations; however, the scored more than 2 and none in the ex pneumonia could just as well be caused by perimental group scored less than 3; one liv secondary infection during the severe cen er scored 5. Because there was no overlap tral nervous system and respiratory depres the differences between the means are high sion, an interpretation favored somewhat by ly significant. the diffuse lesions and by the irregular oc There were no differences in score for the currence of the pneumonia. spleens in the two groups, but the 'kidney The kidney and liver changes described slides of the experimental animals scored by Mastromatteo, et air in rats exposed for significantly less than their controls. 30 minutes to 20, 30 and 40% of vinyl chloride, were not observed here, although Discussion 15 repeated 8-hour exposures to an anesthe tic concentration is also a relatively severe The data from the present investigation stimulus. The findings in the lungs a^j^ confirm the acute effects to be expected from with the relative lack of effect founc^^B various concentrations of vinyl chloride: Mastromatteo et al, in rats exposed foraU concentrations below 1%, when exposure minutes to 10% vinyl chloride, except that is limited to five minutes, cause no ob continued exposure, for days, does result in servable intoxicating effects; five minutes, mortality. The relative lack of pathology as however, is shorter than the time necessary the result of 30 minute exposure at 10% to reach an equilibrium level in the circula found by Mastromatteo et al. would seem tion ; from behavioral observations in the to support the view that lung lesions found rat, it may be estimated that in five minutes after repeated exposures could well arise some two-thirds of the equilibrium level is from the anesthesia and not from some ac reached; consequently a concentration of 0.6 tion peculiar to vinyl chloride. There was to 0.7%, if long continued would not pro no indication at either the 2% or 5% level duce intoxication. As the concentration rises of any untoward or other effects upon the above this level the intensity of the intoxicat lung tissue. ing signs increases until at a concentration of As an overall measure of general health, 7% the righting reflex is lost; at 10% the body weight and rate of growth are sensitive comeal reflex disappears and at concentra indicants. Even in the exposure to a con tions of 15% and above respiratory failure centration of 5%, which at first produces a takes place. Vinyl chloride thus acts as an marked and severe intoxication, the sharp anethetic gas, its depressant action increasing drop in the weight of male experimental with increasing concentration of the gas in animals seen at the start of the exposure was the air breathed, the corresponding increas soon reversed so that by the end of the 19- ing neurological deficits ending in death at dav exposure there was no difference be concentrations greater than !5%. The pur tween experimental and control rats of pose in exposing rats to concentrations caus either sex. Body weight and rate of growth ing anesthesia (8 to 10%) was an attempt similarly showed no differences between the JntKtrial Hygiene Journal 273 groups in the 2% exposure. It must be em view of the wide range that the liver: body phasized again that the growth of the rats weight ratio may encompass (Table VII). during the 5% exposure support the observa Rather this increase may signify a non-spe tion of the rat's behavior which indicated cific response to metabolic derangements oc that there was a rapid development of toler casioned by mild and moderate intoxication ance to the intoxicating effects of this con for daily 8-hour periods. From the data ob centration. tained here, it is not certain that histopatho- Neither the 2% nor the concentrations logical change would have occurred had ex caused changes in the prothrombin time, posures been carried out for longer times. trt-matocrit or hemoglobin values. At both In the paper by Torkelson et a!., histo- ihe 2'7c and the 3% concentrations, the pathological changes in the liver and in white cell count was lowered significantly, creased liver: body weight ratios ate re although still well within the normal range. ported in male rats exposed to 500 ppm The increase in red cells, although signifi vinyl chloride for 4.5 months. That there is cantly elevated in the 5% exposure, was not no causal relation between the reported his- correlated with changes in the hemoglobin topathology and the increases in liver:body content. The increase in concentration of weight ratio is evident from the extensive the vinyl chloride is associated with a greater data gathered by these investigators. Female tall in the white cells and a greater (and rats exposed to 500 ppm vinyl chloride for significant) increase in the red cells at the 4.5 months showed no statistically significant level. Although lymphocytes and neu increase in liver weight but are reported to trophil* are increased, only the change at have histopathological changes in the liver. the 2<i cte nitration unlike the previous Female rats exposed to lower concentrations cell changes, reaches the statistically signifi- (100 and 200 ppm) for six months had sig can^Jevel, It ii difficult to know if these nificantly increased liver:body weight ratios cl^^pfs have an) toxic significance, since no but no pathology. On the other hand, rab tissue changes were seen upon microscopic bits of both sexes exhibited liver pathology examination that would account or be as without showing any increase in liver weight sociated with a drop in the white cells or an after exposure for six months to a concen increase in red cells. The decrease in spleen: tration of 200 ppm. The authors correctly body weight ratio is in a direction opposite to point out that organ:body weight ratios may that usually associated with a severe drop well be artefactual. illustrating the point by in white cells; although the white cell count the significant decrease in kidney weight did not suffer a severe drop, the decrease was substantial at the termination of the ex posure. found in female rats exposed to 50 pptn for six months; such rats exhibited no changes when exposed to higher concentrations. By The only finding suggesting a specific toxic action of vinyl chloride is the increase in liver weight on exposure to 5'yo for 19 days and to 2% over 92 days. The increase in liver weight is not only highly significant statistically but is also substantial, amounting to a 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 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 sta tistically 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. alterations in water, electrolytes and protein There are six possible combinations of the conti-nt of the liver parenchyma, but that presence or absence of pathology and in such i fianget presage the development of creases, decreases and no change in liver actual timologic lesions is not certain in weight. The one combination not observed 136115 (/) by Torkelson, et al. is histopathological 40 times those used by Torkelson. et al. If change associated with a decrease in liver any reliance is to be placed in a dose-effect weight. Obviously, if five of the six possible relationship, pathology of some considerable combinations have been observed in a rela degree should have been found in our ex tively small sample, no causal connection periments. Yet only morphological altera can be said to exist between these two tions of the character already described and measures. pictured were seen, which, in our knowledge Changes in the liver:body weight ratio may well have some toxic significance, but if they are unaccompanied by histopathological alterations, increased fat content or serum transaminase changes it is impossible to conclude that taken alone they signify and experience, are of no pathological signi ficance, Whether the explanation resides in a difference between the rat strains uni^- by us and by Torkelson et al. or elsewh^^. is not known, but without additional data it is impossible to resolve the contradiction. much. Reference to Table VIT will show On the basis of the present data, and the that the liver:body weight ratios of the rats seeming unimportance of the liver weight used as control animals in the 5% exposure changes seen by Torkelson et al., and with were significantly higher than the experi out further evidence, a change of the pres mental animals in the 2% exposure. This ent threshold limit value of 500 ppm seems illustrates well the fact that though control unwarranted. animals are used, unknown and non-specific changes in the environment, time of the year, Summary 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. From 5-minute exposures of human sub jects to concentrations of vinyl chloride rang ing from 0.0 to 2.0%, it is estimated that a prolonged exposure to a level of more than 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 cither the 2% or 5% level between the experimental and control groups. 0,6% is necessary to produce minim symptoms of intoxication. Rats exposed up to two hours to higher concentrations ex hibited moderate intoxication at 5%, lost their righting reflex at 7% and the corneal reflex at 10%. Respiratory failure occurred at 15%. If the exposure to 10% was long Unlike the interstitial and tubular changes in kidneys of rats exposed to 500 ppm of vinyl chloride reported by Torkelson, et al., was the lack of any pathology in our animals exposed to 2% and 5% and the fact that the only morphological alteration in which a continued (two 8-hour daily exposures), mortality increased; death was apparently caused by a pneumonic process, but it was impossible to decide whether this was the result of a primary action or secondary to the anethesia. significant difference between control and experimental animals occurred (kidney at 2%) indicated that the control animals were further from "normal". 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- Rats exposed eight hours daily to 5% for 19 days or to 2% over 92 days did not show any lung involvement. These levels had no effects upon growth rate, hemoglobin, hema tocrit or prothrombin time. At both con centrations there were increases in the liver: body weight ratio and decreases in the white cells; at 2% the spleen:body weight ratio decreased and at 5% there was an increase in red cells. The concentrations to which the rats were No gross or microscopic changes were subjected in these experiments were at least found in any tissue that was correlated with >. . - ri V =*. - Industrial Hygiene Journal l 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 sulm; of 500 ppm for vinyl chloride, one-fortieth of the concentration tested here, seems to offer an adequate margin of safety for human exposure. 275 Acknowledgment It is a pleasure to acknowledge the tech nical assistance of Malcolm Nicholson, Frederick A. Putt and Miss Lillemor Wall- mark. References 1. von OrmNOCN, W. F.; The Halogenated Hydro<rhoru: Toxicity and Potential Dangers. U. St Public Health Service. Publication 414, Government Pnucifls Office, Washington, D. C. (1953). 2. MASntQXornQ, ., A. M, Fisk**, H. CmtiATtE, and H. Danzige*: Acute Inhalation Tonicity of Vinyl Chloride to Laboratory Animals. .-fmrr. Ind. Hyg. Asfoc. J. 21: 394 (Oct. I960). 3. Toaic.tL.AON, T. R., F. Oven, and V. K. Rdwp.: The Toxiaiy of Vinyl Chloride os Determined by Repeated Exposure, of Laboratory AnimaU. An4r, Ind. Hyg, Ajsoc. /, 22: 354 lOet 1961), 4. Smy'tk. H. F.f Jn.* Improved Communication--Hy giene Standards for Daily Inhalation. Amer, lnd. HitAssoc. j. li\ 129 M9a6). 3. PvTTY, F. A., W. P. Yant, and C. P. Wwrt: Acute Response of Guinea Piifs to VaDors of Some New Com* mrrcial Organic Compounds. V. Vinvl Chloride. Pub lic Health Reports (Qj*.) 45: 1963 /Au*. 1930). R&S 136116 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 (>nchiota (^inference Center, Sterling Forest, Tuxedo. New York. Tlie 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 arc 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) 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).