Document Dv58O4Bb81MYy15GmMn818Gbn

Effects of Single and Repeated Exposures of Humans and Rats to Vinyl Chloride D. TESTER, l'li.l).,* I.. A. GREENBERG, l`h.1).,* ami W. ROBERT ADAMS, M.D.f Laboratory of Applied Biodynamics, Yale University and Department of Pathology, School of Medicine, Yale University, S'eiv Haven, Connecticut (of Rais exposed eight hours daily to vinyl chloride at concentrations of 2% for three months and 5% 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, scrum transaminase and hemoglobin, were unchanged, it is suggested that the present threshold limit value of 0.05% need not be lowered. Introduction rTHE TOXICITY of vinyl chloride has been reviewed recently.1,2'3 Because the threshold limit value of 500 ppm is based on limited data* using vinyl chloride less pure than that now obtainable5 the effects of acute exposure in man and rats and long term exposure in rats was investigated by us in 1959 and is described here. Our data do not support the conclusion of Torkelson, vt al,3 that the threshold limit value should be reduced ten-fold; indeed their data indi cate a need for further studies prior to any revision of the threshold limit value. together with a trace of air and carbon dioxide. Tam.?. I Analysis of Vinyl Chloride Monomer Acid Acetylene Aldehyde Iron Sulfur Phenol Non. Vol. Water Color Assay 2.2 ppm 0.0 1.9 ppm 0.0 0.0 60 ppm It ppm 170 ppm w.w 99+% Methods Experimental Material The vinyl chloride monomer was supplied by the Perkins Plant of Solway Process Di vision, Allied Chemical Corporation, in four 100 lb. cylinders with the characteristics shown in Table I, presumably differing ma terially from the vinyl chloride used by Torkelson, el al.3 only in the presence of 60 ppm of the inhibitor, phenol. Gas chro matography of the liquid phase indicated the presence of more than 99% vinyl chloride, Prt^entrc! at the Twenty-third Annual Meeting of the American Industrial Hygiene Association, Washington, D. ('.. May, 1962. This work Mas supported in part by a research grant from Allied Chemical Cnrj>oratiM and in part by U. S. Public ilraltlt Sets iet* Grant C-273H. *l'ieM-itt address: biochemistry and Physiology, Nelson biology l.ahoiaiory, Ruigers-Thc State University, New brtmsMick, New Jersey. * Recipient of (/. S. Public Health Service Senior Research Krllowkhip.No. 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 (lptn) in all but one of the experiments. Five experiments were conducted: (1) a fivc-minutc 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 266 May-Junr, 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 recoded and days and 2f/c for 92 days. re-examined; in this manner a measure of the consistence and reliability of the pathol 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 ease of liver slides, and only slightly less so for 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 cfTcct of long term exposure 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 mining the concentration used during the animal and preserved in 10% buffered for 92-day exposure of rats. malin. All tissues were examined grossly Three men (26, 35, 50 years; 86, 78, 73 both at autopsy and after formalin fixation. Kg.) and three women (25, 40, 55 years; / Following fixation, representative samples of 64, 52, 61 Kg.) were exposed twice each all tissues were processed according to stand day, separated by a 6-hour interval, for ard histological procedure and stained with three successive days to six different con hematoxylin and cosin; separate specimens centrations of vinyl chloride: 0.0, 0.4, O.fl, 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 each sub such a complete examination of the tissues ject to make it possible to factor out any 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 neither the effects to expect from the expos vided as to experimental and control groups ure nor the purpose of the experiment; nor and to sex, exposed lo 2% vinyl chloride. was information vouchsafed as to the con 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% expsotire and the reported kidney changes in exposure to 500 ppnv1 led us to examine these particular tissues with a blind technique, thus excluding the operation of any bias or prejudice from the judgments. centrations that were used at any time. '1 he subjects each sat in a chair separated from the gas mixing equipment by a screen, a simple plastic breathing mask affixed over the face, covering the mouth and nose. 'I he rate of air or air-gas mixture passed through the mask was sufficient (50 lpm) to prevent 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 lime iimucdialely SPI -12754 luilustrial Hygiene Journal 267 prior to putting on the mask; no suggestions of any kind wen: made. The responses of die 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. Table II Responses of Human Subjects to Varying Concentrations of Vinyl Chloride Per Cent Concentration Response 0.0 No differences reported by Subjects 1, 2, 4, 5 and 6. Subject 3: "slightly dizzy". 0.4 No dill't-micc* reported by all subjects. 0.8 No differences reported by Subjects 1, 2, 4, 5 and 6. Subject 3: "slightly heady". 1.2 No differences reported by Subjects 1. 3, 4 and 5. Subject 2 unsure, somewhat dizzy in middle of exposure. Subject 6, reeling, swimming head, "just like netting gas". 1.6 No effect reported by Suhjrct 5. All others report vaiimis degrees of intoxication with dizziness, light* headedness, some nausea, dulling of visual and audi tory curs; theie symptoms disappeared rapidly upon in initiation of the exposure. 2.0 All subjects reported intoxicating effects. Subject 1 reporting a headache that persisted for 30 minutes. These symptoms appeared earlier in the exposure than at 1.6% and the symptoms were more intense than at 1.6%. 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-litcr 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 6% but the righting reflex is still present. This reflex is lost at a concentration of 7%; the corneal reflex disappears at a concentra tion of 10%. On removal from the chamber, the 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 anes thesia within five minutes. Effusion of fluid from the mouth preceded respiratory failure in one rat after 42 minutes; autopsy revealed edema and congestion of the lungs. The sec ond 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, 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 female rats were thus alive at the end of the fifteenth exposure. Female rats exposed 10 and 15 times were autopsied SPI -12755 A 1 4 4J 1 1 -I \ i .1 ;t ,1 i 0 i i > 268 May-]unc, 1968 Tam.k III (Ins'ifiration of Morphologic Changes in Livers C'Ium Description 1 No swelling, no vacuoles, sinusoids visible. 2 Slight swrlling of cells, only a few faint vacuoles or none, sinusoids visible hul roinpiessrd. 3 Xiodcratc swelling of cells, most cells with definite fine to medium vacuoles, sinusoids compressed. 4 Marked swelling of cells, large iriegular `'vacuoles" or elrar spaces, compression of sinusoids. Changes focal in distribution. 5 Similar to 4 but changes more widespread and diffuse. Table IV Classification of Morphologic Changes in Kidneys Class Description 1 Cells writ preserved, no vacuoles, glomeruli normal. 2 Vacuolization and "pykuosis" of some collecting tubules. Otherwise as I. 3 As 2 hut with more extensive vacuolization and "pyknosis" including proximal collecting tubules in cortex. 4 As 3 but with vacuolization and "pyVttosM" extend ing to convoluted tubules. pleural adhesions which were interpreted as at tlic termination of the exposure; tlu- re representing regions of healed pneumonia. maining six rats were autopsied 15 days All other organs and tissues were within later, five of these rats having been exposed normal limits, with no differences between 15 times and the sixth rat 13 times. The experimental and control animals. mortality was greater among the male rats: The experimental animals sacrificed at the only two males survived 15 exposures, the termination of the exposure received a com remaining males, and their replacements, plete histologic examination. The lesions in surviving only an average of eight exposures. the lungs of the three animals in this group The two male rats exposed 15 times were showing gross pleural pathology were seen autopsied at the termination of the exposure. to be due to acute focal necrotizing pneu There was no weight gain in the initial monia in varying stages of organization. days of the exposure to this concentration, al Some pulmonary edema was present as well though after the ninth day some resumption as diffuse infiltrates of mononuclear cells of growth seemed to occur, possibly an in in the alveolar septae. Some areas of meta dication of the development of tolerance to plasia were present in and near the regions the effects of the gas as the exposure con of pneumonia and were interpreted as secon tinued. Upon termination of the exposure, dary to the pneumonia. 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 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. The parasitic liver cysts seen grossly were confirmed microscopically. The liver sections 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 About a third of the animals had parasitic liver cysts. No differences in appearance, color, consistency or degree of congestion Table V Classification of Morphologic Changes in Spleen were observed between the livers of experi Class Description 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 1 Average size follicles, little or no congestion, balance between lymphocytes, germinal epithelium and inter stitial tissue. . , ,. 2 Obvious congestion, no change in fnllsnes or epitheli um, volume nf germinal centers lew titan volume of peripheral lymphocytes. 3 Germinal center* approximately same volume as peri- IiheTa! Ivniphocyte*, moderate congestion, nrreased mnnl>er of lymphocytes, hyperplasia of germinal center* (greater than volume of peripheral lymphocytes). 5 Active hyperplasia: 2 classes of lymphocyte*. (1) a zone immediately around germinal center* containing young lymphocyte* smrounded hv (2) a peripheral zone of small dark lymphocytes. 6 Lympliocvlic hyiwrplasia extending to include most of the inteistitiimi. SPI -12756 Imlusl rial Hygiene Journal 269 Figures 1-5. Rat livers, illustrating the morphologi cal classes described in Table III: (1) Class 1; (2) Class 2; (3) Class 3: (4) Class 4; (5) Class 5. II & E stain, x 200. illustrative of the class descriptions in Table III. There were no significant differences in the scoring of the groups, although there was a class "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 order to assess this feature of its action, to highlight significant pathological features and to avoid the potentiality of damage arising from 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 present 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 Ipm. 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 cx- > SPI -12757 270 May-June, 1963 Table VI Formed Elements of the Blood nt Termination of Exposure to 5 and 2' ,'- Vinyl Chloride Control--6% (9) KxpU. -- () Ml" 1cm than Control -2% (20) KxpU.--2% (29) `V less than nnc XlOVinm1 Mean S.D. 5.13 0.49 5.87 0.76 0. OS 6.60 0.83 6.82 0.83 NS wnc X lO'/mm* Mean S.D. 12.67 2.75 7.96 1.85 0. 01 11.34 2.63 9.12 3.08 0. 01 Lymphocytes Moan 89.4 S.D. 3.7 92.3 8.6 Ns 84.8 4.80 89.7 3.64 0. 01 Neutrophils % Mean 8.D. 9.4 3.2 6.8 8.1 Ns 12.62 4.71 8.02 4.94 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 0.1 M 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 serum transaminase were performed on the blood drawn. Scrum 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 liver: body weight ratio of the experimental animals was significantly elevated. All five male experimental animals ex posed to 5% vinyl chloride had coats which were somewhat thinner than normal; the tails of these animals were scaly. The three fe male 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 Group Vapor Cone. (%> Days on Kxpt. Numlier of Animals Sex Liver % of Body Weight .. - . Spleen Mean S.D. Mean S. D. Control Kapil. 0 89 14 r 3.65 0.36 0.49 0.160 2 92 14 F 4.70* 0.30 0.38< 0.068 Control Kapil. 0 X9 12 M 3.73 0.19 0.38 0.052 2 92 15 M 4.74* 0.30 0.33*1 0.050 Control Kapil. n 19 5 F 6.10 0.23 5 19 3 F 6.95* 0.51 Control KxpU. 0 19 r> 19 4 M 5.17 0.46 5 M G 71** A.40 <> k r* 1`iihtrcil* i < , Kxptl.* KtU i __ _ .......... .... *rAturt`<i fur two wtrk* <H| A. T /* Kov Toxl ___________ 1 * i 4 J__ _ ____ V 4 UH V % IV im M 1______________ **p*,<0.05 rf`V'l.02 5.46 6 09* ...... U v.H i K\ \ 0 57 1.01 ii ----------- -------- SPI-12758 J Industrial Hygiene Journal 271 process was several weeks old and probably chamber to 2.0% vinyl chloride for eight not related to the exposure. Doth experi- hours per day (0(130 to 1630) on Monday mental and control animals had parasitic through Friday for a period of three months. liver cysts. No diHerences in appearance, The control animals were excised 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 cither ex mental and control groups. posure. All rats were weighed at approxi With the exception of the pleural adhe sions in one animal, the microscopic appear ance of all the organs and tissues was nor mal. The gross observation of parasitic liver cystcs in all animals was confirmed micro scopically. Liver sections from all animals 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. 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 experimental groups. The mean score of the control 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" of less than 0.001. No differences in kidney or spleen slides between experimental and control animals 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 were noted. the control animals. No significant differ ences between the groups appeared in the Experiment 5 Because human exposure to vinyl chloride seems unlikely at concentrations of the gas much greater than the concentration causing signs of intoxication, that is, at 1.2 to 1.6%, the long term exposure of rats was conducted at a concentration of 2.0%. Sixty rats, each weighing about 75 grams, were separated randomly into two groups of 15 males and 15 females and placed in eight separate cages. In the week before the ex posure was started, the rats were observed, weighed twice and blood withdrawn for hemoglobin determination. The experimen values for hematocrit and prothrombin. The 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. 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 the 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 tal animals were exposed in the 1100-liter logically were within normal limits, no path SPI -12759 272 May-June, 1963 y ology being evident in cither 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 the 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 arc 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 al.2 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 agree confirm the acute effects to be expected from with the relative lack of effect found by various concentrations of vinyl chloride: Mastromatteo et al. in rats exposed for 30 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 cither 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 lighting reflex is lost; at 10% the body weight and rate of growth arc sensitive corneal 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 antithetic 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 day exposure there was no difference be concentrations greater than 13%. The pur tween experimental and control rats of pose in exposing rats to concentrations caus cither sex. Body weight and rate of growth ing anesthesia (f! to 10%) was an attempt similarly showed no differences between the y SPI -12760 Imlustiial Hygiene Journal 273 ginups in llic 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). (hiring the !)% exposure support the observa Uather this increase may signify a non-spe- tion of the rat's behavior which indicated eilie res|>onse to mrlaliolic derangements oc that there was a rapid development of toler casioned by mild and moderate intoxication ance to the intoxicating ell'ccts 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 5% concentrations logical change would have occurred had ex caused changes' in the prothrombin time, posures been carried out for longer times. hematocrit or hemoglobin values. At both the 2% 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 5c/o exposure, was not In the paper by Torkclson cl al., histopathological changes in the liver and in creased liver:body weight ratios are re ported in male rats exposed to 500 ppm vinyl chloride for 4.5 months. That there is 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 fall in the white cells and a greater (and rats exixised to 500 ppm vinyl chloride for significant) increase in the red cells at the 4.5 months showed no statistically significant 5% level. Although lymphocytes and neu increase in liver weight but are reported to trophils are increased, only the change at have histopathological changes in the liver. the 2% concentration unlike the previous Female rats exposed to lower concentrations cell changes, reaches the statistically signifi (100 and 200 ppm) for six months had sig cant level. It is difficult to know if these nificantly increased liver:body weight ratios f changes have any 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 sociated with a drop in the white cells or an increase in red cells. The decrease in spleen: without showing any increase in liver weight after exposure for six months to a concen 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 found in female rats exposed to 50 ppm for substantial at the termination of the ex six months; such rats exhibited no changes posure. 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% 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 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 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 content of the liver parenchyma, but that presence or absence of pathology and in such changes presage the development of creases, decreases and no change in liver actual histologic lesions is not certain in weight. The one combination not observed J SPI -12761 274 May-June, 1963 by Torkelson, et 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 histopathological alterations, increased fat content or scrum 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 cither 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 reported by Torkclson, ct 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 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- 40 times those used by Torkelson, et al. If any reliance is to be placed in a dosc-cffcct relationship, pathology of some considerable degree should have been found in our expcriincnts. Yet only morphological altera tions of the character already described and pictured were seen, which, in our knowledge and experience, are of no pathological signi ficance. Whether the explanation resides in a difference between the rat strains used by us and by Torkelson et al. or elsewhere is not known, but without additional data it is impossible to resolve the contradiction. On the basis of the present data, and the seeming unimportance of the liver weight changes seen by Torkelson et al., and with out further evidence, a change of the pres ent threshold limit value of 500 ppm seems unwarranted. Summary 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 0.6% is necessary to produce minimum symptoms of intoxication. Rats exposed for up to two hours to higher concentrations ex hibited moderate intoxication at 5%, lost their righting reflex at 7% and the comeal reflex at 10%. Respiratory failure occurred at 15%. If the exposure to 10% was long 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. 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 SPI -12762