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Effects of Single and Repeated Exposures of Humans and Rats to Vinyl Chloride D. LESTER, PUV L. A- GREENBERG, PkD.,* sod W. ROBERT ADAMS, MJJ.f Laboratory of Applied Biodynamics, Yale University and Department of Pathology, School of Medicine, Yale University, New Haven, Connecticut Rat* exposed eight horn* daily to vinyl chloride at concentration* of 2% tor three month* and 5% for 19 day* exhibited change* In liver and spleen weight and in red and white cell count*. Except in the case of liver,, tissue alteration* 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 i* suggested that the present threshold limit value of 0.0596 need not be lowered. Introduction HTHE TOXICITY of vinyl chloride ha* been reviewed recently.1'1-* Because the threshold limit value of 500 ppm is based on limited data4 using vinyl chloride less pure than that now obtainable* 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, et aL* 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. Tails I Analysis of Vinyl Chloride Monomer Acid Acetylene Akkhrd* Iron Sulfur Phenol Non. VoI W,,,, Color Amt 2.2 Ppm 0.0 1.9 Mm 0.0 0.0 0 ppm II 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 mas tcrially from the vinyl chloride used by Torkelson, et al.* 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, Praaootod M tba Twtair-tbird Aoauol Maafcur et Aw Amnicaa Industrial Hvama Awuri--Uo. Wariuuftoa, D. C.. Mar. 1962. Tbit wu sippartcd la pan br a raaaarab grant fnra Allivd Cbmiesl Corporation aod is part br U- S. Public Health Sarvica Gnat C-27SS- -Pru,,i tdbra: Biocbwoirtry ud npiiobe, Nahaa Biolorr Laboratory. Rutgara-Thn Slot* Umviratty, Now Brunt-rich, New Jcnor* 1 tRceipicat et u. S. Public Haaltb Strain Sonin Rorauch Fcilowtbip 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 5Q 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 hour* with concentrations up to 15%; and (3, 4, and 5) 265 ! 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 recoded and days and 2% for 92 days. re-examined; in this manner a measure of the consistence and reliability of the pathol 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 ogist's technique was obtained. This con sistency was nearly perfect in the case of liver slides, and only slightly less so for spleen and kidney. The results of these examina tions are 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 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 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 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 control animals surviving the 3% exposure; and 20 randomly selected rats, equally di of suggestibility could be made. Until its conclusion, the subjects were told 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 to 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% expsoure and the reported kidney changes in exposure to 500 ppm* 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 centrations that were used at any time. The 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. The rate of air or air-gas mixture passed through the mask was sufficient (50 1pm) to prevent any dilution effects from the atmosphere. After five minutes of breathing the mixture, the exposure was terminated and the sub jects were asked to compare their feelings at this time to the time immediately Ucc 002675 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. TasuII Response* of Human Subjects to Varying Concentrations of Vinyl Chloride Per Csat CoMntntidg RpOW 0.0 No diifcrcaev repotted bf Sobjteti 1, 2, 4, 3 ond 6. Subject St "tlifbtit ditty1*. 0.4 Q.t No dinertftoM reported by otf gubraet*. No fUfferaacot reported by Subject* 1, 2> 4* 5 *nd 6- Subject ft Md!(K<ly 1.2 No differences reported by Subjects 1* 3, 4 and 5- Subject 2 UBwn,# mtwlpt ditty it middle i aponirt. Subject 6, reeling, iwimaiotf hood, "hw like cenifii fa*'\ 1.6 No effect reported by Subject 5. AH otJien report vmnoue dfnet of iatocicotiou with dioifteei, light- heededness, some neie>t dulling of viwil and udi* lory cues; these lyapicm disappeared rapidly upoa termination of the spawn. 2.0 AH tvbjeets reported intoxicating effects. Subject 1 reporting t hndsdii that persisted lor 30 minutes. These tvjBptoau appealed rarUer in the expawn then et 1.69k and the symptoms were more intense than et 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-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 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<7c. 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 13% 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-Iiter 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 dally 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 13-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 13 times were autopsied 268 May^Jum, 1963 ' Table III Clarification of'Morphologic Change* in Liven Clin Description 1 No moiling, no vnotolos, limrtAicfi vitihie. 2 Slight iweumi of cdU. only few flint vwioln r none, tiousoim visible but compressed. ... 3 Moderate moiling of cells, most cells with definite fine to medium vaeuoles, sinusoids eompmird. 4 Marked swelling of cells, lirge irregular "vaeuoles" or clear spaces, compression of sinusoids. Changes focal in distribution. .. 3 Similar to 4 but changes aoro widespread and diffuse. Tam* IV ClassificatioB of Morphologic Change* in Kidney* dam DTM=riptjm 1 Cell, mil leeumJ. m naela, flomeruli normal. 2 \ acuoiicntioo and 'pykoan" of mm eoDectie* tubules. Otherwise as 1. 3 As 2 but with more extrmiie WrtdiniioB and "pyknosu" includiBg |inmimal rullsriin* tnbuks in cortex. 4 As 3 but with vaeuolisatioo and ''pyfcoen" estmdinf to convoluted tubules. pleural adhesions which were interpreted as at the termination of the exposure; the re maining six rats were autopiied 15 days later, five of these rats having been exposed 15 times and the sixth rat 13 times. The representing regions of healed pneumonia. All other organs and tissues were within normal limits, with no differences between 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 arc About a third of the animals had parasitic liver cysts. No differences in appearance, color, consistency or degree of congestion Tails V Classification of Morphologic Changes in Spleen were observed between the livers of experi CUas mental and control animal*. The lungs of three experimental animals sacrificed im __________m MHel, littl* Or no cm.winn. boboir between, limphnryff, germinal cpithelium and mtvr- 1 qS&amilwiin. ao efcutfe in foUfclet or epitfarfi- 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 um, volume <d germinal center* lem than value* * peripheral lymphocyte*. , 3 Germinal enter* approximately nm volume at prr* pheral lymphocyte*, moderate comnom. . 4 Increased munhar of lymphocytes, hyparpimo m terminal cmto** (greater than slums of prripfcmi 3 ^Rahypriplw; 2 damn of lymphocyte*. (U cone immediately around germinal enter* nwm young lymphocytes surrounded by (Z) a penphttd sane of smaN dark lymphocytes. 6 Lymphocytic hyperplasia extendi** to include mm of the iuteitririum. ...... .. a*1 * 3 4 * 6 ucc 002677 illustrative of the clan 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 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- 270 MayJune, 1963 TaBLS VI Formed Elements of the Blood et Termination of Expoasr* to 6 and 2% Vinyl Chloride posure period revealed no difference between experimental and control group*. On the twentieth day experimental and control animal* 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 cell*, differential white cell* and serum transaminase were performed on the blood drawn. Serum transaminase, hematocrit value* 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 phil* 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 animal* was significantly elevated. All rive male experimental animals ex posed to 5% vinyl chloride had coats which were somewhat thinner than n ratal; 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 ride; the fibrous nature of these adhesions suggested that the Table VII Liver and Spleen Weight* of Rata Exposed to Vinyl Chloride 002679 Industrial Hygiene Journal 271 process was several weeks old and probably not related to the exposure. Both experi mental and control animals had parasitic liver cysts. No differences in appearance, color, consistency or degree of congestion were noted between the livers of the two groups. The other organs and tissues were within normal limits in their gross appear ance, with no differences between experi mental and control groups. 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 cystes in all animals was confirmed micro scopically. Liver sections from all animals were stained for fat, but none revealed evi dence of increased fat nor were there any differences in intracellular fat between the two groups. 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 4.36, 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 were noted. 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 tal 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. The control animals were exposed to 0.0% of the test gas,' that is, to a How 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 the two groups. All other organs and tis sues were similarly normal, no- differences between the groups bring apparent. All the organs and tissues examined histo logically were within normal limits, no path- 272 May-June, 1963 ology being evident in either experiment*] or control animals. The parasitic liver cysts seen, grossly were confirmed microscopically. Liver sections stained for fat revealed normal variation, but no animals had increased intracellular fat nor were there differences between the experi mental and control animals. Graded in accord with the morphology in Table III, the liver slides revealed differences between the groups. The mean score of the control group was 1.58, that of the experimental group 3.63, No liver in the-control group scored more than 2 and none in the ex perimental group scored less than 3; one liv er scored 5. Because there was no overlap the differences between the means are high ly significant. There were no differences in score for the spleens in the two groups, but the kidney slides of the experimental animals scored significantly less than their controls. Discussion The data from the present investigation confirm the acute effects to be expected from various concentrations of vinyl .chloride: concentrations below 1%, when exposure is limited to five minutes, cause no ob servable intoxicating effects; five minutes, however, is shorter than the time necessary, to reach an equilibrium level in the circula tion; from behavioral observations in the rat, it may be estimated that in five minutes some two-thirds of the equilibrium level is reached; consequently a concentration of 0.6 to 0-7%, if long continued would not pro duce intoxication. As the concentration rises above this level the intensity of the intoxicat ing signs increases until at a concentration of 7% the righting reflex is lost; at 10% the corneal reflex disappears and at concentra tions of 15% and above respiratory failure takes place. Vinyl chloride thus acts as an anethetic gas, its depressant action increasing with increasing concentration of the gas in the air breathed, the corresponding increas ing neurological deficits ending in death , at concentrations greater than 15%. The pur pose in exposing rats to concentrations caus ing anesthesia (8 to 10%) was an attempt to produce some singular or characteristic pathology. It cannot be said that this goal was achieved, the results being questionable and uncertain. Lung lesions were certainly present but these could not be ascribed with certainty to any irritant properties of the gas since they might well have arisen from the long-continued anesthesia; there was an ap parent regression of these lesions in rats al lowed a 14-day recovery period. The pres ence of pneumonia certainly raises the pos sibility of an acute toxic effect on lung tis sue at these concentrations; however, the pneumonia could just as well be caused by secondary infection during the severe cen tral nervous system and respiratory depres sion, an interpretation favored somewhat by the diffuse lesions and by the irregular oc currence of the pneumonia. The kidney and liver changes described by Mastromatteo, et al* in rats exposed for 30 minutes to 20, 30 and 40% of vinyl chloride, were not observed heje, although 15 repeated 8-hour exposures to an anesthe tic concentration is also a relatively severe stimulus. The findings in the lungs agree with the relative lack of effect found by Mastromatteo et al. in rats exposed for 30 minutes to 10% vinyl chloride, except that continued exposure, for days, does result in mortality. The relative lack of pathology as the result of 30 minute exposure at 10% found by Mastromatteo et al. would seem to support the view that lung lesions found after repeated exposures could well arise from the anesthesia and not from some ac tion peculiar to vinyl chloride. There was no indication at either the 2% or 5% level of any untoward or other effects upon the lung tissue. As an overall measure of general health, body weight and rate of growth are sensitive indicants. Even in the exposure to a con centration of 5%, which at first produces a marked and severe intoxication, the sharp drop in the weight of male experimental animals seen at the start of the exposure was soon reversed so that by the end of the 19day exposure there was no difference be tween experimental and control rats of either sex. Body weight and rate of growth similarly showed no differences between the wmmm ucc 002681 Industrial Hygiene Journal 273 group* in the 2% exposure. It must be em phasized again that the growth of the rats during the 5 % exposure support the observa tion of the rat's behavior which indicated that 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, 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 5% exposure, was not correlated with changes in the hemoglobin content. The increase in concentration of the vinyl chloride is associated with a greater fall in the white cells and a greater (and significant) increase in the red cells at the 5% 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 know if these changes have any toxic significance, since no tissue changes were seen upon microscopic 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: body weight ratio is in a direction opposite to that usually associated with a severe drop in white cells; although the white cell count did not suffer a severe drop, the decrease was substantial at the termination of the ex posure. 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 alterations in water, electrolytes and protein content of the liver parenchyma, but that such 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). Rather 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 times. In the paper by Torkelson et 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* topathology 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 but 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 artefactual, illustrating the point by the significant decrease in kidney weight found in female ran exposed to 50 ppm for six months; such rats exhibited no changes when exposed 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 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. 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 274 May-Jvne, 1963 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 ur ex l tively small sample, no causal connection periments. Yet only morphological altera l 4l t can be said to exist between these two tions of the character already described and measures. pictured were seen, which, in our knowledge 1 and experience, are of no pathological signi Changes in the liver:body weight ratio ficance. Whether the explanation resides in may well have some toxic significance, but a difference between the rat strains used by if they are unaccompanied by histopatho- us and by Torkelson et al. or elsewhere is logical alterations, increased fat content or not known, but without additional data it serum transaminase changes it is impossible is impossible to resolve the contradiction. to conclude that taken alone they signify much. Reference to Table VII 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, Summery temperature, diet, etc., may be responsible From 5-minute exposures of human sub i for changes in organ:body weight ratio with jects to concentrations of vinyl chloride rang out at the same time producing pathological ing from 0.0 to 2.0%, it is estimated that a l alterations in the organ. 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 either the 2% or 5% level between the experimental and control groups. 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 Unlike the interstitial and tubular changes in kidneys of rats exposed to 500 ppm of vinyl chloride reported by Torkelson, et til., 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 ancthesia. 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 > *......... him * * 1 *>,**.. % <JCC 02683 1 Industrial Hygiene Journal 275 the change* 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, m the long term exposures, it was 5 . .. 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 Lillemor Wallmark, n. *, nJ O- s. public Hntnvlu Saeermviccei,i Pruuobluicaantiown 414. Govcmncat Priutiag uOmfficeet, Wnansuhinngimton,, uD>. uC. u(19w55i)- 2. MAAmmooMtUATTTito, E., A-. M-. FFii*thK*txt, H. CChwauimL And CHh. lorDidxexttutoon:tUbAwciuMteryInAiufltA_ii_tuiolk_n. JT--Aomxictri,ty tlm.Jod%f VuHinyyt-l Aim. /J.. i2l1l: 394 (Oct. j1y9O6U0J)*. 3- Tooulmn, T. 1C, F. Oykn, And V. K. Row*; The Toxicity of Vinyl Chloride u Determined by Repeated Exposure of tumtoiy Anioik. Am*r. Ind. H-n- Aim. J. 22: 354 (Oct. 1981), ^ 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 widi 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?[round so that all administrative and technical persons having responsibilities or 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) 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). m ucc 002684