Document wDGXRv0dbowDMdQakdB8LQV7Q

021564 (/) VINTL CHLORIDE: RESULTS OF TWO SHORT-TERM INHALATION STUDIES K. A. Johnson and H. 0. Yakel Reviewed by: M. J. McKenna Final P,,eport February 3, 1981 Toxicology Research Laboratory Health and Environmental Sciences, USA Dow Chemical U.S.A. Midland, Michigan 43640 'ABLE OF CONTENTS SUMMARY INTRODUCTION MATERIALS AMD METHODS............................................................................................ General Study Design................................................................................... Test Material................................................................................................. Animals.................................................... Chambers, Vapor Generation, and Analysis.................................. Exposure Regimen............................................................................................ Microbiological Determinations........................................................... Necropsy and Gross Pathology................................................................ Histological Preparations .................................................................... 3 3 3 4 4 3 5 3 7 RESULTS . ......................................................................................................................... 8 Chamber Analysis............................................................................................. 8 Animal Observations ................................................................................... 8 Microbiological Determinations........................................................... 9 Gross Pathology............................................................................................. 9 Hlstopathology....................................................................................................... 11 DISCUSSION............................................................................................................................... 17 SIGNATURE PAGE. . . . ......................................... -..................................................23 QUALITY ASSURANCE STATEMENT .............................................................................. 24 REFERENCES......................................................................................................................... 25 TABLES 1-10 27 J3 B CO 0 >N4> 01 O) Ul SUMMARY A thirty-day cooperative study designed to examine Che pulmonary effects of vinyl chloride (VC) inhalation exposure in male CO-1 mice, Golden Syrian hamsters, and Sprague-Dawley rats was terminated after only 4 exposures due to unexpected early mortality in male mice. Animals were exposed to 0, 1000, or 5000 ppm VC (nice and hamsters only), 6 hours/day. By the fourth exposure, 1 of 30 mice exposed to 1000 ppm were dead and 2 more were moribund. There was no mortality in either of the ocher groups or species. VC-induced hepatic necrosis was determined to be the cause o: death based upon viral serology, gross, and histopathologic examination. A second study utilizing only five male 0-1 mice ac each of the same exposure levels for 5 exposures produced similar results. Clinically, several mice exposed to either 1000 or 5000 ppm VC were lethargic and had distended abdomens. Gross necropsy examination disclosed ascites, a variable degree of subcutaneous edema, and liver lesions. The livers were generally swollen, congested, and had variable sized necrotic areas. Histopathuloglcal examination disclosed centrllobular to massive hepatic necrosis. Lesions considered secondary co the he^cic necrosis were noted involving the lymphoid and circulatory systems. Mice sur viving the exposures and allowed co recover for up co 13 days had evidence of hepatic reparative ai i regenerative processes. Generally, the hepatic lesions were more severe in mice exposed co 1000 ppm chan in mice exposed to 5000 ppm VC. However, there was wide vari ability within each exposure group. In contras*, to mice, there were no significant lesions noted in either hamsters or rats. The results o: these two studies should be considered when evaluating results from experiments with vinyl chloride utilizing mice. -2- INTXODUCTION As part of a cooperative study on the pulmonary effects of Inhaled vinyl chloride (VC), male mice and hamsters were exposed to concentrations of 0, 1000, or 5000 ppm VC by the Inhalation Toxicology Section of the Toxicology Research Laboratory. To make efficient use of the inhalation chambers, male rats from a study on molecular mechanisms of carcino genesis were included in the 0 and 1000 ppm exposure groups. The exposures were originally scheduled to last for 30 days but early mortality and other adverse effects were noted, necessitating the pranacure termination of the study after u exposures. Because preliminary investigations indicated that the mortality was possibly due to unexpected scute hepatoto::ic effects of VC at these exposure levels, a second small probe study, utilizing only male mice at each of the above exposure levels, was performed. This second study was designed to test the reproducibility of the previous observations and to obtain better necropsy specimens for histopachological characterization of acute lesions Induced by VC inhalation. This report will present- the results o^ these two studies. Emphasis will be placed upon the histopachological description of the lesions. R&S 021567 MATERIALS A1ID METHODS General Studv Design The initial study reported herein was a continuation of collaborative research pursued jointly with Dr. Yasunosuki Suzuki, Research Professor of Coseunicy Medicine, Mt. Sinai School of Medicine, Hew York, New York under a grant he received from the NIH. The grant was for investigation into the pulmonary effects of vinyl chloride exposure in nice and hamsters. This was a cooperative effort wherein the exposures to VC were to be conducted at the Dow Toxicology Research Laboratory and the animals were to be shipped alive to Dr. Suzuki at the completion of the exposure period for specimen collection, processing and evaluation. This study contained 30 male mice and 10 male hamsters at exposure levels of 0, 1000, or 5000 ppm VC. In addition, 10 male rats were included la the 0 and 1000 ppa exposure groups as part of a separate study on molecular mechanisms of carcinogenesis (Stott, 1979). For purposes of this report, this cooperative study will be termed Study A. This study was scheduled to last for 30 days but was terminated after 4 exposures. The second study, hereafter termeo Study a, consisted of 5 male mice at each of the three above exposure levels. This study was scheduled, and lasted, for 5 consecutive dally exposures. J Test Material The vinyl chloride was supplied by The Dow Chemical Company and assayed for purity by gas chromatography by The Dow Chemical Company Analytical Laboratories, Midland, Michigan. The sample was found to.be 99.92 pure (Cortes, 1979). Traces of methyl*chlorine and vlnylidene chloride were found to b* the only impurities. R&S 021568 r f rf The sice used Is both Study A end Study S were calc CD-I alee, 5-6 weeks old (Charles River Breeding Laboratories, Inc., Portage, Michigan). Hamsters were 4-6 week old male Golden Syrian hamsters (Engle Laboratory jy Animals, Inc., F&rmersburg, Indiana). The rats were male Sprague-Dawley rats weighing about 225 grams (Spartan Laboratories, Haslett, Michigan). Y'r The animals were acclimated to the laboratory for at least 11 days prior ~ v to the initial exposure to VC. The animals were randomized from a single lot for each species (separate lots for mice in Study A and Study B) by use of a computer generated randomization procedure. The mice and rats were individually identified by a numbered metal ear tag. The hamsters vere individually identified by cage and rack numbers. The animals were housed (and exposed) 1 animal/cage in stainless steel mesh cages. The animals were housed in rooms designed to maintain a controlled environment of humidity (40 to 60Z), temperature (22*2*C), a 12 hour photocycle, and 12 air change$/hour. Food (Ralston Purina rodent chow) and water were available ad libitum except during exposure. The animals in Study A were all weighed the day prior to the initial exposure to VC but subsequent weights were not taken. All animals vere observed for signs of toxicity as well as changes in appearance and demeanor. Chambers, Vapor Generation, and Analysis 3 The exposure chambers were 1 n stainless steel and glass Rochester-type chambers. Airflow through the chambers was maintained at 150 liters per minute. The air supply system is designed to provide chamber temperatures of approximately 21*C and 5QZ relative humidity. The VC gas was metered through a calibrated flowmeter at a controlled rate into the main chamber air supply and further diluted to the desired concentration. 20 fi (/> o ro Ul o> <0 mssam m I MSS The analytical concentration of VC In each chamber was determined by Infrared spectophotoaetry using a Miran I infrared spectrophotometer equipped with a variable pachlengch gas cell. Analyses were performed at a wavelength of 10.at least 10 times dally for each exposure chamber. Standards for the analyses were made by Injecting a known volume of VC gas into a 100 liter Saran gas sampling bag filled with a known volume of air. Exposure Regimen The groups of animals as detailed in the general study design were exposed to 0 (control), 1000, or 5000 ppm VC for 6 hours per day. These exposures were to be performed 5 days/veek. However, in Study A, the exposures vcre terminated after 4 consecutive days. In Study B, the animals were exposed for 5 consecutive days. Microbiological Determinations Blood samples were collected from anesthetized animals from Study A for serological determination of antibodies to several murine viruses commonly found in laboratory rodents. Samples from representative animals from each exposure level/species were taken 5 days after termi nation of the exposures. Additional (convalescent) serums were collected iron mice 13 days after the termination of exposures. The serums were sent to Microbiological Associates, Bethesda, Maryland for their standard panel of murine virus antibody determinations. Included in this panel were pneumonia virus of mice, reovirus 3, encephalomyelitis virus, K virus, polyema, minute virus, ectromelia, Sendai, mouse adenovirus, mouse hepatitis, lymphocytic choriomeningitis, and rodent corona. Necropsy and Cross ?tof.hologv All animals presented to necropsy alive ware anesthetized with methaxyflurane and the animal was killed by decapitation. Immediately after R8tS 021571 -6- decapltatlon, the eyes were examined In situ by gently pressing a glare slide against the cornea and examining the eye under fluorescent light Illumination. A gross necropsy examination, both externally and internally, was performed with all alterations recotded. Representative portions of the following organs and tissues were preserved in 101, neutral, phosphatebuffered formalin from each mouse: adipose tissue adrenal glands accessory sex glands aorta bone with bone marrow (thoracic vertebral column) brain epididymides esophagus eyes gal lb-Ladder heart kidneys lacrimal (Harderlan) gland large intestine (cecum and coion) larynx liver lower Jawlungs lymph nodes (thoracic, mesenteric) mammary tissue pancreas peripheral nerve (sciatic) pituitary g-ta-nd salivary gland (submandibular) skeletal muscle skin small intestine spinal cord (thoracic) spleen sternum stomach testes thymus thyroid (with parathyroid) tongue trachea urinary bladder all gress lesions Only livers and kidneys were preserved from three of the nomsters and rats. For Study A, two mice/exposure group were sacrificed thrae days after termination of exposures. Two days later three aniaals/xoecies/exposure grout- were sacrificed. The remaining animals were held until 13 days after the termination of exposures, a- which time they were sacrificed by decapitation following methoxyflurane anethesia. Because of the lack of further clinical signs of disease or mortality considered due to VC expose: e, necropsies were not performed on these ariaals. However, the abdominal and thoracic viscera from all mice :*o.-e exposed and exam ined and the. livers from six mice were retai.-.ec m formalin. R&S 021572 An external and internal necropsy examination was performed for all animals that died spontaneously during the course of the studies. This consisted of four mice exposed to 1COO ppm VC dying after the third or tourtr. exposure in Study A, one mouse ex,' vsed to 5000 ppm dying 13 days alter termination of exposures in Study a, and one mouse exposed to 1000 ppm dying after 2 exposures in Study B. These animals were refrigerated rrom the time death was noted until necropsy. The tissues and organs listed above were retained in 10S, neutral, phosphate--buffered formalin. Histological Preparations Hematoxylin and eosin-stained sections of paraffin-embedded tissues were prepared by the standard operating procedures of the histology laboratory. An extensive set of tissues, intended to include all the major soft tissue organs, was prepared from the six moribund or dead mice after 3 or 4 exposures in Study A. The liver, gallbladder, thymus, and kidneys were prepared for hlstopathological examination from all mice from both studies except chose mice from Study A terminated at 3 and 13 days_ following the final (fourth) exposure. From these mice, only the livers with gallbladders were prepared for histopathologieal examination. The four largest lobes of the liver (left lobe, right lobe and boch portions of the medial lobe) were, routinely prepared for histological examination. The livers, gallbladders (hamsters), thymus, ard kidneys were prepared for histopathologieal examination from all rats and hamsters necropsied. ror selected tissues, special-stained histological preparations (Giemsa stain. Von Kossa calcium stain, Dahl's calcium stain) were prepared by standard histopathology laboratory procedures (Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, 1968). RESULTS Chamber Analysis The daily mean analytical concentrations of VC were calculated from multiple daily chamber analyses. For Study A, these analytical concen trations were 968t33 ppm and 5137tl65 ppm VC for the 1000 and 5000 ppm groups, respectively. For Study B, these values were 999i45 ppm and 5112*173 ppm VC. Animal Observations The morbidity and mortality data from these two studies are summarized in Table 1. Study A: After 3 exposures, some of the mice exposed to either 1000 ppm or 5000 ppm VC were lethargic. Their coats were roughened and some had distended abdomens. On the morning following the third exposure (prior to the fourth exposure) 3 mice exposed to 1000 ppm were dead and 3 more were moribund. During the fourth exposure 1 of these moribund nice died. The hamsters and rats appeared normal. At this time, the expo sures were terminated and all animals were held for further observation. The two moribund and four dead mice exposed to 1000 ppm VC were presented for necropsy examination. Three days after termination of the exposures, two more mice from each group were necropsied. Clinically, these animals were all active but one mouse exposed to 5000 ppm VC had a distended abdomen. Fiv< days after termination of the exposures, three more mice and three rats and hamsters from each group were necropsied. These mice were active but two of the animals exposed to 5000 ppm VC had distended abdomens. The remaining animals were observed for 13 days. During this interval, clinical abnormalities were not noted; however, one mouse exposed to 5000 ppm VC was found dead thirteen days after the final exposure. R&S 021574 -9- The rats'and hamsters regained r.oraal throughout the 2 week observation period. Three animals/exposure level/species were sacrificed five days after the fourth exposure. Study 3: The clinical observations of the mice in Study B were similar to those noted in Study A. One mouse exposed to 1000 ppm died after two exposures. Some mice exposed to 1000 ppm and 5000 ppm VC were lethargic and had roughened pelage after three exposures. Distended abdomens were noted after three or four exposures for 3 of the 4 remaining mice exposed to 1000 ppm and 2 of the 5 mice exposed to 5000 ppm VC. Microbiological Determinations To investigate the possible presence of viral infectious disease, serological examinations were performed on specimens collected from mice, rats, and hamsters from all exposure groups in Study A five days after the last (fourth) exposure. Convalescent titers from mice were analyzed on samples taken 13 days after the last exposure. The titers are presented in Table 2. Antibo lies to 12 different murine viruses were not detected in mice at any exposure level at either sampling time. Hamsters and rats had antibodies to pneumonia virus of mice (PVM) and Sendai virus. The antibody level in rats to these viruses was low. In hamsters, antibody titers to Sendai virus were high, whereas those to PVM were low. Gross Pathology The gross lesions noted in Study A are presented in Tables 3 (mice), 4 (hamsters), and 5 (rats). The gross lesions noted in mice from Study B are presented in Table 6. -10- The only gross lesions considered to be directly related to the Inha lation of vinyl chloride were noted in mice from both studies. Because of the irregular schedule of termination vith the accompanying variable, but small, group size and also because the lesions noted were similar in both studies, the results of the gross pathologic examination will be presented without regard to study but with interpretation allowing for the temporal responses of the recovery period. Externally, a low number of mice exposed co either 1000 or 5000 ppm VC developed distended abdomens. This was noted in animals presented alive to necropsy after 4 or 5 exposures and was still present in some animals 3 to 5 days after termination of exDOSure; however, this was not noted in animals dead or moribund after 2-4 exposures. One of the five animals exposed to 5000 ppm VC for 4 exposures and allowed a 5-day recovery period also had ventral subcutaneous edema noted externally. The animals dying spontaneously aftei 3 or 4 exposures to 1000 ppm VC had reddened nares considered to represent agonal congestion. Internally, some mice exposed co either 1000 or 5000 ppm VC had gross lesions suggesting toxic.'.tv to the liver, lymphoid system, and possibly cardiovascular system. In the affacte'd mice, the livers were usually enlarged and had a variety of color changes. Frequently there was hepatic congestion that varied from generalized co a unique pattern of central or hilar congestion vith pale or blanched margins. The hepatic lobular pattern was often accentuated, apparently due to congestion. Foci compatible with acute necrosis were noted. These varied from large foci up to several mm in size, occupying most of a liver lobe and varying in color from pale to red, to multiple pinpoint pale foci. These necrotic-appearing areas were generally larger and involved more of the liver in those mice dying or moribund after 2-4 exposures to 1000 ppm VC than in mice surviving the exposure periods. R&S 021575 Clear to yellow ascitic field and subcutaneous edema was also generally notea in the alee having liver lesions. The alee exposed to 1000 ppn VC had, generally, slightly aore severe fluid accumulations than those exposed to 5000 ppn VC. The animals dying spontaneously frequently had pulmonary lesions charac terized by slight pulmonary edema and slight to moderate accumulations of serous fluid in the thoracic cavity. Since this was not noted in animals surviving the exposure period, it Is believed that this repre sents agonal cardiovascular collapse. A slight decrease in the size of the thymus was noted in all four mice surviving five exposures to 1000 ppm VC (Study 3) and 3 of 5 surviving five exposures to 5000 ppm VC. The other gross lesions presented in Tables 2 and 5 are considered to be either secondary non-specific lesions commonly noted in moribund animals, changes associated with the necropsy procedures, or spontaneous lesions frequently noted In mice of this age and strain. The one mouse found dead 13 days following termination of exposure to 5000 ppm VC was moderately autolytic but no other lesions were noted. The gross observations noted in hamsters (Table 4) and rats (Table 5) from Study A were all considered to be artifacts induced by necropsy procedures or spontaneous lesions frequently noted for these species. Histonathology The histopathologic observations made in Study A are presented in Tables 7 (mice), 3 (hamsters), and 9 (rats). For the mice from Study B, the histopathologic observations are presented in Table 10. The histopathologic examination of tissues from these animals confirmed the gross observations of liver, lymphoid, and possibly cardiovascular toxicity in mice. As was done in the section on gross pathology, the R&S 021576 \ f histopathologic observations will be discussed by combining both studies. The lesions were similar in the two studies when allowance 1$ made for the varying time period from termination of exposure until necropsy. Mice dying spontaneously or moribund after 2-4 exposures to 1000 ppm VC were characterized by acute hepatic necrosis affecting a large portion of the liver. In the seven mice with this exposure history, about onefourth of the liver area was necrotic in one mouse, between onefourth and one-half was necrotic in four mice, between one-half and threefourths was necrotic in one mouse, and the remaining mouse had necrosis of over three-fourths of the liver as judged from histologic sections of the four major liver lobes. When the necrotic areas were small, they were centrilobular; however, they were frequently massive and involved confluent lobules with only a thin rim of viable hepatocytes around the portal triads. Frequently, minute basophilic intracellular granular structures were noted in swollen necrotic hepatocytes as well as viable hepatocytes at the margins of the necrotic areas. These were demon strated to contain calcium by special staining techniques. There was minimal inflammatory reaction noted at the margin of the necrotic areas in these mice dying spontaneously. The few inflammatory cells present were predominantly macrophages. There were narked hepatic vascular chances noted in this group of mice. There was usually intense con gestion within and at the margins of the necrotic areas. In the areas of viable hepatic parenchyma there was often severe dilatation of the centrilobular vein with compression of adjacent parenchyma along with focal areas of sinusoidal dilatation scattered throughout the hepatic lobule. These areas often appeared as vascular "lakes" among the parenchyma. The viable areas in the livers of these animals had centri lobular or periaecrotic hepatocytes generally characterized by swelling with vesicular or vacuolated cytoplasm. In some livers, these centri lobular or periaecrotic hepatocytes had increased cytoplasmic homogen eity and basophilia with occasional mitotic figures. A low number of small, eosinophilic, sinusoidal masses were frequently seen f t * if. I 23 Oo CO 8 mt, Ol "NNi y\-':' -..r.V anong these cells, while this material could represent necrotic cellu lar debris, these masses were considered more iiicely to be microthromboses. The livers of mice surviving u or 5 exposures to 1000 or 5000 ppm VC had narked histopathological variability. The four mice sacrificed immediately after completion of 5 exposures to 1000 ppn VC (Study E) had acute centrilobular hepatic necrosis. While this was qualitatively similar to that noted for chose animals dying spontaneously at this exposure level, it was much less in degree -- being about one-tenth of the liver in three of these mice and one-fourth in the other one. These necrotic foci were often present in a subcapsular location. The reaction to the necrotic foci was similar to that described for those animals dying spontaneously but the vascular reaction was less dramatic and the inflammatory reaction included slight connective tissue proliferation. In centrilobular areas lacking necrosis, the hepatocytes often were enlarged with increased cytoplasmic homogeneity and basophilia and occasionally cytoplasmic vacuolization and mitotic figures. Hyaline sinusoidal masses consistent with microthrombi were seen in some of the centrilobular areas. The five mice sacrificed immediately after completion of 5 exposures to 5000 ppm VC (Study B) had marked variation in histopathologic changes. Three of these mice had only microvesiculation of the centrilobular hepatocytes but lacked any significant amount of necrosis other than the scattered microscopic foci typically noted also in control animals. The ocher two animals were similar to chose animals dying spontaneously in Chat they had large areas of acute necrosis (between one-fourth and one-naif of the liver in one mouse and between one-half and three-fourths in the ocher). The inflammatory and vascular events roughly paralleled those noted in the mice dying spontaneously with comparable liver lesions. R&S 021578 -14- fi /> 021579 With 3 to 5 days of recovery following completion of four exposures to vinyl chloride, there was again variability in the histopathologic changes in the liver. Three of five mice exposed to 1000 ppm VC and 2 of 5 mice exposed to 5000 ppm VC had changes of the centrilobular hepatocytes with either enlargement and cytoplasmic vealculaclon or cytoplasmic homogeneity, basophilia, and occasional mitotic figures. In one mouse in each exposure level this reaction was more severe with marked cytoplasmic swelling and veslculation, scattered necrotic indi vidual hepatocytes and sinusoidal thrombi and dilatation. In one mouse exposed to 1000 ppm VC and 2 nice exposed to 5000 ppm VC, there were multifocal areas of centrilobular hepatic necrosis involving about onetenth of the liver parenchyma. These were surrounded by a slight subacute inflassnatory reaction in which fibroblasts were noted. In one mouse exposed to 5000 ppm VC, some multinucleate giant cells were noted in the reaction. By 13 days after completion of the exposures, the livers of 4 of 6 mice in each exposure group were similar to controls or- had only slight microveslculation and enlargement of centrilobular hepatocytes. One mouse in the 5000 ppm VC exposure group had marked centrilobular hepatocyte enlargement with -occasional mitotic figures and sinusoidal microthrombi. Two mice exposed to 1000 ppm VC and one mouse exposed to 5000 ppm VC had multifocal areas of fibrosis with mononuclear inflammatory cells and, characteristically, multinucleate giant cells containing a deeply basophilic structure. This was identified as calcified material by appropriate staining techniques. These fibrotic areas were centri lobular and often subcapsular. These livers also had slight periportal accumulations of mononuclear inflammatory cells and slight bile duct hyperplasia was noted in one mouse exposed to 1000 ppm VC. Foci of dilated sinusoids surrounded by thin fibrous connective tissue walls were noted throughout the liver, particularly subcapsularly. The thymus had histopathologic lesions that closely paralleled the degree of hepatic necrosis. Mice chat were moribund or died sponta neously had loss of the thymic lymphoid cells chat varied but was R&S 021580 -15- usualiy moderate to marked in severity. These also had karyorrhectic nuclear fragments in the medulla of the thymic lobules. The thymuses of mice that survived the exposure period were frequently normal. When depletion of thymic lymphocytes was noted in surviving mice it was present in the mi-.e having hepatic necrosis. The severity of thymic lymphoid depletion roughly paralleled the severity of hepatic necrosis. In the mice dying spontaneously, the other lymphoid tissues examined (spleen and lymph nodes) had decreased lymphoid cells similar in degree to that noted in the thymus. Histopathologically, the kidneys of mice dying spontaneously frequently had tubules with sloughing epithelium, pyknotic nuclei, and hypereoslnophillc cytoplasm. As these changes were not noted in properly fixed kidneys from sacrificed mice they were attributed to autolysis. Three of the five mice exposed to 5000 ppm VC (Study B) had slight to moderate cytoplasmic cicrovesiculation of the renal cortical tubules. All other nistopachologic observations made on the kidneys of treated or control mice were considered to be spontaneous entitles and not due to exposure to vinyl chloride. The hearts of 3 of the 6 mice moribund or dead after 1 exposures to 1000 ppm VC (Study A) had isolated focal areas of minute basophilic structures within degenerating cardiac myocytes. This was shown to be calcified material by use of special histologic staining techniques. Other organs examined from mice dying spontaneously (Study A) had occa sional lesions. These were generally considered to be either secondary to the agonal condition of the mouse or spontaneous entities not due to VC exposure. The histopathologic observations noted for the liver, gallbladder, kidneys, and thymus from hamsters (Table 8) and rats (Table 9) exposed to vinyl chloride were considered to be spontaneous lesions with no lesions ascribed to VC exposure. ,,;V'v m -16- Histologic examination of the liver and kidneys from che one mouse fcund dead 13 days after termination of exposure to 5000 ppm VC disclosed moderate autolytlc changes. No lesions similar to chose present in the affected nice were noted. Although a cause of death was not determined, it was felt that this mouse did not die from VC-induced hepatotoxlcity and it was not included in the gross and hlstopathology tables (Tables 3 and 7). Oto tn oo R&S -17- DISCCSSION The acute deaths la alee exposed to 1000 ppm VC were first suspected to be of infectious origin. Early results from histopathological exam ination combined with results of viral serology failed to support this presumption. Neither vlrai inclusion bodies nor bacteria were noted on histopathological examination. Mice failed to have titers to numerous viral pathogens, including those concaonly associated with hepatic necrosis (ectromelia, mouse hepatitis virus, and reovlrus). Although antibody titers to two viruses were present in both rats and hamsters, the titers were relatively low and the viruses both produce pneumonia rather than hepatic disease. These findings suggested that VC may be acutely toxic to the male mouse. Although hindered by the low numbers of mice, the marked variation in severity of lesions, and the variable degree of aucolytic change, the staggered necropsy times following termination of VC exposure allowed a morphologic assessment of the nature and progression of lesions in mice following short-term inhalation exposure to VC. The major organ affected was the liver. The first histological changes appeared to be vesiculacion, vacuolation and swelling of the centrilobular hepatocytes. This was noted in most of the animals exposed to either 1000 ppm or 5000 ppm VC. In a low proportion of animals this lesion was markedly more severe with marked centrilobular and midzonal cellular enlargement and vacuolization and compression of the sinusoids. In these areas small hyaline masses were frequently noted among the swollen cells. These masses could represent necrotic hepatocytes but are considered more likely to be microthromboses. Following the Initial cytoplasmic changes, the centrilobular and midzonal hepatocytes in some livers had homogeneous eosinophilic cytoplasm with changed staining affinity characterized by a slight basophilic tinge. r R&S 021582 -16- These cells were enlarged, sometimes markedly, and mitotic figures were often noted. As this change was more prominent in the animals sacri ficed after several days of recovery, this was considered a regenerative response. Next in the apparent progression of lesions was centrilobular necrosis. This vas characteristically coagulation necrosis with only a very slight peripheral inflamatory reaction. The inflammatory reaction that ensued consisted primarily of macrophages and fibroblastic elementsThe progression from the centrilo >ular hepatocyte swelling to necrosis was unclear. Frequently the perinecrotic hepatocytes were either vacuolated or contained eosinophilic intracytoplasmic man; is assumed to be degenerated cystosol proteins. However, these changes in the hepatocytes adjacent to necrotic areas were of no greater severity than those present in cells in non-necrotic centrilobular areas. Thus, degree of vacuolar change alone did not appear to correlate with the necrosis. Of interest are the minute basophilic structures noted in some of the necrotic hepatocytes and perinecrotic hepatocytes. These were demon strated to contain calcium via the use cf special histological stains. It is speculated that these structures represent mineralized mito chondria. Mitochrondrial calcification is commonly noted by agents prim-'.rily attacking the cell membrane (Trump and Arstila, 197 >. In all mice dying after 2-4 exposures to 1C00 ppm VC, there was progression from multiple small centrilobular necrotic areas to massive confluent areas of necrosis. Most animals dying spontaneously had necrosis of about 50% of the hepatic area on the four liver sections, examined. Survivors generally had necrosis of 25% or less of thu liver area. With mice exposed to 5000 ppm VC, there were no deaths ascribed to VC and the amount of liver necrosis was generally less. However, in two mice exposed to 5000 ppm VC for 5 exposures (Study B), there was massive necrosis (up to 75%) and it is speculated that death may have been imminent had the animal not been sacrificed. R&S 021583 The role of che vascular system in che pathogenesis of the liver necrosis is uncertain. In che animals dying spontaneously there was dramatic congestion in and adjacent to che necrotic areas. There was frequently marked congestion of the central vein and focal areas of sinusoidal dilatation with compression of adjacent hepatocytes. There was slight to moderate pulmonary congestion and edema; however, ocher abdominal organs (spleen, kidneys, gastrointestinal tract) had little evidence of vascular congestion to suggest generalized heart failure. Although sinusoidal microthrombi were present, larger thrombi were not noted. Survivors frequently had small sinusoidal dilatations surrounded by fibrous connective tissue, but lacked ocher vascular changes. The dramatic vascular congestion noted in the livers of moribund or dying animals was considered to be a probable secondary reflection of the liver necrosis as well as terminal heart failure. In a recent paper reviewing human hepatic angiosarcomas due to vinyl chloride and ocher causes. Popper <it al. (1978) note a precurs ir stage characterized by hepatocellular proliferation along with sinusoidal dilatation and sinusoidal lining cell proliferation. Their early lesions are similar to chose described in this report; however, their report does not note hepatic necrosis, but rather the progressive atrophy and disappearance of the hepatocytes with increasing prolif eration of che sinusoidal lining cells leading to angiosarcoma. Also prominently affected by short-term inhalation exposure to VC was the lymphoid system, particularly reflected by che degree of thymic lymphoid necrosis and atrophy. The degree of thymic lymphoid depletion generally paralleled the degree of liver necrosis but appeared to lag temporally slightly behind the liver necrosis. This was noted espe cially in survivors in which there were slight thymic changes in spite of liver necrosis. It is speculated that the lymphoid depletion is secondary to the liver necrosis, possibly due to elevated levels of adrenal corticosteroids produced in response to systemic stress due to the massive hepatic necrosis. -20- Therc were several changes suggestive of possible disturbed nineral Betabolisa in the mice with liver necrosis. In addition to the intracycoplasmic mineral noted in and adjacent to the necrotic liver areas, nineral deposits were noted in foci in the myocardium and larger bodies in necrotic areas of the thymus. It is speculated that the mineral ization is dystrophic due to cellular injury rather than a reflection of calcium-phosphorous imbalance. The kidneys had only slight changes considered to be insignificant in causing the death of these nice. The tubular changes noted in the mice dying spontaneously were regarded as artifacts due to autolysis. With sacrifice and prompt fixation, the only lesion attributed to VC exposure was slight to moderate microvesiculation of the cortical tubular epithe lium. Surprisingly, the acute hepatotoxic effect of VC in mice has apparently not been generally recognized. Early short-term inhalation experiments noted that 30 minute exposures to 300,000 ppm VC were lethal to mice and rats, but 100,000 ppm produced only narcosis (Mastromatteo et al. 1960) . Prodan t al_. (1975) calculated the LC.q for white mice as 11.75- VC in a ventilated chamber. A dominant lethal test in CD-I male mice (the same strain as that used in this report) exposed by inhalation to 3,000, 10,000, or 30,000 ppm VC for 5 daily 6-hour exposures was reported by Anderson et al. (1976). These doses were selected on the basis of their unreported probe tox icity studies which determined 30,000 ppm to be in the toxic range (the criteria of toxicity were not stated). In the published work, Anderson al. had significant mortality (55-) at the 30,000 ppm exposure level and 51 and 101 mortality at the 10,000 ppm and 3,000 ppm exposure levels, respectively. The cause o: death was not stated. "here have been several subchronic or chronic toxicity studies utilizing nice exposed to VC at levels close to those used in this report. Winell al. (1976) exposed NMRI (albino) ri.ce to 50 or 500 ppm VC. After 40 weeks the serum levels of lactate dehydrogenase and alkaline phosphatase were increased. Although these enzymes may indicate liver damage, they were apparently increased only after liver tumors were present. There is no indication of any acute deaths due to VC exposure in their report. Carcinogenesis studies on VC in mice have been reported by Maltoni and Lefemlne (1975), Keplinger e al^ (1975), and Lee t al. (1977). In all these studies, the mouse was markedly more sensitive than the rat. Maltoni and Lefemine exposed Swiss mice 4 hours/day, 5 days/week for 30 weeks to levels of 50, 250, 500, 2500, 6000, or 10,000 ppm VC. Although no acute lethal effects of VC were presented, there appears to be slight excess mortality in male mice at the higher exposure levels as Judged by Che number of survivors at 16 weeks. Keplinger ^ al, exposed CD-I mice to 50, 200, or 2500 ppm VC for 7 hours/day, 5 days/week for 9 months. The interim data reported after 8 months of exposure indicated several mice with tumors but there was no indication of acute hepatoxicity. The only published report noting acute hepatotoxiclty in mice is that of Lee al., in which CD-I mice were exposed to 50, 250, or 1000 ppm VC for 6 hours/day, 5 days/week for 12 months. They report observations strikingly similar to those noted in this report. Two males and one female (from group size of 36/sex) were found dead between the third and ninth exposure to 1000 ppm VC. Histopathology disclosed acute toxic hepatitis characterized by diffuse coagulation necrosis beginning in the centrilobular areas and focal to marked congestion. They also reported renal tubular necrosis characterized by pyknosis and eosinophilic granulation of the cytoplasm. Their research protocol included the sacrifice of 4 mice/sex at the end of 1, 2, and 3 months. Review of 33 CO o M (71 00 o> I their progress report (No. 8 submitted to NILKS) discloses no reported histological lesions for these 24 alee other than minor lesions also present In the control animals. The studies reported herein confirm the acute hepatotoxicity of VC to CD-I mice as first noted by Lee jet al. and present a more detailed histopathological description of the associated lesions. Several note worthy aspects of this response are presented Including: 1. VC appears to be acutely hepatotoxlc at these relatively low concentrations only in the mouse, particularly the CD-I strain of mice. 2. There is a consistent association of greater severity of toxicity by virtually any of the parameters effected (mortality, gross lesions, or histologic lesions) In the lower exposure level of 1000 ppm VC as opposed to those in the higher exposure level (5000 ppm). 3. There is wide variability within a treatment group; i.e., up to 752 of the liver necrosis and death in some mice exposed to 1000 ppm VC while others survive with virtually undetectable histological lesions. It is concluded chat the. observations reported herein need consideration when evaluating research involving vinyl chloride exposure of mice. -23- This report was prepared and submitted by the following Staff Members: Keith A. Johnson, D.V.M., Ph.D. biplomate, American College of Veterinary Pathologists Project Leader _______________________________ Hi oT 'Ld'kel, 3.S. Senior Research Biologist Inhalation Toxicology Reviewed by: M. J.-'McKenna, Ph.D. Dlplomate, American Board of Toxicology Croup Leader, Inhalation Toxicology Date: Data: //f*/ -24- QUALITY ASSURANCE STATEMENT VINYL CHLORIDE: RESULTS OF TWO TITLE OF STUDY: SHORT-TERM INHALATION STUDIESHET-K-1711-(29) In compliance with Good Laboratory Practice Regulations, the study phases were inspected by the Quality Assurance Unit and the results of these inspecticns reported to Management and the Study Director on the dates listed below. The report accurately reflects the data generated in accordance with the regulations and standard operating procedures of the laboratory. All data and the reports are located at the submitting laboratory. S t if f' t t Study Started: Dates of Inspection: 30 Oct. 1979 19 Oct. 1979 1 Nov. 1979 31 Jan. 1980 3 June 1980 16 Jan. 1981 Report Issued Date: Date of Report: 3 Feb. 1981 19 Oct. 1979 2 Nov. 1979 5 Feb. 1980 5 June 1980 22 Jan. 1931 \ 30 &S 021589 f /?$/ W. E. HooverD^te Quality Assurance Toxicology Research Laboratory Healt.n s Environmental Sciences 1SC3 3uilding Dow Chemical U.S.A. Mid lane. MI 4864Q -25- Ri';SESCiS Anderson, D., Hodge, M.C.E., and Purchase, I.F.H. Vinyl Chloride: Dominant Lethal Studies in Hale CD-i Hice. Hut. Res. 10:359-370, 1976. Cortes, H. J. Purity of Vinyl Chloride for Animal Inhalation Study. Analytical Report, 1979, Midland Analytical Laboratories, The Dow Chemical Co. Keplinger, M. L., Goode, J. V., Gordon, D. E., and Calandra, J. C. Interim Results of Exposure of Rats, Hamsters, and Mice to Vinyl Chloride. Ann. N.Y. Acad. Sci. 216:219-224, 1975. Lee, C. C., Bhandari, J. C., Winston, J. H., House, W. 3., Peters, ?. J., Dixon, R. L., and Woods, J. S. Inhalation Toxicity of Vinyl Chloride and Vinylidene Chloride. Environ. Health Perspect. 21:25-32, 1977. Malconi, C., and Lefemine, G. Carcinogenicity Sioassays of Vinyl Chloride: Current Results. Ann. N'.Y. Acad. Sci. 216:195-218, 1575. Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. L. G. Luna, editor. McGraw Hill Book Co., New 'fork. 3rd ed., 1968. Mascromatteo, E., Fisher, A. M., Christie, H-, and Danziger, H. Acute Inhalation Toxicity of Vinyl Chloride to Laboratory Animals. Am. Ind. Hyg. Assoc. J. 21-.39U-398, 1960. Popper, H., Thomas, L. B., Telies, N. C., Falk, H., and Selikorr, I. J. Development of Hepatic Angiosarcoma in Man Induced by Vinyl Chloride, Thorotrasc, and Arsenic. Am. J. Pathol. 92:319-376, 1978. -26- 30 (/) O ro in (O REFERENCES (Continued) Prodan, L., Suciu, 1., Fislaru, V., Ilea, E., and Pascu, L. Experimental Acute Toxicity of Vinyl Chloride. Ann. N.Y. Acad. Sci. 246:154-158, 1975. Scott, h. T. Protocol for the Investigation of the Mechanism of Tumor Initiation by Vinyl Chloride, 1,4-Dioxane, and 1,3-Hexachlorobutadiene, 1979. The Dow Chemical Co., Midland, Michigan. Trump, B. F.. and Arstila, A. U. "Cellular Reaction to Injury" in Principles of Pathobiology. pps 9-96, 2nd ed., (1975). Edited by LaVia, M. ?. and Hill, R. B. Oxford University Press, New York. Winell, M., Holmberg, B., and Kronevi, T. Biological Effects of Vinyl Chloride: An Experimental Study. Environ. Health Perspect. 17:211-216, 1976. TABLE 1 VINYL CHLORIDE: RESULTS OF TWO SKORT-TERM INHALATION STUDIES Morbidity and Mortalit*/ Exposure Level (ppm) STUDY A - 4 exposures Mice - Number Exposed Number Died (Number of exposures before death) Number moribund or with distended abdomens (Number of exposures) Hamsters - Number Exposed Number Died Number Moribund Rats - Number Exposed Number Died Number Moribund 30 0 10 0 0 10 0 0 L000 30 u (3,3,3,1) 2 (^,4) 10 0 0 10 0 0 5000 30 1 (ia) h2 (l,lb) 10 0 0 STUDY 3-5 exposures Mice - Number Exposed Number Died (Number of exposures before death) Number with distended abdomens (Noted at necropsy after 5 exposures) 3 0 55 10 (2) 3 This animal was found dead 13 days after exposures were terminated. These animals were noted with distended abdomens 3 and 5 days after the exposures were terminated. - Indicates not applicable. > r *> </) o t.oa tn <0 to MaMIHC Ifc'IHl* IlnUKC Hihiii* Jl.lM'it i` 1 Haas! tr tl.mslrr Rjl Hat fc* piuru i,vi'ti)>in) 0 I.1KII1 Villi) i> Hum Mlltll 0 loot) soon 0 iooo TABl.r 7 vinyi. mtmiDKi umin.ts or ivi> sinirt-thoi inmaiation si nous Viral AitllbiHly Tlltft rr Kit*, U>Miria, n,<J R,<| Antibody Titer to: Tint; of Hrpallet* KoJent S.iap le rvH Hfo 1 cnv it Jt Pii 1 yowl Hlrulf K*t rowl I* S nnl* 1 Adeito Hmiit IOI (Imuiia 5 -- ._ _ _ -- S -- -- - . - -. -_ 5 "- -- - - - -- - -- 11 - - J _. . Ml 1] - - -- - - -- _ Nil 11 - - -- - - - -- - - Mi S .5 40 _ 5 20 - _ _ -- _ _ - - _ ^|0 >40 - 40 - .: - -- S *0 . b 70 - _ -- _ _ . 10 _ __ _ 10 - - - - n|ly!( rolltiwliif, u-rnlniil lu ut mpuNtirr*. - Jmllcitc* ii< .mt IIhhIli*u in [ In: ntiiIi-il vlni* wrrc |il*-jn'tit, Nil - tbit Ikiiu -sz C6S130 S9d mm -srr _ mm VI MY*. Clll JJKJ Ut; R1.SIJI.T* Of TWO SlKlRT-TCKH tMI'AMTlON ST^UtS !** IhologIc OturrvAllofl - Study A Hlt'r u vi 1 4iaii^j . Hcrtapby Cl oeji_ _ ___ ___ ___ Ifatiuf gl 1 n Croup_____________ _____ __ __ Q ) m ) Hi)ft Recovery ___ _ 5___ Moribund or Dead alter ^ 3-4 |juKmrcP 6 JOOCJ 3 or 3 Itaye Recovery ___ 3 13 llaya Recovery 4' CXTbRNAI. OBSIKVATIOtt* Ki> vlelhlc teflon* allMliMCn SuWuUiHHiiia udi-au Ki'JJvaaI iuru focitt caniMballai IHTLKNAL OBStHVATlOHS Nt viHlt.lv k'altms Crnaul Vet'll ml Niikut.nii'tMia rJatfd - MOdcl.tli' Atfvilvtf - lU'ji jIkIimIiuI fluid 4Mn**e - toJl'IJli! InMlttfJ tobill'll Liver General Ilol iJiLirsuaeiil nr swelling UtUf ui large ureua of color change, eoggeallng tu croafls (either pale or reddened) I*U|ni|ii1 1*jIv toeI# multiple, suggesting focal necrobU GJilgtriit Jun ui rcJdctted Accentual t'd r*<JJetilug ol lillur or van ot Multiple lul>c* Pale tohar aai^lua, tuial ui dHliMc Multiple mliI I fal loci Increased lobular pattern CctHiralJiid jfctfleticfctf 3 0 0 0 0 1 0 0 0 () 0 0 1 0 0 0 Q ) I 0 0 4 I 0 0 0 1 3 2 0 4 0 2 1 3 0 lUta prevent rd are ruHshcr of alu> having lira at a led observation. Grouped by day* after final (fourth) eaiHjaura when necropalrd. b Include* 1 dead after three exposure* and | dead and 2 Moribund after four exposure#. 3 0 0 fl 0 0 0 0 0 0 0 I 1 1 1 2 0 6 0 11 0 0 3 n n 0 tl 0 ) 0 0 0 0 0 0 VMNI J r 4 f>jyn Recovery 5* 11 llayu Ma'ovci y 6 2u 1u I0 0n 00 3 n t 11 00 [} 0 l 1 11 0 (1 1u 00 0n 1 ii 00 f6SL30 S9U __ *- - 3 Tf- Tv" ` - % 4'*` 1 ` A*'*-*`jtr- ;t TABLE 3 <Continued) vuni. aiijonrut: results of tub somt-toti imuiatioh studies Croat Putlmltittlc Oiaemtloii - Sihdjr A Nice S* * /- l!l|iU)ilMla ltVI*l __ Not 1 oil My l'i ou|i Hnnliot nl tljt p In Crm*p in , ^ _ _ , jiin^K Congenled, dllfnati Mli " atight Kocul Jii-ai of c*phy*temj Multiple tctl foot ron* la lout with asp Imt ml Mood IUm Cjv t ly Clear flolJ - m 11 r*1ti in iMHlrraiu Hemnl cty f^lt'Hbt SliMkhli IUm'Omo dtnli'iilliiM (*oiii<vri i*tl tir i oMcintl S*aM Intestine rongcHietl or fcdilnttHl iHri* Teased ln^i'Ktd f tdiley til reddened Thyua iVlm tilil toMM*! rli.igv 11 ) or 1 ILiya Ht-4 nveiy ^ S 0 0 0 1 n - (i 0 0 0 0 1 0 Unllwid nr Ikid after K 3-4 Ewpoaur ca _ 6 1000 ) III 1 Djjra Aeeovery j 1 1 Ikiya Am overy b 4 00 1 00 1 a0 U s0 4 i0 1 u0 1 L0 I n0 1 00 I 00 0 u0 0 10 t>.ila jiroiii'tilwl otu nunln't of atkv having tire Mated observation. - ^CrtMjjJvtl by days after 1 Init ffourth) m|ioiurc vlurA necropalcd. Jneltidea 1 iIimJ if let lliri't' eM|Hjuoi us and 1 dead and 2 nor1bond after four expoauras. UKM) a .* r - 3 or 3 iMy* Recovery ^3 1 3 U*ys Arrow t y * 00 10 00 3n 0 ft (T 0 0n l) u 00 0n 00 00 S6S130 S9U TA3LE i VINYL CZJORIDE: RESULTS OF TVO SHORT-TERM INHALATION STUDIES Gross Pathologic Observations - Hamsters3 Exposure Level0 Nuaoer Examined external observations No visible lesions Firm enlarged boney prominence on lower left rear leg INTERNAL OBSERVATIONS No visible lesions Lungs Multifocal red areas consistent with aspirated blood at necropsy Focal, depressed, dark red area, isolated Liver Slight accentuation of lobular pattern Tibia Firm smooth boney mass, distal tibia; consistent with healed fracture 3 3 0 1 1 1 0 0 1000 3 3 0 0 2 0 2 0 5000 3 2 1 1 1 0 1 1 Data presented is number of hamsters with stated observation. Hamsters were necropsied 5 days after fourth exposure to vinyl chloride. TABLE 5 VINYL CHLORIDE: RESULTS OF TWO SHORT-TERM KiHALATION STUDIES Cross Pathologic Observations - Rats'* Exposure Level (pon)____________________________ Number Examined _3 EXTERNAL OBSERVATIONS No visible lesions 0 3 1QQQ 3 3 INTERNAL OBSERVATIONS No visible lesions Thvmus Multiple petechial hemorrhages lungs Multifocal red areas consistent with aspirated blood 02 11 20 Data presented is number of rats with stated observation. aRats were necropsied 5 days after the fourth exposure to vinyl chlo 33- tA3L* 3 'I'.rc. CX3tst: USCL~3 OF 7V0 SnCZL-TZM VSiUdJCIlZM S Grata Pathologic Obtarvatiena - Study 3 MI;* re r.rroaut* Lvl (pm) ::*crpp*v Crouo (unbar of Mlc* la Crsur 0 Pinal Tamisaticn1 f tXTELSAL OlSERVAItCSJ "a vtiibl* la* ten* Cjra**i clou41n**, fscil unllnfrij ZLsctodad ibdotw Sail! ill* - Pllataral Clinically dapraiian to 0 Xr 0 0 3 ".tcswai. oBsatVArrcss vitibi* iaaiona nral Ventral subeucanaoui 4o* - urud - aodarat* - alight Aicltts. clur ibdoaliil fluid - =irUi - nodaraca Cacranad abdominal fat J 3 0 0 0 0 0 llvar Canaralizad anlargacanc Lobar or larg* araaa of color chant* (alct-ar pal* or raddanad) Pinpoint pal* foci, ailtlpl* Acctncuictd fdUn: of hil-ir 4ru of sultlpl* lobva with, >*J. lob*r urfla* Accactuatad lobular pat tarn 0 0 0 0 0 3 lunga Congaatad. conatataat vlch hypoatatlc congat tlon Pinpoint pink foci. law. tad I* tine t hultipl* rad foci cocatacant vlth laptratad blood 0 I 1 5 comae* Suboucoial adaaa. jl*ocular portion tllght yailttfocal pinpoint dark brovn foci, glanaular aucoaa 0 0 Snail Znrascln* Zacraatad Inga* ca 0 Cacua :acr*B4 confUCt&cy ?f 0 Thvtut 3*cr**a*d 112a - flight 0 Vrttora* Proolaant tnoraelc lacarvartasral dlaca 0 2 cxooauras ,b 1000 Pin*L Tarairjtlon 4 4 00 00 03 0' 00 3 0 31 0 *4 0 03 00 0 3 0n 03 1- 0 *1 i1 0 * 40 00 33 3> 00 0 0 0i 00 3aca praaantad ara nrmhr of uuulJ having th* atatad ob*rvcioa. *'l*cropgl*d after !lv upomu. b Antoil died Zollovlng cvo *xpo*ur*e. 5000_______ Inal lamination 5_____________ L 0 4 0 1 0 0 0 0 1 1 4 0 z 2 0 0 0 0 1 0 0 3 l 4 : ; ,4 / 2D CO N> -a cn <0 ao VIHTL 011,0*1 UP r RESULTS OP TWO S1IORT-TTIU1 1HKA1.ATJOH STUDIES llltoplholi)|lc Obervillon - Study A Mice - rn|n!;i'rr liVi'l necropsy lifuMji'1 _ _ Huftbir of llire lii tirotip 0 3 or 5 iMyi Herovery Hurlknd or DeiJ after ^ )< Ei|io>urei 6 UK*! ) r r i Diijra 13 Days Kernvery ]___ t__ 1.1 ver (nuulKrr tiishii'J otcrinirnplcal 1y) Ku vUlMe lesions Acute roj|;iiUttiin iiecrosla, - foe.il* rentrtlobular, about IOt of area of aeclloua e*alned, often *"heapmi tar - focal, cent rl tabular to eonfluent, lnvn|vhK I5-23X til area of section* examined - local, emit I loliwlor to continent, Involving 26*SOX of area of sect limit outlined - final, tent rl lobular to confluent, Involving 5I-25X of nr on uf sections e xan I no*] - hihulvc, l.ugc arena of liver, greater th.in 7SX ol area of sections esaalned HI note, fnt t .ice f lu Inr baa oj'li I M c alrmlurea at n.tigld!* ul lied otic .n i'.h l'*-t f lift i til 11' I of l.iMwnlory tel la, nixed, - vviy all gbt In alt gilt IV11 nee i m Ic imlunitc Inf lawoat ury ami connective lt5*uecello - slight Per Inerrot Ic swollen, vjciihJ Rt ctl hepalocylrs CVni r t litbiilnr hcpai ocylev, Mlt rovealcu lar change - very alight toalight - Marked 5 0 0 0 0 0 D I? 0 0 U 2 0 6 0 1 0 0 0 0 0 0 2 0 3 0 Data |i resented are lumber of iinlakilr having the at a ted observation, Vi ou|> j by days after final (fourth) exposure wlien necropslrd. ^Includes 3 dead after three exposures and I dead and 2 mot I bund after four exposures. __________5000 3 or 5 Daya 13 Hoys lecovvry___ Recovery I) tl 0 22 1 04 1I Mr 66SIZ0 SSU TAttl t I (Cotil 1 nucd) VIHY1 UtiUHtUi:: ktStllTS ilf 1W0 SIH^T-TLS* IffilAIATlnN SllihJI.S lljUiL>li|<|c f)|ikUVJl Ion - Sii*ly A Mhv Ktp<l*ntC twet ^_ ___ (1 Hvcr muty iifuup JtooCrr d( Hite In l!rou| 3 oi i iKiyn _ Kccoyc i y___ 3 j_lvi-r (Cimitmicd) CctiltMulmhr and oldzonal hepatocylea cnliriis), cytopianai of * lightly Incrcaacd haaopUl lid, occna Iona I kllmei focal Bukjpftiilar hrpatnryto dr general I on with and hyaline n4tcrUl MillliniK lealv slant cell a cmtlalnlns t alt ll Ic4 SlnuaoMjl ronsaatlfni and h*orr lijp.t', In and ncai necrotic irvia - wlrrale - aui Ltd Ml i I ltl>ii I jt vUn runstfallnn, ajiVrd, tonpruoaloo of adjacent tiepalorytea VaacuUt ectaala, apparently nldtunal, wilt* coaprebaton of adjacent hcpjtocytea K*>cat telac Hectaa la Snail hyaline uiaei In I nuaoldii, Marglna of nect otic foci or In areaa of mm lien hepatorylca Hlcrovcatcular change of pcdpnrUl liepatocyiea - hIJ ghl thill I final !croacnpIc loci of hepulM'yla degeneration or necruafa with BommMclear lof I amatory cell reaction - very alight nr alight - noderule Per * ,*oi c .it ihiiiiNiiii'lt!<ir J nf lainaalor y cel la alight 111 le duel liyperplaalu - alight 1 it 0 0 0 0 0 0 0 2 A 0 0 0 HotIhund or iWd after ^ 3-4 Eipoaorea l KtfftO _ _ 3 or 3 Uiya Kecoyery V 11 fhiya Itec overy ___ 6___ Ikiia presented are nuadtur uf ><iIi*hIi having the atated ulivfrvalloit. Grouped ly day a jIUt final (fouith) uxpoanri! wlien ncctopalcd* h Includes 3 dcod after three CApoanrca jihI 1 dcid and 2 atn I bun J after four eapoaurea. 31)00 3 or 3 fhiye Recovery _ 5` II lUyM tnowry im 3i 1) LI 1 II Cl LI t 11 00 20 )2 0J , f'H ' ^aaMa - --* i ,*5 0091-30 S9U 1ABI.E 1 (Continual) VMrrt. ntijORiDEi rcsui.ts or wo short-term imuotion rnnurs Nlaiopathalogtc Obaervatlona - Study A Hire tn[H)Sul t! I.ivr 1 _ ______ _____^ ________ Nceiopty (Uimh _ ______ __________________ . uf Hit* In Ciohp CaMMmMcr (imnhrr rMaliicit ali riiHiuiptcilli) vleJM* limithi a SiihmiruMl oilt'ihi - alfftlit jiHliiim oii.it |fIonm*tnry eel la, aired* focal - very nl Iglit El Jury (uinln'r e4>lnnl alcr<ti|i|ia1 ly) Ku via Mile Ira Iona Irictrascd cyloyltMlc granularity, colt leal lobules - very atl|lil tu alight HlcrovcalcutxlIon, cart teal lulwlca - alight lobular eos tno|ilil 1 la or ntcruveaIculalloo and nuclear |>ykuoKla; conalaluiit wllli auto) yt Ic rtiungc Tubular casta.Inolated - very allgtit Coi Ileal tiilHilar cyat, laolated - very alight I'ot i, a of husoyli 11 Ic t oho lea - very alight I ol ci at 11 (ill uoiHiiHo'Icai 1 oil anaantory eel I a, local - vci y alight I'rlvli dilatation - alight 0 1 or 1 Hays Recovery s 4 1 0 1 1 3 0 0 0 0 0 I) I O IVirl bund or Dead after ^ 1-4 Exposures 4 1000 1 or 5 Days Recovery i 1) llaya Reroscry 4 4 40 4 i0a 0i 4 . 10 0 1- I0 0 1A 0- 1000 1 M 1 Ihtya Rncovar.y 1 4 1 i o 11 Itifi Reruvary ~4~ 1 1 n o 10 1 0 0 0 0 1- o 1 l- 0 00 0 0-1 0 0- 1 Il.ila incscntcd arc lumber of anlniln having the a laird observation. Ilroufied by days jfter final (fourth) tiyoaurt when nee tops led. Includes ) dead alter three i-a|>osiiri`a nml 1 dead and 1 nortbond alter four exposures. - I ml lent ca not a|>|*l I cable. -c a' r i * 109120 S9U TABLE 7 (Conilmied) VINYL QfUfllliLi KLSULTS TWO SHOUT-TEAM IMIAIATION SIHfUES HI(o|ij(tM)lo|1c Obaervst |on - StuJy A Mice Eipoimc IfvcI Qm>) ____ _ HU OpIiJF CfOQp* Nunliet ol hTci in Cioup Thymus (iiualir vkmIiiinJ iilctUHUiplccIly) No visible lesions Atrophy, Jos* of curl leak lyMjitiucyUii, with UiyunlMctlc ducUt - til Ighl - noJerat* - MrUit KlaryoitWcttc nuclei t medulla - alight - Moderate Mrkvd lopMUc riMiikJ bodies In norlvx (alnerel lied JWla) Spleen (lumber chmIrvi} nlcruacopJcalIy) No visible lesions JyMphulJ depletion - slight * f 4) I V - MrUJ LAC ranedul lot y hcjuj|Hites J s - slight to Moderate Heacnlet lc I jf^ph NoJe (miaber exJoined ildoscoptciiry) No visible lesions l.yMphol J depletion - Moderate - SJikeJ D 1 or S luyt Recovery__ 3 3 0 0 0 0 0 0 u I) Kirlbuod or Deed after ^ 3-A llaposurea * 1000 3 or S Pope 13 Ihiys Becovety Rrrovery ___________ " 3 30 0) 6 lha* p rusen I rd Jiu matte I of mi I tula having Ih* slot* J observation. LroupcJ by days after final (fourth} exposure uWn necropvled. Include* 3 dead after thfee ex|H>*uiea and I dead and 2 nor I bund alter four exposures. " Indicates not applicable. 3000 3 or 5 buy* ft covety * 13 buys Recovery r < 1 309120 S9U Mm m,ssa TABU 7 (Continued) VI KYI. CKl-ORIDEl RESULTS OF TWO SHORT-TERM INHALATION STUDIES IIIs topatliologIc Obmvittona - Study A Mice Kji|Mtiiuj e I jppn) __ Ktfg ii a f Croup | Nu4t of Mice In (*fip ) or 5 Daya Recovery tier lbuad or Dead after VA hpsawte8 A Thoracic typli Node (mtaber naelned alcroacoplcallf) No visible lesions Acute aubciipitular Iteaorrbagt! Heart (nusdier vnailtteJ Icroacopldlfy) Ho visible leu Ion* Uolaivd local ateu of Imite granular hasopbl 1 Ia {Incralltatlon) of ayocyta ayloplas*, eterpacopk - very alight UngM fnuwber (*xae1iwd Microscopically) Ni visible lesions fjfeM - very slight to slight - prrlvaacular and per(liroiiclilaI alveolar Alveolar uacrupli.igcs,, Increased * very slight Cocpu (mjoher examined Microscopically) Ho visible loalous Siihasicossl edest, diffuse - soderatc Depletion of cells of'lmina propria very 1Ight Colon (mmber exes filed si rroscoplcnl ly) Ho visible lesions Sobsuicossl edeaui * slight Data paeaented are nusher of anlisals having the stated observation. o* Cropped by days after final (fourth) exposure when necropeted* b Includes ) dead after three exposures and I dead and 2 nor (bond after four exposures. - Indltalca not applicable. 1000 1 or 1 Days Bee over t 13 Days Recovery 4 HiWWlMii 09ISO S9U **" ihhi'IUMBMP1 r:;i/ . It-a TABte J (Cant Inunl) vitm. cmakiw:-. hesults or too shout-tehh imiaiatiun studies H lelofallmloglc Obeervat Ion* - Study A Hite Etpafcnrr Itvvl (________ _____ JieClujiny Cl W]I __ Hunber ol hic In Croup twufHi (itmliti emlneJ 1 cronroji leal ly) Ho vlatble llni U'ctaiud luUUl separation, eu*gcalt*e 3 or 3 Diijii Httiiwi y Kor I bund or Dead after ^ 1-1 Erpoaurea _________4 6 S 1 I (HK) ) or 1 Uaya recovery S 1) Raya recovery 6 $;<.. I Tln (lumber (Ukilnwl nlcroacoplcalIjf) Ho vlalble Itilaiu Scattered tperamlJ giant cello Epldldynle (iHiREtr vuilih'J olcroacoplcatly) Ho vlalble leolono Focol nt'croolo of nucoea - very allgbl IHcrcwi'il aprraJtoioa 111 iuorn Fur lit* fulluwjng tteouee, no visible IcoIimib (moii'l oilnl nlcroacoplcaIty)I Aar t Urdu Cmjulii |ii| Claud Kao|dia|ue I*iyn faralhyiold Claud ferlplieral Hrrvn (aclalle) Mtullory Cland Prostate Sjllvary Clanda Sonina I Vealcleo Skeletal Hoscle Snail Inteellne St onJell Ttiyruld (II undo Trarliea Uiluary >1ad<*~; 0 0 u A 0 0 n o o o o o o 0 0 0 0 A 4 J 3 6 I A A 6 6 5 I 6 4 4 A 4 Hal a pr a willed aie lumber of an taut a bavins f.bo Mated oboer vat Ion, aCrou|>ed by deya aflor find] (fourlli) ei|H)M<ro when nocropaled, I nr) odea ) dead oflar I hrvo ea|>oaurei and 'I dead end 2 nor I bund after four eapoouree. - Indicator not appllcahlo. A 0 0 0 A 0 0 0 0 A 0 0 0 0 0 0 0 0 0 0 0 0 0 A 0 0 0 0 0 0 0 0 0 0 frOJHZO S?H ________ MHIO 3 nr 3 Uaya Hecovety V 13 Uaya recovery ___ 4___ oA a A 0A II II 0 (I 0A 0 II 0 II 0 (I 0A 00 0A A0 0A 00 0a 0A 00 0 II 0A `flWSAfUdflf -40- TABLE 8 VINYL CHLORIDE: RESULTS OF TWO SHORT-TERM INHALATION STUDIES Histopathologic Observations - Hamsters8 Exposure Level (ppm) Liver (number examined microscopically) No visible lesions Hepatocyte microveslcular change, centri- lobular and midaonal - very slight to slight Multifocal microscopic aggregates of mono nuclear and reticuloendothelial cells - very slight Isolated small focus of subcapsular coagulation necrosis with mixed Inflammatory cell reaction Periportal lnflasasatory cell accumulation, mixed type - slight - moderate Hemosiderin-laden macrophages, periportal - slight Gallbladder (number examined microscopically) No visible lesions Submucosal mononuclear cells, primarily lymphoid, focal ' - slight diffuse - very slight Adventitial mononuclear cells - slight Kidneys (number examined microscopically) No visible lesions Dilatation of multiple cortical tubules and epithelial vacuolization - very slight Eosinophilic hyaline casts - very slight Thymus (number examined microscopically) No visible lesions 0 1000 5000 33 3 000 202 12 2 010 210 11 1 100 12 3 0 12 0 10 00 1 10 0 3 33 3 32 0 01 0 01 33 3 33 3 Data presented is number of hamsters with the stated observation. aHamscers necropsied 5 days after fourth consecurive exposure to vinyl chloride. R&S 021605 VINYL CHLORIDE: RESULTS OF TWO SHORT-TERM INHALATION STUDIES Histopathologic Observations - Rats* Exposure Level (ppm) Liver (number examined microscopically) No visible lesions Microscopic mononuclear and reticulo endothelial cell aggregates, multifocal, occasionally adjacent to isolated degenerated hepatocytes - slight Periportal inflammatory cells, mixed type, - slight - moderate Kidney (number examinedmicroscopically) No visible lesions Scattered tubules with eosinophilic casts - slight Cortical Interstitial mononuclear cell accumulation, focal ~ veryslight Focus of fibrosis and chronic inflammation - slight ~ Focus of mononuclear cells, primarily lymphoid, beneath pelvic epithelium - very slight Isolated cortical focus of hypertrophied, basophilic tubules - very slight Thymus (number examined microscopically) No visible lesions Petechial hemorrhage of medulla - very slight Anterior Mediastinal I.vmoh Node (number examined microscopically) Lymphoid hyperplasia and sinusoidal hemorrhage 0 3 0 1 2 1 3 0 1 2 1 0 1 3 3 0 0 1000 3 0 3 3 0 3 1 1 1 0 1 0 3 1 1 1 Data presented is number of rats with stated observation. *Rats were necropsied 5 days after the fourth consecutive exposure to vinyl chloride. - Indicates not applicable. R&S 021607 -v* fs^v?- - mf.'.. . , TABLE 10 vim nnnoii eesults or tho shou-tol". lstuiatiom stvoies Histopathologic Observations - Study B Mix* Exposure Level room) Necropev Croup Number of Mice in Croup 0 Final Termination 3 Mo visible las ions Acuta coagulation necrosis, - focal, eentrllobular, about 101 of area examined, oftoo aubcspeulir - focal, cancrllobular to confluent, involving 231 of axaa of aac cions examined - focal, cancrtlobular to confluaat, involving 26-301 of araa of aoctlons examined - focal, csntrllobular to confluanc. Involving 31-73: of araa of auctions examined Isolated focal nacrotic araa - slight Hlouta, Intracellular basophilic a true Curas at margins of nacrotic areas parloserotic loll amatory calls, sixad - vary alight to alight Ferlntcrotlc subacute Inflammatory and - connective tissue i - alight ferinacrocic swollen, vacuolated hapatocytaa Cantrllobular hapatocytaa, alcrovaelcular change - vary alight to slight - narked Cancrllobular end aid tonal hapatocytaa enlarged, homogeneous cytoplsso of slightly increased baeophllla, occasional nltoees local aubcapeular bepatocyta degeneration with vacuolaclon and hyaline aatarlal Sluaoldal congestion and hmorrhaga, in and near nacrotic areas - moderate - narked Canrrilobular vain congestion, compression of adjacent hapatocytaa - narked Focal telangiaccasla Small hyaline masses In slraiaolds, aarglna of nacrotic foci or In areas of swollen hapatocytaa Multifocal microscopic foci of hapatocyta degeneration or necrosis with aonotaiclear inflametory call reaction - vary slight to alight 3 1 Callbladder (ouaber axialnad microscopically) Mo visible lesion.* 1000 Fin, J 2 Exposures Termination 1 4 14 00 3000 Final Tarnination 5 3 0 Data presented are number of animals having the tea ted observation. *Nacropsiad after five expo auras, bA. nimal died attar two exposures. * * * 3 t ... v?-.- ` ,; R&S 021608 IfeT: -'v te1 r^v , , .-",v,, := '''v ' ; TAILS 10 (Coorinuad) , ran' cHLorat:v iisms or tvo sacst-tsek nniAiAiicst stvoiis Hlatopachaloflc OSaarvatioaa - Study 5 Stlea '^ J t A ;'Jk -<l K" . u S!..j. * EExposure Laval (teal' -v . L 1 X' aeropsv Croup ' , . i , . ' " " r................... 0 riaal TtniaACioc 1000 .. Final 2 SxDOiurii `lamination 3000^1 Final: '* Tarsination Nuabtf of Mlc* is Crouo s Is 4 3 ^iSS$rC-- nr " \i-T- : '*. m v y fs 'K':' v. 'Cr'^vr--'--. . Kldcav (suxbor rraolnaH olcroacsplcally) ' ,5 Ho visible las Iona ; 3 Idarjed calla, acattarad. lunar cortical cubulaa . ' ' y - allsbt . ' Mlcrovaainulaclon. cortical cubulaa - ' - illjhc co sodarata Focua of baaopnille tubulaa : - vary alicht Intaraticlal aoooiatclaar Laf Lama cory call*. focal ... - vary iil*hc Hadullary caata. Iso La tad, occasionally minarallxac - - sli|btV .... `1 Thveus (maoar axaalnad alcrofcoplcaliy)- 0 <- Ho vlillIt laaiona ' . .k Atrophy, loaa of cortical lyaphocycn, vlch : karyorr&axie oudal - flight i - narked laar.phllic round bod la la cortax (dnarallnod dabrli) Thyrofloiaal duct cyat, laolacad, alccoacsplc 4 ' '3 i 3 ` fJ- 0 'i. 0 0 "1 1 3 0 51'V- 0o 0 o; > <t j. ' : 'Y l o v" l i' o .i \ WV. Sata praaaetad ara aiabat of aalsals havlcf cb* stated ebacrvatlon. *liacropalad altar fiva nxposurs. b An laa I dlad altar two exposures. . " ^:v i., iifV.'J",. j . -" '. . i