Document 0L4BQ75qrpLoJr30oQQ0Nv18J

R&S 114496 < BIO-MEDICAL* RESEARCH 4 DOCUMENT DESCRIPTION FORM 63 68 69 76 Duplicate .in all cards:--> /f/7 year as-1961- File number [Right justify [Numeric only] 77 78 r~p^n Sub-Index Code Author(s), as Last Name FS (No Punctuation) and coden for journal as JAMA preceeded by one blank, space " r ' % SI 62 11 12 13 Title of Report; end with space-hyphen-hyphen-space. Follow with Index Terms, separated from each other with comma-space. Avoid other punctuation; do not abbreviate. 12 1--------------------------- ------------- :--------------------- " 61 62 21 ! ' `M^L /- 7v-r" -' W./ irj ^ 22 '/Lr# -r~l?2/*cr------- AfW' f://9 l/C f37~ y 23 24 Source (Journal, Vol., Number, Pages, Date ) 12' soaiLr .,/*- \ V7 7: rJf fs- & jJ ` F F2 3. ! if/ 1 62 31 32 Brief Summary 12 10 SUMMARY: 61 62 61 62 63 64 Ws/f hi *' i\,f tyF ,7J V * ` t% ` DOW CONFIDENTIAL INFORMATION R & D REPORT CRI number DOW CHEMICAL U.S.A. R&D REPORTS SHOULD REMAIN ON THE PREMISES OF THE DOW CHEMICAL COMPANY laboratory report code HET-K-1711-(29) DATE ISSUED February 3, 1981 CEP ARTMENT Health and Environmental Sciences, USA TITLE- VINYL CHLORIDE: RESULTS OF TWO SHORT-TERM INHALATION STUDIES L A b- PROBLEM NO. .g.l III 0 i 01 0 i 2 | 7 | 3 43 PAGES IN FULL REPORT A L THOR IS) CRI NUMBER R&S 114497 WITH CONCLUSIONS: A thirty-day cooperative study designed to examine the pulmonary effects of vinyl chloride (VC) inhalation exposure in male CD-I 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 (mice and hamsters only), 6 hours/day. By the fourth exposure, 4 of 30 mice exposed to 1000 ppm were dead and 2 more were moribund. There was no mortality in either of the other groups or species. VC-induced hepatic necrosis was determined to be the cause of death based upon viral serology, gross, and histopathologic examination. A second study utilizing only five male^CD--1 mice at 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 exami nation disclosed ascites, a variable degree of subcutaneous edema, and liver lesions. The livers were generally swollen, congested, and had variable sized necrotic areas. Hlstopathological examination disclosed centrilobular to massive hepatic necrosis. Lesions considered secondary to the hepatic necrosis were noted involving the lymphoid and circulatory systems. Mice surviving the exposures and allowed to recover for up to 13 days had evidence of hepatic reparative and regenerative processes. Generally, the hepatic lesions were more severe in mice exposed to 1000 ppm than in mice exposed to 5000 ppm VC. However, there was wide variability within each exposure group. In contrast to mice, there were no significant lesions noted in either hamsters or rats. The results of these two studies should be considered when evaluating results from experiments with vinyl chloride utilizing mice. / !/ DISTRIBUTION: i DEPARTMENT FILES RAD ADMINISTRATION CENTRAL REPORT INDEX - 4 COPIES (566 Bid*. - Midland) FORM C-4300 PRINTED R-3-80 Distribution list is continued on attached page. t t VINYL CHLORIDE: RESULTS OF TWO SHORT-TERM INHALATION STUDIES K. A. Johnson and H. 0. Yakel Reviewed by: M. J. McKenna Final -Report February 3, 1981 Toxicology Research Laboratory Health and Environmental Sciences, USA Dow Chemical U.S.A. Midland, Michigan 48640 R&S 114498 TABLE OF CONTENTS Page SUMMARY........................... 1 -INTRODUCTION.............................................................................................................. 2 MATERIALS AND 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 5 5 5 7 RESULTS ....................................................................................................................... 8 Chamber Analysis....................................................................................... 8 Animal Observations.................................... 8 Microbiological Determinations. . ........................ .... . . . 9 Gross Pathology....................................................................................... 9 Histopathology................................................................................................. 11 DISCUSSION........................................................................................................................17 SIGNATURE PAGE........................................................................................... 23 QUALITY ASSURANCE STATEMENT . . .. v--.... .....................................................24 REFERENCES........................................................................................................................ 25 TABLES 1 - 10 . 27 30 CD 4* CO CO oosnv SUMMARY A thirty-day cooperative study designed to examine the pulmonary effects of vinyl chloride (VC) inhalation exposure in male CD-I 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 (mice and hamsters only), 6 hours/day. By the fourth exposure, 4 of 30 mice exposed to 1000 ppm were dead and 2 more were moribund. There was no mortality in either of the other groups or species. VC-induced hepatic necrosis was determined to be the cause of death based upon viral serology, gross, and histopathologic examination. A second study utilizing only five male CD-I mice at 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. Histopathological examination disclosed centrilobular to massive - hepatic necrosis. Lesions considered secondary to the hepatic necrosis were noted involving the lymphoid and circulatory systems. Mice sur viving the exposures and allowed to recover for up to 13 days had evidence of hepatic reparative and regenerative processes. Generally, the hepatic lesions were more severe in mice exposed to 1000 ppm than in mice exposed to 5000 ppm VC. . However, there was wide vari ability within each exposure group. In contrast to mice, there were no significant lesions noted in either hamsters or rats. The results of these two studies should be considered when evaluating results from experiments with vinyl chloride utilizing mice. R&S 114501 2- - INTRODUCTION 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 premature termination of the study after 4 exposures. Because preliminary investigations indicated that the mortality was possibly due to unexpected acute hepatotoxic 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 repr jducibility of the previous observations and to obtain better necropsy sp scimens for histopathological characterization of acute lesions induced ay VC inhalation. This report will present the results-of_these two studies. Emphasis will be placed upon the l Lstopathological description of the lesions. -3- MATERIALS AND METHODS General Study Design The Initial study reported herein was a continuation of collaborative research pursued jointly with Dr. Yasunosuki Suzuki, Research Professor of Community Medicine, Mt. Sinai School of Medicine, New 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 mice 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 in the 0 and 1000 ppm 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 A exposures. The second study, hereafter termecLJitudy B, consisted of 5 male mice at each of the three above exposure levels. This study was scheduled, and lasted, for 5 consecutive daily exposures. 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 99.9% pure (Cortes,*1979). Traces of methyl chloride and vinylidene chloride were found to be the only impurities. R&S 114502 -4- Animals The mice used in both S tidy A and Study B were male CD-I mice, 5-6 weeks old (Charles River Breei ing Laboratories, Inc., Portage, Michigan), Hamsters were 4-6 week did male Golden Syrian hamsters (Engle Laboratory Animals, Inc., Farmersb>vfrg, Indiana). The rats were male Sprague-Dawley rats weighing about 225 grams (Spartan Laboratories, Haslett, Michigan). The animals were acclima t ed to the laboratory for at least 11 days prior to the initial exposure to VC. The animals were randomized from a single lot for each spec Les (separate lots for mice in Study A and Study B) by use of a computer generated randomization procedure. The mice and rats were individually i ientified by a numbered metal ear tag. The hamsters were individua1 Ly 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 60%), temperature (222C), a 12 hour photocycle, and 12 air c ranges/hour. Food (Ralston Purina rodent chow) and water were available ad libitum except during exposure. The animals in Study A ufei;re all weighed the day prior to the initial exposure to VC but subset[uent weights were not taken. All animals were observed for signs of tokc:icity as well as changes in appearance and demeanor. Chambers, Vapor Generati?n, and Analysis The exposure chambers wi elm stainless steel and glass Rochester-type chambers. Airflow throu ;h the chambers was maintained at 150 liters per minute. The air supply system is designed to provide chamber temperatures of approximately 21C an I 50% relative humidity. The VC gas was metered through a calibrated flo meter at a controlled rate into the main chamber air supply and f irther diluted to the desired concentration. 33 So (/) -P* cn o 00 -5- *OSHV S *U The analytical concentration of VC in each chamber was determined by infrared spectophotometry using a Miran I infrared spectrophotometer equipped with a variable pathlength gas cell. Analyses were performed at a wavelength of 10.9u at least 10 times daily 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. i 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/week. However, in Study A, the exposures were 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 re_ presentative animals from each exposure level/species were taken 5 days after termi nation of the exposures. Additionai~4onvalescent) serums were collected from 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, polyoma, minute virus, ectromelia, Sendai, mouse adenovirus, mouse hepatitis, lymphocytic choriomeningitis, and rodent corona. necropsy and Gross Pathology All animals presented to necropsy alive were anesthetized with methoxyflurane and the animal was killed by decapitation. -Immediately after -6- R&S 114505 decapitation, the eyes we :e examined in situ by gently pressing a glass slide against the cornea md examining the eye under fluorescent light illumination. A gross necropsy examinat Lon, both externally and internally, was performed with all altera :ions recorded. Representative portions of the following organs and tiss les were preserved in 10%, neutral, phosphatebuffered formalin from ea ;h mouse: adipose tissue adrenal glands accessory sex glands aorta bone with bone marrc w (thoracic vertebral column) brain epididymides esophagus eyes gallbladder heart kidneys lacrimal (Harderian) gland large intestine (cecum and colon) larynx liver lower jaw lungs lymph nodes (thoracic, mesenteric) mammary tissue pancreas peripheral nerve (sciatic) pituitary gland salivary gland (submandibular) skeletal muscle skin small intestine spinal cord (thoracic) spleen sternum stomach testes thymus thyroid (with parathyroid) tongue trachea urinary bladder all gross lesions Only livers and kidneys \ ere preserved from three of the hamsters and rats. For Study A, two mice/exj osure group were sacrificed three days after termination of exposures, Two days later three animals/species/exposure group were sacrificed. ' he remaining animals were held until 13 days after the termination of exposures, at 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 exposure, necropsies t ere not performed on these animals. However, the abdominal and thorac: c viscera from all mice were exposed and examined and the livers from six mice were retained in formalin. R&S 114506 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 1000 ppm VC dying after the third or fourth exposure in Study A, one mouse exposed to 5000 ppm dying 13 days after termination of exposures in Study A, and one mouse exposed to 1000 ppm dying after 2 exposures in Study B. These animals were refrigerated from the time death was noted until necropsy. The tissues and organs listed above were retained in 10%, 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 histopathological examination from all mice from both studies except those mice from Study A terminated at 3 and 13 days following the final (fourth) exposure. From these mice, only the livers with gallbladders were The four largest lobes of the liver (left lobe, right lobe and both portions of the medial lobe) were routinely prepared for histological examination. The livers, gallbladders (hamsters), thymus, and kidneys were prepared for histopathological examination from all rats and hamsters necropsied. For 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). -8- +. R&S 114507 RESULTS Chamber Analysis The daily mean analytics: concentrations of VC were calculated from multiple daily chamber a alyses. For Study A, these analytical concentrations were 968+33 ppm and 5137+165 ppm VC for the 1000 and 5000 ppm groups, respectively. Fi r Study B, these values were 99945 ppm and 5112173 ppm VC. Animal Observations The morbidity and mortal: .ty data from these two studies are summarized in Table 1. Study A: After 3 exposu es, some of the mice exposed to either 1000 ppm or 5000 ppm VC were leth rgic. Their coats were roughened and some had distended abdomens. On :he morning following the third exposure (prior to the fourth exposure) : mice exposed to 1000 ppm were dead and 3 more were moribund. During t ie fourth exposure 1 of these moribund mice died. The hamsters and ats appeared normal. At this time, the exposures were terminated an i all animals were held for further observation. The two moribund and fou dead mice exposed to 1000 ppm VC were presented for necropsy examination Three days after termination of the exposures, two more mice from each ;roup were necropsied. Clinically, these animals were all active but one louse exposed to 5000 ppm VC had a distended abdomen. Five days afte termination of the exposures, three more mice and three rats and hamst ;rs from each group were necropsied. These mice were active but two of t le animals exposed to 5000 ppm VC had distended abdomens. The remaining animals were observed for 13 days. During this interval, clinical abnor lalities were not noted; however, one mouse exposed to 5000 ppm VC w ls found dead thirteen days after the final exposure. -9- The rats and hamsters remained normal throughout the 2 week observation period. Three animals/exposure level/species were sacrificed five days after the fourth exposure. Study B: 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. Antibodies 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. R&S 114508 -10- The only gross lesions o nsidered to be directly related to the inhalation of vinyl chloride were noted in mice from both studies. Because of the irregular schedul : of termination with the accompanying variable, but small, group size am also because the lesions noted were similar in both studies, the result of the gross pathologic examination will be presented without regard to study but with interpretation allowing for the temporal responses o the recovery period. Externally, a low number of mice exposed to either 1000 or 5000 ppm VC developed distended abdo' ens. 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 af ter termin l tion of exposure; however, this was not noted in animals dead or morib1 rnd after 2-4 exposures. One of the five animals exposed to 5000 ppm VC f r 4 exposures and allowed a 5-day recovery period also had ventral iubcutaneous edema noted externally. The animals dying spontaneou ily after 3 or 4 exposures to 1000 ppm VC had reddened nares considere to represent agonal congestion. .Internally, some mice ex >osed to either 1000 or 5000 ppm VC had gross lesions suggesting toxic ty to the liver, lymphoid system, and possibly _ cardiovascular system, n the affected mice, the livers were usually enlarged and had a varie :y of color changes. Frequently there was hepatic congestion that rarled from generalized to a unique pattern of central or hilar congest .on with pale or blanched margins. The hepatic lobular pattern was oftei accentuated, apparently due to congestion, Foci compatible with acu e necrosis were noted. These varied from large foci up to several ram in size, occupying most of a liver lobe and varying in color from pa e to red, to multiple pinpoint pale foci, These necrotic-appearing areas were generally larger and involved more of the liver in those mi :e dying or moribund after 2-4 exposures to 1000 ppm VC than in mice surv Lving the exposure periods. R&S 114509 R&S 114510 -ll- Clear to yellow ascitic fluid and subcutaneous edema was also generally noted in the mice having liver lesions. The mice exposed to 1000 ppm VC had, generally, slightly more severe fluid accumulations than those exposed to 5000 ppm 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 B) 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. Histopathology The histopathologic observations made in Study A are presented in Tables 7 (mice), 8 (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 114511 histopathologic observatj|ons will be discussed by combining both studies, The lesions were similar in the two studies when allowance is made for the varying time period rom termination of exposure until necropsy. ----- Mice dying spontaneously or moribund after 2-4 exposures to 1000 ppm VC were characterized by aict te hepatic necrosis affecting a large portion of the liver. In the se1 en mice with this exposure history, about onefourth of the liver area was necrotic in one mouse, between onefourth and one-half was necroti in four mice, between one-half and threefourths was necrotic in ne mouse, and the remaining mouse had necrosis of over three-fourths of the liver as judged from histologic sections of the four major liver lob s. When the necrotic areas were small, they were centrilobular; howe rer, they were frequently massive and involved confluent lobules with o ay a thin rim of viable hepatocytes around the portal triads. Frequent y, minute basophilic intracellular granular structures were noted in swollen necrotic hepatocytes as well as viable hepatocytes at the margi is of the necrotic areas. These were demonstrated to contain calc i xm by special staining techniques. There was minimal inflammatory re,afction noted at the margin of the necrotic areas In these mice dying spon zaneously. The few inflammatory cells present were predominantly macro xhages. There were marked hepatic vascular changes noted in this gi *up of mice. ...There was usually intense congestion within and at tl 2 margins of the necrotic areas. In the areas of viable hepatic parent^;,yma there was often severe dilatation of the centrilobular vein with compression of adjacent parenchyma along with focal areas of sinusoids dilatation scattered throughout the hepatic lobule. These areas oft en appeared as vascular "lakes" among the parenchyma. The viable areas in the livers of these animals had centrilobular or perinecrotic hepatocytes generally characterized by swelling with vesicular or vacuo! ated cytoplasm. In some livers, these centrilobular or perinecrotic hepatocytes had increased cytoplasmic homogeneity and basophilia wit! occasional mitotic figures. A low number of small, eosinophilic. sinusoidal masses were frequently seen -13- among these cells. While this material could represent necrotic cellu lar debris, these masses were considered more likely to be microthromboses. The livers of mice surviving 4 or 5 exposures to 1000 or 5000 ppm VC had marked histopathological variability. The four mice sacrificed immediately after completion of 5 exposures to 1000 ppm VC (Study B) had acute centrilobular hepatic necrosis. While this was qualitatively similar to that noted for those 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 mlcrovesiculation of the centrilobular hepatocytes but lacked any significant amount of necrosis other than th scattered microscopic foci typically noted also in control animals. The other two animals were similar to those animals dying spontaneously in that they had large areas of acute necrosis (between one-fourth and one-half of the liver in one mouse and between one-half and three-fourths in the other). The inflammatory and vascular events roughly paralleled those noted in the mice dying spontaneously with comparable liver lesions. R&S 114512 With 3 to 5 days of reco\ e;ry following completion of four exposures to vinyl chloride, there was again variability in the histopathologic changes in the liver, Tlr ree of five mice exposed to 1000 ppm VC and 2 of 5 mice exposed to 500C ppm VC had changes of the centrilobular hepatocytes with either Ailargement and cytoplasmic vesiculation or cytoplasmic homogeneity, aasophilia, and occasional mitotic figures. In one mouse in each exposurfe level this reaction was more severe with marked cytoplasmic swellip;g and vesiculation, scattered necrotic indi- vidual hepatocytes and s Hiusoidal thrombi and dilatation. In one mouse exposed to 1000 ppm VC at 1 2 mice exposed to 5000 ppm VC, there were multifocal areas of cent:r|Llobular hepatic necrosis involving about one- tenth of the liver parenc|ryma. These were surrounded by a slight subacute inflammatory re; a in which fibroblasts were noted. In one mouse exposed to 5000 ppm VC, some multinucleate giant cells were noted in the reaction. R&S 114513 By 13 days after completi >n of the exposures, the livers of 4 of 6 mice in each exposure group we e similar to controls or had only slight microvesiculation and enllrgement of centrilobular hepatocytes. One mouse in the 5000 ppm VC ixposure group had marked centrilobular hepatocyte enlargement with occ LSional mitotic figures and sinusoidal microthrombi. Two mice exposei to 1000 ppm VC and one mouse exposed to 5000 ppm VC had multifocal are s of fibrosis with mononuclear inflammatory cells and, characteristic, lly, multinucleate giant cells containing a deeply basophilic structu: e. This was identified as calcified material by appropriate staining t. chniques. These fibrotic areas were centrllobular and often subcapsi lar. These livers also had slight periportal accumulations of mononucli ar inflammatory cells and slight bile duct hyperplasia was noted in ne mouse exposed to 1000 ppm VC. Foci of dilated sinusoids surroum ed by thin fibrous connective tissue walls were noted throughout the liver, particularly subcapsularly. The thymus had histopatho ogic lesions that closely paralleled the degree of hepatic necrosi Mice that were moribund or died sponta- neously had loss of the t ymic lymphoid cells that varied but was -15- usually 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 mice 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 hypereosinophilic 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 microvesiculation of the renal:cortical tubules. All other histopathologic observations made on the kidneys of treated or control mice were considered to be spontaneous entities and not due to exposure to vinyl chloride. % The hearts of 3 of the 6 mice moribund or dead after 4 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. R&S 114514 -16- Histologic examination o the liver and kidneys from the one mouse found dead 13 days after termi ation of exposure to 5000 ppm VC disclosed -- - moderate autolytic chang s. No lesions similar to those present in the _ affected mice were noted Although a cause of death was not determined, it was felt that this mo se did not die from VC-induced hepatotoxicity and it was not included n the gross and histopathology tables (Tables 3 and 7). 37 CO -b cn --i Cl R&S 114516 -17- DISCUSSION The acute deaths in mice 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 viral inclusion bodies nor bacteria were noted on histopathological examination. Mice failed to have titers to numerous viral pathogens, including those commonly associated with hepatic necrosis (ectromelia, mouse hepatitis virus* and reovirus). 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 autolytic 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 vesiculation, 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&S 114517 -18- These cells were enlargec , sometimes markedly, and mitotic figures were often noted. As this ch^n;ge was more prominent in the animals sacrificed after several days of recovery, this was considered a regenerative response. Next in the apparent pro gjression of lesions was-centrilobular necrosis, This was characteristics!1y coagulation necrosis with only a very slight peripheral inflammatory xjeaction. The inflammatory reaction that ensued consisted primariljy of macrophages and fibroblastic elements, The progression from the entrilobular hepatocyte swelling to necrosis was unclear. Frequently the perinecrotic hepatocytes were either vacuolated or contained e 3 sinophilic intracytoplasmic masses assumed to be degenerated cystosol proteins. However, these changes in the hepatocytes adjacent to erotic areas were of no greater severity than those present in cells in non-necrotic centrilobular areas. Thus, degree of vacuolar chang alone did not appear to correlate with the necrosis. Of interest are the minutfe basophilic structures noted in some of the necrotic hepatocytes and serinecrotic hepatocytes. These were demonstrated to contain calciub via the use of special histological stains. It is speculated that the;e structuresrrepresent mineralized mitochondria. Mitochrondrial calcification is commonly noted by agents primarily attacking the c ill membrane (Trump and Arstila, 1975) . In all mice dying after 2 4 exposures to 1000 ppm VC, there was progression from multiple small centrilobular necrotic areas to massive confluent areas of necros Is. Most animals dying spontaneously had necrosis of about 50% of he hepatic area on the four liver sections examined. Survivors gene rally had necrosis of 25% or less of the liver area. With mice exposed :o 5000 ppm VC, there were no deaths ascribed to VC and the amount of 1 .ver necrosis was generally less. However, in two mice exposed to 5000 >pm VC for 5 exposures (Study B), there was massive necrosis (up to 7 >%) and it is speculated that death may have been imminent had the an1 la1 not been sacrificed. R&S 114518 -19- The role of the vascular system in the pathogenesis of the liver necrosis is uncertain. In the animals dying spontaneously there was dramatic congestion in and adjacent to the 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, other 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 other 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 other causes. Popper et^ al. (1978) note a precursor stage, characterized by hepatocellular proliferation along with sinusoidal dilatation and sinusoidal lining cell proliferation. Their early lesions are similar to those 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 the sinusoidal lining cells leading to angiosarcoma. Also prominently affected by short-term inhalation exposure to VC was the lymphoid system, particularly reflected by the 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. There were several chang :s suggestive of possible disturbed mineral metabolism in the mice w .th liver necrosis. In addition to the intra cytoplasmic mineral note in and adjacent to the necrotic liver areas, mineral deposits were no :ed in foci in the myocardium and larger bodies in necrotic areas of the thymus. It is speculated that the mineralization is dystrophic du i to cellular injury rather than a reflection of calcium-phosphorous imba .ance. The kidneys had only sli ;ht changes considered to be insignificant in causing the death of the se mice. The tubular changes noted in the mice dying spontaneously were regarded as artifacts due to autolysis. With sacrifice and prompt fix l tion, the only lesion attributed to VC exposure was slight to moderate ml'crovesiculation of the cortical tubular epitheHum. Surprisingly, the acute lepatotoxic effect of VC in mice has apparently not been generally recog lized. Early short-term inhalation experiments noted that 30 minute exp >sures to 300,000 ppm VC were lethal to mice and rats, but 100,000 ppm pr >duced only narcosis (Mastromatteo et al. 1960). Prodan et al. (1 >75) calculated the LC,.^ for white mice as 11.75% VC in a ventilate L chamber. A dominant lethal test ii CD-I male mice (the same strain as that used in this report) exposed >y inhalation to 3,000, 10,000, or 30,000 ppm VC for 5 daily 6-hour expos i:res was reported by Anderson et al. (1976). These doses were selecte I on the basis of their unreported probe toxicity studies which dete rmined 30,000 ppm to be in the toxic range (the criteria of toxicity wer i not stated). In the published work, Anderson et al. had significant m >rtality (55%) at the 30,000 ppm exposure level and 5% and 10% mortality at the 10,000 ppm and 3,000 ppm exposure levels, respectively. T le cause of death was not stated. 3J fio W b --cnt <o -21- There have been several subchronic or chronic toxicity studies utilizing mice exposed to VC at levels close to those used in this report. Winell et al. (1976) exposed NMRI (albino) mice 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 Lefemine (1975), Keplinger et al. (1975), and Lee jet 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 the number of survivors at 16 weeks. Keplinger et d. 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__ 20 Co The only published report noting acute hepatotoxicity in mice is that of CO Lee et al., in which CD-I mice were exposed to 50, 250, or 1000 ppm VC JX for 6 hours/day, 5 days/week for 12 months. They report observations tn to o 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 R&S 114521 -22- their progress report (Nc 8 submitted to NIEHS) discloses no reported histological lesions for these 24 mice other than minor lesions also present in the control al; i Lmals. The studies reported here Ln confirm the acute hepatotoxicity of VC to CD-I mice as first noted iy Lee .et al. and present a more detailed histopathological description of the associated lesions. Several noteworthy aspects of this re s-ponse are presented including: 1. VC appears to bn acutely hepatotoxic at these relatively low concentrations inly in the mouse, particularly the CD-I strain of mice. 2. There is a cons Lstent association of greater severity of toxicity by vi.r rually any of the parameters effected (mortality, gro s lesions, or histologic lesions) in the lower exposure Level of 1000 ppm VC as opposed to those in the higher e :posure level' (5000 ppm). 3. There is wide va iabllity within a treatment group; i.e., up to 75% of the live necrosis and death in some mice exposed to 1000 ppm VC whi e others survive with virtually undetectable histological le ions. It is concluded that the >bservatlons reported herein need consideration when evaluating research .nvolving vinyl chloride exposure of mice. R&S 114522 This report was prepared and submitted by the following Staff Members: f^.sXL A. ______ Keith A. John6bn, D.V.M., Ph.D. Diplomate, American College of Veterinary Pathologists Project Leader H: 0. X^kel, B.S. Senior Research Biologist Inhalation Toxicology Date: =? Fj'jfl'Si Date .. vW. ^ /ts-/ Reviewed by: - -___________________ M. J-. ^McKenna, Ph.D. Diplomate, American Board of Toxicology Group Leader, Inhalation Toxicology Date:_ *//?7 /7 -_ - -24- qua: ity assurance statement VINYL CfLORIDE: RESULTS OF TWO TITLE OF STUDY: SHORT-'t: EM INHALATION STUDIES HET-K-1711-(29) In compliance with Good aboratory Practice Regulations, the study phases were inspected by the Qu lity Assurance Unit and the results of these inspections reported to anagement and the Study Director on the dates listed below- The repor accurately reflects the data generated in accordance with the regu ations and standard operating procedures of the laboratory- All data an the reports are located at the submitting laboratory. R&S 114523 Study Started: of Inspection: 30 Oct. 1979 Report Issued Date: 3 Feb. 1981 19 Oct. 1979 1 Nov. 1979 31 Jan. 1980~~ 3 June 1980 16 Jan. 1981 Date of Report: 19 Oct. 1979 2 Nov. 1979 . 5 Feb. 1980 5 June 1980 22 Jan. 1981 2d t ^ 6'-cis<,/V 3 W. E. Hoover Quality Assurance Toxicology Research Laboratory Health & Environmental Sciences 1803 Building Dow Chemical" U.S.A. Midland, MI 48640 Dctte $/ -25- REFERENCES Anderson, D., Hodge, M.C.E., and Purchase, I.F.H, Vinyl Chloride: Dominant Lethal Studies in Male CD-I Mice, . Mut. Res. 40:359-370, 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. W., Gordon, D. E., and Calandra, J. C. Interim Results of Exposure of Rats, Hamsters, and Mice to Vinyl Chloride. Ann. N.Y. Acad. Sci. 246:219-224. 1975. Lee, C. C., Bhandari, J. C., Winston, J. M., House, W. B., Peters, P. J,, Dixon, R. L., and Woods, J. S. Inhalation Toxicity of Vinyl Chloride and Vinylidene Chloride. Environ. Health Perspect. 21:25-32. 1977. Maltoni, C., and Lefemine, G. Carcinogenicity Bioassays of Vinyl Chloride: Current Results. Ann. N.Y. Acad. Sci. 246:195-218, 1975. Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. L. G. Luna, editor. McGraw Hill Book Co., New York. 3rd ed., 1968. Mastromatteo, E., Fisher, A. M., Christie, H., and Danziger, H. Acute Inhalation Toxicity of Vinyl Chloride to Laboratory Animals. Am. Ind. Hyg. Assoc. J. 21:394-398, 1960. Popper, H., Thomas, L. B., Telles, N. C., Falk, H., and Selikoff, I. J. Development of Hepatic Angiosarcoma in Man Induced by Vinyl Chloride, Thorotrast, and Arsenic. Am. J. Pathol. 92:349-376, 1978. R&S 114524 -26- R E F ] R E N C E S (Continued) prodan, L., Suciu, I., P: slaru, V., Ilea, E., and Pascu, L. Experimental Acute Toxicity of V: nyl Chloride. Ann. N.Y. Acad. Sci. 246:154-158, 1975. Stott, W. T. Protocol f< r the Investigation of the Mechanism of Tumor Initiation by Vinyl Chloride, 1,4-Dioxane, and 1,3-Hexachlorobutadiene, 1979. T1 e Dow Chemical Co., Midland, Michigan. Trump, B. F., and Arstil. , A. U. "Cellular Reaction to Injury" in Principles of Pathol iology, pps 9-96, 2nd ed., (1975). Edited by LaVia, M. F. and Hi: 1, R. B. Oxford University Press, New York. Winell, M., Holmberg, B.; and Kronevi, T. Biological Effects of Vinyl Chloride: An Exper: mental Study. Environ. Health Perspect. 17:211-216, 1976. R&S 114525 -27- TABLE 1 VINYL CHLORIDE: RESULTS OF TWO SHORT-TERM INHALATION STUDIES Morbidity and Mortality Exposure Level (ppm)___________________ STUDY A - A 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 __ 0 30 0 0 10 0 0 ~ 10 0 .0 1000 30 A (3,3,3,A) 2 (A,A) 10 0 0 10 0 0 5000 b2 (A,Ab) 10 0 0 0 STUDY B - 5 exposures Mice - Number Exposed 5 Number Died 0 (Number of exposures before death) Number with distended abdomens 0 (Noted at necropsy after-5 exposures)------ 55 10 (2) 32 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. SSfcf tn to Spec1es Mouse Mouse Mouse Mouse Mouse Mouse Hamster Hamster Hamster Rat Rat Exposure Level (ppm) 0 1000 5000 0 1000 5000 0 1000 5000 0 1000 TABLE 2 VINYL CHLORIDEt RESULTS OF TOO SHORT-TERM INHALATION STUDIES Viral Antibody Tltera From Mice, Hamsters, and Rats imiiDoay ncer toj Time of Hepatitis Rodent Sample PVM Reo 3 C0V II K Folvoma Minute Extreme11a Sendai Adeno Mouse LCM Corona 5_ 5 -- /- - - - - -- - - 5 - - I- - - - - -- - -- 113 _ _ 1 - m. _ _ _ ND 13 - - -- - - -- - -m 13 - - -- - - - -- - - ND 5 5 5 40 20 - _ --- _ - _ - _ _J80 - ^80 - - 40 - .. _ - -- -- 5 40 5 20 - --- _ _ _ .. 10 * __ _ 10 -- - _ _ aDays following termination of exposures. - Indicates no antibodies to the stated virus were present. ND - Not Done l IZStn S2H 82St?U. s^?y TABLE 3 VINYL CIIL0K1DE: RESULTS OE TWO SHORT-TERM INHALATION STUDIES Cross Pathologic Observation - Study A Mice Exposure Level (ppm) Necropsy Croup0__________ Number nf Mice In Croup 0 3 or 5 Days Recovery 5 Moribund or Dead after ^ 3-4 Exposures0 6 1000 3 or 5 Days Recovery S 13 Days Recovery 6 EXTERNAL OBSERVATIONS No visible lesions Distended abdomen Subcutaneous edema Reddened narcs focal cannaballsa INTERNAL OBSERVATIONS No visible lesions Ceneral Ventral subcutaneous edema - moderate to marked Ascites - clear abdominal fluid - moderate to marked Obese Liver Generalized enlargement or aveiling Lobar or large areas of color change, suggesting necrosis (either pale or reddened) Pinpoint pale foci, multiple, suggesting focal necrosis Congestion or reddened Accentuated reddening of hilar areas of multiple lohes Pale lobar margins, focal or diffuse Multiple small red foci Increased lobular pattern Generalized paleness S 0 0 0 0 X / 0/ 0f 0 0 0 1 0 0 0 0 X 2 0 0 4 1 0 0 0 1 s 2 0 4 0 2 1 5 0 Data presented are number of mice having the stated observation, ^Grouped by days after final (fourth) exposure when necropsled. ^Includes 3 dead after three exposures and 1 dead and 2 moribund after four exposures. .5 0 0 0 0 0 .0 0 0 X 0 0 X 1 X 1 2 0 6 0 0 0 0 5 0 0 0 0 0 1 0 0 0 0 0 0 5000 i | 3 or 5 Days 13 Daya ! Recovery Recovery i '5 i 6 2 3 X 0 0 1 2 .2 0 0 1 1 0 1 0 0 3 0 6 0 0 0 0 5 0 0 0 0 X 0 0 0 0 0 0 0 NIIo TABLE 3 (Continued) VINYL CHLORIDE: RESULTS OF TWO SHORT-TERM INHALATION STUDIES Cross Pathologic Observation - Study A Mice Exposure Level (ppm) Necropsy Croup Number of Mice in Croup Lungs Congested, diffuse Edema - slight Focal areas of emphysema Multiple red foci consistent with aspirated Mood nitllJLlU LdVllV Clear fluid - slight to moderate Mesentery Edema Stomach Caseous distention Congested or reddened Small Intestine Congested or reddened Decreased ingestu Kidney Congested or reddened Thymus Petechial hemorrhage 0 3 or 5 Days Recovery 5 0 0 0 2 0 0 0 0 0 0 1 0 Moribund or Dead after ^ 3-4 Exposures 6 1000 3 or 5 Days Recovery 5 13 Days Recovery 6 40 10 10 05 61 10 10 20 10 20 0 0 01 0 0 0 0 0 0 0 0 0 0 0 0 Data presented are number of mice having the stated observation. aCrouped by days after final|(fourth) exposure when necropsled. ^Includes 3 dead after three exposures and 1 dead and 2 moribund after four exposures. ezsfru s$u i 5000 3 or 5 Days Recovery 5 13 Days Recovery 6 00 1 (1 00 30 00 00 00 00 00 00 00 00 1 TABLE 4 VINYL CHLORIDE: RESULTS OF TWO SHORT-TERM INHALATION STUDIES Gross Pathologic Observations - Hamsters Exposure Level Number Examined EXTERNAL OBSERVATIONS No visible lesions Firm enlarged boney prominence on lower left rear leg 0 1000 5000 3^ 3 3 3 32 0 01 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""~---------- 1 1 1 0 0 0 2 0 2 0 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. R&S 114530 R&S 114531 -32- TABLE 5 VINYL CHLORIDE: RESU TS OF TWO SHORT-TERM INHALATION STUDIES Gross Pat idoglc Observations - Rats a Exposure Level (ppm) Number Examined EXTERNAL OBSERVATIONS No visible lesions 0 -1--0-0- 0j 3 INTERNAL OBSERVATIONS No visible lesions Thymus Multiple petechial hemo: rhages Lungs Multifocal red areas consistent with aspirated blood Data presented is number < f rats with stated observation, Rats were necropsied 5 di ys after the fourth exposure to vinyl chloride. R&S 114532 TABLE 6 VINYL CHLORIDE: RESULTS OF TWO SHORT-TERM INHALATION STUDIES Gross Pathologic Observations - Study B Mies Exposure Level (com) Necroosv Group Number of Mice in Group 0 Final Termination4 S EXTERNAL OBSERVATIONS No visible lesions Corneal cloudiness, focal unilateral - bilateral Distended abdomen Small size Clinically depressed 4 0 1 0 0 0 INTERNAL OBSERVATIONS No visible lesions General Ventral subcutaneous edema - marked - moderate - slight Ascites, clear abdominal fluid - marked - moderate Decreased abdominal fat 3 0 0 0 0 0 0 Liver Generalized enlargement Lobar or large areas of color change (either pale or reddened) Pinpoint pale foci, multiple Accentuated reddening of hilar area of multiple lobes with pale lobar margins Accentuated lobular pattern Generalized paleness 0 0 . _0 0 0 o - Lungs Congested, consistent with hypostatic congestion Pinpoint pink foci, few. Indistinct Multiple red foci consistent with aspirated blood 0 1 1 S tomach Submucosal edema, glandular portion - slight Multifocal pinpoint dark brown foci. glandular mucosa 0 0 Small Incestine Decreased ingesta 0 Cecum Increased consistency of ingesta 0 Thvmus Decreased size r slight 0 Vertebrae Prominent thoracic Intervertebral discs 0 2 Exposures lb 1000 Final Termination 4 11 00 00 03 01 00 00 0 0 0 0 0 o *: 1 0 0 1 1 ___ O' 1 1 1 3 0 1 3 2 3 0 2 2 10 00 03 01 00 10 1 0 04 00 Data presented are number of animals having the stated observation* *Necropsled after five exposures, b . Animal died following two exposures. 5000 Final Termination 5 2 1 0 2 0 1 0 0 0 0 1 1 4 2 2 0 2 2 0 0 0 2 0 1 0 0 3 1 TAISLF. 7 VINYL CIII.0RIDE: RESULTS OF TWO SIIORT-TERH INHALATION STUDIES Histopathologic Observations - Study A Mice Exposure Level (ppm) Necropsy Group Number of Mice In Group 0 3 or 5 Days Recovery 5 Moribund or Dead after 3-4 Exposures'* 6 1000 3 or 5 Days Recovery S 13 Days Recovery 6 Liver (number examined microscopically) No visible lesions Acute coagulation necrosis, - focal, centrllobular, about 10X of area of sections examined, often subcapsular - focal, centrllobular to confluent, 5 0 0 0 6 56 0 00 0 11 1 00 examined - focal, centrllobular to confluent. Involving 26-50Z of area of sections examined - focal, centrllobular to confluent. involving 51-75Z of area of sections examined - massive, large areas of liver, greater than 75X of area of sections examined Minute, intracellular basophilic structures at margins of necrotic areas Peri necrotic inflammatory cells, mixed, - very slight to slight Perlnecrotlc subacute Inflammatory and connective tissue cells - slight Perlnecrotlc swollen, vacuolated hepatocytes Centrllobular hepatocytes, talc rove slcular change - very slight to slight - marked 0 oi i 00 11 0 0 0 2 0 3 00 1 00 1 00 5 00 4 00 0 12 5 10 Data presented are number of animals having the stated observation. Grouped by days after final (fourth) exposure when necropsled. ^Includes 3 dead after three exposures and 1 dead and 2 moribund after four exposures. 5000 3 or 5 Days Recovery 5 13 Days Recovery 6 56 00 21 00 00 00 00 00 0 2 1 0 2 0 I cesfru svu i TABLE 7 (Continued) VINYL CHLORIDE: RESULTS OP TWO SHORT-TERM INHALATION STUDIES Cross Pathologic Observation - Study A Mice Exposure l,evel (ppu) 0 Necropsy Group Number of Mice In Croup 3 or 5 Days Recovery 5 Liver (Continued) Centrllobular and midzonal hepatocytea enlarged, 1 homogeneous cytoplasm of slightly Increased basophilia, occasional mitoses Focal subcapsular hepntocyte degeneration with 0 vacuolization and hyaline material Multinucleale giant cells containing 0 calcified material Sinusoidal congestion and hemorrhage, in and near necrotic areas - moderate 0 - marked 0 Centrllobular vein congestion, marked. 0 compression of adjacent hepatocytea Vascular ectasia, apparently midzonal, with 0/ comprunsion of adjacent hepatocytea | Focal telangiectasia 01 Small hyaline masses in sinusoids, margins of 0 necrotic foci or in areas of swollen hepatocytea 1 Hlcroveslcular change of periportal hepatocytea - slight 2 Multifocal microscopic foci of hepatocyte degeneration or necroBls with mononuclear inflammatory cell reaction - very slight or alight A - moderate 0 Periportal mononuclear Inflammatory cells - slight 0 Bile duct hyperplasia - slight 0 Moribund or Dead after ^ 3-4 Exposures ( 1000 3 or 5 Days Recovery 5 13 Days Recovery 6 2 21 1 11 0 02 0 01 6 00 4 00 2 00 0 20 1 21 0L 2 3 0 23 0 10 0 01 0 01 Data presented are number of animals having the stated observation. aCroupe`d by days after final (fourth) exposure when necropsled. ^Includes 3 dead after three exposures and 1 dead and 2 Moribund after four exposures. 5000 3 or 5 Days Recovery 5 13 Days Recovery 6 32 00 11 10 00 10 00 20 12 02 32 11 01 00 TABLE 7 (Continued) VINYL CHLORIDE: RESULTS OF TWO SHORT-TERM INHALATION STUDIES Histopathologic Obsatvations - Study A Mice Exposure Level (ppm) 0 Necropsy Group3 Number of Mice in Group 3 or 5 Days Recovery 5 Gallbladder (number examined microscopically) No visible lesions Submucosal edema - slight Submucosal Inflammatory cells, mixed. focal - very slight 4 3 0 1 No visible lesions Increased cytoplasmic granularity. cortical tubules - very slight to alight Hlcroveslculation, cortical tubules - Blight Tubular eosinophilla or mlcroveslculatlon and nuclear pyknosls; consistent with autolytic change Tubular casts. Isolated - very slight Cortical tubular cyat, isolated - very slight Focus of basophilic tubules - very slight Interstitial mononuclear Inflammatory cells, focal - very slight Pelvic dilatation - slight 3 2 0 0i 0 1 I 01 0 0 1 0 Moribund or Dead after . 3-4 Exposures0 6 6 6 0 0 6 0 1000 3 or 5 Days Recovery 5 4 3 0 1 3 1 13 Days Recovery 6 0 - _ 0 2 0- 0 14 0- 0 1- 1 1- 0 1- 0 000 Data presented are lumber of animals having the stated observation. *Crouped by days after final (fourth) exposure when necropsled. ^Includes 3 dead after three exposures and 1 dead and 2 moribund after four exposures. - Indicates nut applicable. sooo 3 or 5 Daye Recovery 5 13 Days Recovery 6 43 33 10 00 1 0 00- 00" 01 1_ I TAULE 7 (Continued) VINYL CHLORIDE) RESULTS OR TWO SHORT-TERM INHALATION STUDIES Histopathologic Observations - Study A Mice Exposure Level (ppa)_____________________________ Hecropay Croup____________________________________ Number of Mice in Croup _____________________ ThymuB (number examined microscopically) Ho visible lesions Atrophy, loss of cortical lymphocytes, with karyorrhectlc nuclei - slight - moderate . - marked Karyorrhectlc nuclei In medulla - slight - moderate - marked Basophilic round bodies in coctex (mineralized debris) Spleen (number examined microscopically) No visible lesions I Lymphoid depletion - slight - moderate - marked Extramedullary hemopoiesis - slightto moderate Mesenteric Lymph Node (number examined microscopically) Ho visible lesions Lymphoid depletion - moderate - marked ______ 0 3 or 5 Days Recovery ______ 5 3 3 0 0 0 0 0 0 0 0 - 0 - Moribund or Dead after . 3-4 Exposures1' 6 5 0 1000 3 or 5 Days Recovery 5 3 3 13 Days Recovery 6 0 10 2 020 1 0_ 2 02 01 0-- 6 00 0 -3 -2 -1 -- 4 -- 2 00 0 -1 -1 - Data presented are number of animals having the stated observation, Grouped by days after final (fourth) exposure when necropsied. ''includes 3 dead after three exposures and 1 dead and 2 moribund after four exposures. - Indicates not applicable. 9SSUI- SSU ' 5000 ' 3 or 5 Days | Recovery i5 13 Days Recovery 6 i4 3 0 0 1 0 .0 0 0 0 00 - - - - 0 - TABLE 7 (Continued) VINYL CHLORIDE: RESULTS OF TWO SHORT-TERM INHALATION STUDIES Histopathologic Observations - Study A Mice Exposure 1-evel (ppm) Necropsy Croup Number of Mice In Group 0 3 or 5 DayB Recovery 5 Moribund or Dead after ^ 3-4 Exposures0 6 Thoracic Lymph Node (number examined microscopically) No visible lesions Acute subcapsular hemorrhage 0 - - 1 0 1 Heart (number examined microscopically) No visible lesions Isolated focal areas of minute granular basophilia (mineralization) of myocyte nytoplasm, microscopic - very slight Lungs (number examined microscopleallv) No visible lesions Edema - very slight to slight - perivascular and peribronchial - alveolar Alveolar macrophages. Increased - very slight 0 - 1I I _1 - 5 3 ( 0 3 1 2 Cecum (number examined microscopically) No visible lesions Submucosal edema, diffuse - moderate Depiction of cells of'lamlna propria . - very slight 0 - _ - 1 0 I 1 Colon (number examined microscopically) No visible lesions Submucosal edema - slight 0 - - 6 5 1 Data presented are ^number of animals having the stated observation. Grouped by days after final (fourth) exposure when necropsled. Includes 3 dead after three exposures and 1 dead and 2 moribund after four exposures. - Indicates not applicable. 1000 3 or 5 Days Recovery 5 :o - 0 0 - 0 - 0 - 13 Days Recovery 6 0 0 0 - 0 - 0 - - sooo ' 3 or 5 Days Recovery 5 13 Days Recovery 6 0 0. _ -- 00 -00 -- _ _ -00 _ __ -00 - __ i iest?H s'sy 8csfru s^y TABLE 7 (Continued) VINYL CHLORIDE t RESULTS OF TWO SHOKT-TERH INHALATION STUDIES His topatliologic Observations - Study A Mice Exposure Level (ppm) Necropsy Croup* Humber of Mice in Group 0 3 or 5 Days Recovery 5 Moribund or Dead after ^ 3-4 Exposures* 6 Pancreas (number examined microscopically) No visible lesions Increased lobular separation, suggestive of edema 0 - - 6 5 1 Testes (number examined microscopically) No visible lesions Scattered spermatid giant cells Epididymis (number examined microscopically) No visible lesions Focal necrosis of mucosa - very slight Decreased spermatozoa in lumen 0 - - G - / 6 4 2 6 4 i 1 For the following tissues, no visible leslonB (number examined microscopically)! Aorta Brain Coagulating Gland Esophagus Larynx Parathyroid Gland Peripheral Nerve (sciatic) Pituitary Cland Prostate Salivary Glands Seminal Vesicles Skeletal Muscle Small Intestine Stomach Thyroid Glands Trachea Urinary Bladder I I 0 0' 0 0 G 0 0 G 00 0 0 0 0 0 0 0 11 1 4. 6 3 5 6 2 4 4 6 6 5 1 6 6 6 4 5 Data presented are number of animals having the stated observation. aCrouped by days after final (fourth) exposure when necropsied. Includes 3 dead after three exposures and 1 dead and 2 moribund after four exposures. - Indicates not applicable. I 1000 3 or 5 Days Recovery 5 0 - .- 0 - - 0 - - 0 0 0 '0 0 0 0 0 0 0 0 0 0 0 0 0 0 13 Days Recovery 6 0 - - 0 - - 0 - 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5000 3 or S Days Recovery 5 13 Days Recovery 6 00 _ 00 00 i f 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 -40- TABLE 8 VINYL CHLORIDE: RES ILTS OF TWO SHORT-TERM INHALATION STUDIES Histopathologic Observations - Hamsters a Exposure Level (ppm) Liver (number examined m:j croscopically) No visible lesions Hepatocyte microvesicular change, centri- lobular and midzonal - very slight to slight Multifocal microscopic aggregates of mono- nuclear and reticulo iidothelial cells very slight Isolated small focus of subcapsular coagulation necrosis tfith mixed inflammatory cell reAction Periportal inflammatory cell accumulation, mixed type slight moderate Hemosiderin-laden macrc|phages, periportal slight Gallbladder (number examined microscopically) No visible lesions Submucosal mononuclear cells, primarily lymphoid, focal slight diffuse very slight Adventitial mononucleai cells slight Kidneys (number examined nicroscopically) No visible lesions Dilatation of multiple cortical tubules and epithelial vacuolizattLcon very slight Eosinophilic hyaline ca^ts very slight Thymus (number examined n Lcroscopically) No visible lesions 1000 5000 333 000 2 10 111 123 012 0 10 001 333 332 001 001 333 333 30 (/> J* CJl 03 (D Data presented is number if hamsters with the stated observation, hamsters necropsied 5 da rs after fourth consecutive exposure to vinyl chloride. -41- TABLE 9 VINYL CHLORIDE: RESULTS OF TWO SHORT-TERM INHALATION STUDIES 4 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 examined microscopically) No visible lesions Scattered tubules with eosinophilic casts - slight Cortical interstitial mononuclear cell accumulation, focal - very slight Focus of fibrosis and chronic inflammation - slight Focus of mononuclear cells, primarily lymphoid, beneath pelvic epithelium 1 - 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 Lymph 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 2 1 1 1 Data presented is number of rats with stated observation. aRats were necropsied 5 days after the fourth consecutive exposure to vinyl chloride. - Indicates not applicable. o v s H I S'8* -42- R&S 114541 TABLE 10 VINYL CHLORIDE: RESUIfS OF TOO SHORT-TERM INHALATION STUDIES Hiscopacholog .c Observations - Study B Mice Exposure Level (ppm) Necropsy Group Number of Mice la Group Final Termination 5 Liver (number examined microscopically) No visible lesions Acute coagulation necrosis, - focal, centrilobular, about 102 of examined, often subcapsular - focal, centrilobular to confluent. Involving 252 of area of sections examined - focal, centrilobular to confluent. Involving 26-502 of area of sectl examined - focal, centrilobular to confluent, involving 51-752 of area of secti examined Isolated focal necrotic area. - slight Minute, intracellular basophilic struc at margins of necrotic areas Perinecrotic inflammatory cells, mixed - very slight to alight Perinecrotic subacute Inflammatory and connective tissue cells - slight Perinecrotic swollen, vacuolated hepati cytes Centrilobular hepatocytes, microvesicu. a.:r change - very slight to slight - marked Centrilobular and midzonal hepatocytes enlarged, homogeneous cytoplasm of slightly increased basophilia, occasional mitoses Focal subcapsular hepatocyte degenerat; oa with vacuolatlon and hyaline material Sinusoidal congestion and hemorrhage, necrotic areas - moderate and near - marked Centrilobular vein congestion, compresjion of adjacent hepatocytes - marked Focal telangiectasis Small hyaline masses in sinusoids, marf tna of necrotic foci or in areas of swoU sn hepatocytes Multifocal microscopic foci of hepatocy b degeneration or necrosis with mononuc Lear inflamina tory cell reaction - very slight to blight Gallbladder (number examined microscopic: Lly) No visible lesions 1000 Final 2 Exposures Termination ,b 5000 Final-Termination Data presented are number of animals havi ig the seated observation. ^ecropsied after five exposures. Animal died after two exposures* ff. -43- R&S 114542 TABLE 10 (Continued) VINYL CHLORIDE: RESULTS OF TWO SHORT-TERM INHALATION STUDIES Histopathologic Observations - Study B Mice Exposure Level (ppm) Necropsy Group Number of Mice in Group 0 Final Termination 5 Kidney (number examined microscopically) No visible lesions Enlarged cells, scattered. Inner cortical tubules - slight Mlcrovesiculatlon, cortical tubules - slight to moderate Focus of basophilic tubules - very slight Interstitial mononuclear inflammatory cells, focal - very slight Medullary casts, isolated, occasionally mineralized - slight Thymus (number examined microscopically) No visible lesions Atrophy, loss of cortical lymphocytes, with karyorrhexlc nuclei - slight - marked Basophilic round bodies In cortex (mineralized debris) Thyroglossal duct cyst, isolated, microscopic 5 3 0 0 1 1 1 5 5 0 0 0 0 1000 Final 2 Exposures Termination lb 4 5000 Final' Termination 5 14 13 00 01 00 00 00 5 0 1 3 1 0 0 3 3 0 2 1 1 Data presented are number of animals having the stated observation. aNecropsiad after five exposures. bAnimal died after two exposures. DISTRIBUTION ADCOCK, L.D., 2020 Dow C nter AXE, F.D., Pittsburg, CA BEARDEN, C.R., Granville BRADLEY, T.D., 834 Bldg. BRANSON, D.R., 1803 Bldg BRAUN, W.H., 1803 Bldg. 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