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<sr MANUFACTURING CHEMISTS ASSOCIATION '1825 CONNECTICUT AVENUE. N.W. WASHINGTON. D. C. 20009 (202) 483-6126 CHEMICALS' RESEARCH MAY 1 3 t975 May 14, 1975 TO: Technical Task Group on Vinyl Chloride Research Gentlemen: Attached for your perusal is Industrial BIO-TEST Laboratories' May 9, 1975 report on the "Chronic Vapor Inhalation Study with Vinyl Chloride (Ethylene Derived) in Albino Rats." Sincerely, (f\v\ Milton Freifeld, Project Manager MF/etv Attachment VVC 000004458 %i&uA&uqJ BIO -TEST Jlak&bGJkWAzA', 3no. 1810 FRONTAGE ROAD NORTHBROOK, ILLINOIS 60062 REPORT TO MANUFACTURING CHEMISTS ASSOCIATION GROSS AND HISTOPATHOLOGIC FINDINGS ONE-YEAR INTERIM SACRIFICE CHRONIC VAPOR INHALATION STUDY WITH VINYL CHLORIDE (ETHYLENE DERIVED) IN ALBINO RATS MAY 9, 1975 IBT NO. 663-03222 vvc OOOOO4459 Sn&uAt/itid BIO -TEST Jjc&yuztoueA', Dnc. 1810 FRONTAGE ROAD NORTHBROOK. ILLINOIS 60062 May 9, 1975 Mr. Milton Freifield Project Manager, Vinyl Chloride Research Manufacturing Chemists Association 1825 Connecticut Avenue, N.W. Washington, D. C. 20009 Dear Mr. Freifield: Re: IBT No. 663-03222 - Chronic Vapor Inhalation Study with Vinyl Chloride (Ethylene Derived) in Albino Rats - Gross and Histopathologic Findings - One-Year Interim Sacrifice We are submitting herewith our laboratory report prepared in connection with the above study. Very truly yours. JCC: mcb J. C. Calandra President vvc 000<K460 yndttd&U''1 B I O - T E S T Xalt&iahyu&L, Due. REPORT TO MANUFACTURING CHEMISTS ASSOCIATION GROSS AND HISTOPATHOLOGIC FINDINGS ONE-YEAR INTERIM SACRIFICE CHRONIC VAPOR INHALATION STUDY WITH VINYL CHLORIDE (ETHYLENE DERIVED) IN ALBINO RATS MAY 9, 1975 IBT NO. 663-03222 I. Introduction At the request of the Manufacturing Chemists Association, 40 rats (5 males and 5 females) from the control and test groups were sacrificed on September 5, 1974, to conduct cytogenetic studies on bone marrow specimens obtained at time of sacrifice. The purpose of this report is to present the gross and histopathological findings in other tissues from these animals. VVC 000004461 jndUdtn' ' B I O T E S T Ja6o*a&ue, 2*tc. 2 II. Summary Treatment-related gross and/or histopathological findings were confined to the liver of test animals at exposure levels of 50, 200 and 2,500 ppm. Gross pathologic findings ascribed to vinyl chloride were confined to the liver of 2 animals of the 200 ppm level (group T-II) and the liver of 5 animals of the 2,500 ppm level (group T-III) . Findings consisted of solitary, dark red, nodular foci in the subcapsular region of single liver lobes. These nodules varied in size from approximately 4 mm to 4 cm in diameter and, on cross-section, they contained blood-filled cystic spaces. Upon histopathological examination, the above lesions were identified as sites of hepatic angiosarcoma. In addition, there was focal hypertrophy and hyperplasia of hepatocytes in the liver sections from test animals at each exposure level. The incidence and relative severity of this finding could be correlated with the level of exposure to the test material. No extra-hepatic angiosarcomas were observed. Materials and Methods All animals, selected at random for sacrifice, were rendered unconscious in a closed chamber by exposure to carbon dioxide gas and exsanguinated, and a complete necropsy examination was performed. The organs were weighed. Representative specimens of the following organs and tissues were placed in 10% neutral buffered formalin for fixation: VVC 000004462 Undudift/'1 BIO-TEST J.a&QteUvu&i, 9nc. Adrenal Glands All Gross Lesions Bone (femur and sternum) Bone Marrow (sternal) Brain and Spinal Cord Esophagus Eye Gonads (testes and ovaries) Kidneys Large Intestine (caecum and colon) Liver Lungs Lymph Nodes (tracheobronchial, cervical and mesenteric) Optic Nerve Pancreas Parathyroid Gland Pituitary Gland Prostate Gland Salivary Gland Seminal Vesicles Skin Small Intestine (duodenum, jejunum and ileum) Spleen Stomach Thymus Thyroid Gland Trachea Urinary Bladder Uterus The above tissues from all animals were processed by conventional methods, embedded in Paraplast*. sectioned at 4-6 microns, stained with Hematoxylin and Eosin and examined by light microscopy. The gross pathologic findings are presented in Tables I through II. The histopathological findings are presented in Tables III and IV. The pathologist's report is presented on pages 4 through 6. Respectfully submitted, INDUSTRIAL BIO-TEST LABORATORIES, INC. Report prepared by: Donovan E. Gordon, D.V.M., Ph.D. Section Head, Pathology Report approved by: V/. L. Kepi Manager, Toxicology mcb * Sherwood Medical Industries, St. Louis, Missouri 63103. VVC 000004463 !)tidt4&&u'`l B I O - T E S T 2ahyu2bvue6, Vhc. 4 IBT No. 663-03222 Manufacturing Chemists Association February 27, 1975 A gross and histopathological examination was conducted on a series of tissues and organs from rats of the 12-month interim sacrifice of IBT No. 663-03222. Treatment-related gross pathologic changes were confined to the liver of 2 T-II females (Nos. 550 and 599), 1 T-III male (No. 694) and 4 T-III females (Nos. 717, 732, 735 and 742). The lesions consisted of solitary, dark red nodules within the subcapsular region of 1 liver lobe. The nodules measured approximately 4 mm to 4 cm in diameter and contained blood-filled cystic spaces . There were fibrous adhesions between the capsular surface of some of these nodules and adjacent visceral structures. In 1 animal (No. 735), there were 2 different liver lobes with solitary nodular foci similar to the above. Upon histopathologic examination of the hepatic nodules, they were found to be angiosarcomas. A small amount of hemorrhagic fluid was present in the abdominal cavity of 1 animal (No. 59 ) as a result of hemorrhage from the surface of the liver tumor. Treatment-related histopathologic findings were confined to the liver of 5 T-I animals and all of the T-II and T-III animals. There was an exposurerelated incidence and severity of hypertrophy and hyperplasia of hepatocytes throughout the liver sections . The hypertrophied hepatocytes had an increase in cytoplasmic mass which had a light, eosinophilic staining appearance with a few basophilic condensations. In some sections, these cells formed small 9ttdn&fo' ' B I O - T E S T JlaA&iat&Uei, 9*ux 5 nodules with very little compression of the adjacent parenchymal cells of the liver. Some of the hyperplastic hepatocytes had prominent nuclei and nucleoli. They also formed small, discrete nodules in some areas. In addition, these cells often had a slightly increased cytoplasmic basophilia. In regard to the angiosarcomas, the early and more advanced stages of the tumor could be identified. In the early lesion, there was focal sinusoidal dilatation accompanied by hyperplastic and/or hypertrophied endotheliallike lining cells. The areas usually communicated with frank areas of angiosarcoma. The latter were characterized by the presence of blood-filled cystic sinusoidal spaces (peliotic foci) in which there were papillary pro jections of cords of liver cells that were covered by large, hyperchromatic to pleomorphic lining cells on the sinusoidal surface. The hepatocytes in the area adjacent to the tumor were also invaded by these tumor cells. In some areas, there was complete destruction of the enveloped liver cords and replacement by solid sheets of sarcomatous tumor cells which contained numerous mitotic figures and had anaplastic features. In most instances, the tumors were situated just beneath the capsular surface of the affected liver lobule and there was focal invasion of tumor cells into the capsule with hemorrhage into the peritoneal cavity at these sites. There were also fibrous adhesions to the adjacent visceral organs at the capsular surface of the tumors which correlated with the gross observations. In 2 animals (Nos. 663 and 668) , the tumor foci were too small to be detected microscopically and they were in the early stages of development. In this study, there were no metastatic VVC 000004465 Unduit/i' ' B 1 O - T E S T JtaJtMaitvU&> 9*ux 6 tumor foci found in other organs, however; tumor emboli were present in some of the vessels of the larger liver tumors. All liver lesions, observed by light microscopy, summarized in Table III. The other changes described in the liver and other tissues (Table IV) were regarded as lesions of naturally occurring diseases. The lesions were present, in most instances, among both control and test animals. The small (1-3 mm), red, subcapsular foci observed at necropsy in the liver of both control and test animals could be correlated with scattered foci of hepatocellular necrosis observed histologically in these animals. Donovan E. Gordon, D.V.M., Ph.D. Diplomate, American College of Veterinary Pathologists VVC 000004466 9ndu4bua 'M O - T E S T laliMotosuei., !)ac. 7 TABLE I TEST MATERIAL: Vinyl Chloride (Ethylene Derived) Chronic Vapor Inhalation Study - Albino Rats One-Year Interim Sacrifice Individual Gross Pathologic Findings in the Liver - Males Group Animal Number Gross Pathologic Findings Control 39 54 55 69 84 1 mm dark red foci in all lobes None Few 1 mm dark red foci in all lobes Numerous 1 mm dark red foci in all lobes None T-I 205 256 264 279 299 Few 1 mm dark red foci in all lobes 1-3 mm dark red foci in all lobes None None Numerous 1-3 dark red depressed foci in all lobes T-II T-I1I 404 433 459 469 489 609 639 663 668 694 Numerous 1-5 mm dark red depressed foci in all lobes Few 1-3 mm dark red foci in all lobes Few 1 mm dark red foci in all lobes 1 mm dark red foci in all lobes 1 mm dark red foci in all lobes, slight pale discoloration None Numerous 1 mm dark red foci in all lobes Granular surface with numerous 1 mm dark red foci in all lobes 1 mm dark red focus in 1 lobe 0.5 cm dark red subcapsular nodule in 1 lobe with capsular adhesions to spleen Vvc Qooo^ 67 9ndu4&ual S I O - T E 5 T la&QKit&Uei', 9*ic. 8 TABLE I continued TEST MATERIAL: Vinyl Chloride (Ethylene Derived) Chronic Vapor Inhalation Study - Albino Rats One-Year Interim Sacrifice Individual Gross Pathologic Findings in the Liver - Females Group Animal Number Control 106 149 160 178 200 Gross Pathologic Findings 1-3 mm dark red foci in all lobes Few 1 mm dark red foci in all lobes None Numerous 1 mm red foci in all lobes Few 1 mm red foci in all lobes T-I 303 349 369 381 397 Few 1 mm dark red foci in all lobes 1-3 mm dark red depressed foci in all lobes 1-3 mm dark red foci in all lobes 1-3 mm dark red foci in all lobes Few 1-2 mm dark red foci in all lobes T-H 524 550 569 594 599 Few 1-3 mm dark red foci in all lobes 0.5 cm dark red nodule in 1 lobe 1 mm dark red foci in all lobes 1-2 mm dark red foci in all lobes 4 cm subcapsular nodular focus in major lobe, capsular adhesions to diaphragm, spleen and serosal surface of small intestine T-III 717 728 732 735 742 0.5 cm dark red focus in 1 lobe with capsular adhesions to mesentery None 1 cm dark red focus in 1 lobe 3 cm nodule in 1 lobe; 0.5 cm dark red nodule in another lobe; focal capsular adhesions between nodule, small intestine, stomach, spleen and mesentery 4 mm dark red depressions in 1 lobe, 1-5 mm dark red foci in all other lobes VVC 000004468 Lung Kidneys Ovaries Adrenal glands Uterine horns <. < o o o O o o O' TABLE II TEST MATERIAL: Vinyl Chloride (Ethylene Derived) Chronic Vapor Inhalation Study - Albino Rats One-Year Interim Sacrifice Summary of Gross Pathologic Findings in All Tissues Except Liver Findings Group: Number per Group: Control 10/10 Male/Female Incidence T-I 10/10 Male/Female Incidence T-II 10/10 Male/Female Incidence T-III 10/10 Male/Female Incidence Focal adhesions to diaphragm or thorax Few pin-point red foci in all lobes Focal consolidation of 1 or more lobes Abscesses in parenchyma 3/0 1/0 0/1 0/0 1/0 0/0 0/0 1/0 2/0 0/0 0/1 0/0 0/0 1/0 1/0 0/0 Unilateral hydronephrosis Bilateral hydronephrosis Unilateral or bilateral follicular cysts 0/0 1/0 2 1/0 0/0 2 0/0 0/0 0 0/0 0/0 1 Small (1 nun) blood-filled cysts in cortex of 1 gland Enlarged and dark red color Hydrometra 0/1 0/0 2 0/0 0/0 0 0/0 0/1 0 0/0 0/0 0 `v w r ffo w iv j 1 S 3 1 " 0 I vO M O -T E S T TABLE II continued TEST MATERIAL: Vinyl Chloride (Ethylene Derived) Chronic Vapor Inhalation Study - Albino Rats One-Year Interim Sacrifice* * Summary of Gross Pathologic Findings in All Tissues Except Liver Organ Findings Group: Number per Group: Con trol 10/10 Male/F emale Incidence T-I 10/10 Male/Female Incidence T-II 10/10 Male/Female Incidence T-III 10/10 Male/Female Incidence Pituitary gland Spleen Hemorrhagic focus Few adhesions to mesentery Enlarged (3 times normal size) 0/0 0/0 0/0 1/0 0/0 0/0 0/0 1/0 0/1 0/0 1/0 0/0 Abdominal cavity Small amount of blood in peritoneal cavity 0/0 0/0 0/1 0/0 < n< o o oo o * 4- -w o o UncLt&Uial B ! O T E S T JtUxyia&yueA, --y A2 *? '1 s! ! u5 -r3 c3 <3 TJ* mj5 2> i |S||i .3 > 22_'su o u J*J nV s2* =2 U33 -U3 a,> 3 2,1 >v _ <J .2" - 533 33 --ej a"t "3Vi a. a. a. 11 22252 (S .N IN *' N -- (SI -- rm '1 tM N 2 22 N N N IN l*J !M .'J 3yjyl *--3 'w;)j i S3 C h ro m e V iijur In lia la tiu n S lm ly - A lb in o Uats G ra tlm # Syau-in and A b b re v ia tio n s *! V t5 11 a* ?3:J 0 5 .3 = <S Z3 --- art* ivd Kutl J(? M i/( 4 <r os j. ^ N3 .A 4CJ I<>N SN' 2^--a 9 ^ O3' <O"' *^03 -43-0) VVc 000004471 TABLE ill continued TEST MATlilUAL: Vinyl Chloride (Ethylene Derived) Cl ironic Va|inr Inhalation Study - Albino Rats One-Year Interim Sacrifice Summary of Liver Lesions* - Females Group anti Animal Nutiiiier Bile Duct Proliferation Portal to Sinusoidal Extramedullary li cma tupoiesis Necrosis ol llepalocvtes Cytoplasmic Hypertrophy l.lpld Vacuoles of in llL-paiocvtes llepalocvtes Hypertrophy and Hyperplasia of liepatocvtcs Sinusoidal Dilatation Subcspsular Throughout Reuion Only Section 106 149 160 i?a 200 T-l iOi 349 369 381 397 T 11 524 550 569 594 599 T-11E 717 728 732 735 742 1 1 1 1 1 1 I 1 1-3 1-2/M 2 11 1-2 (few) 1 1-2 1 1-2 12 1 1-2 1 1 1-2 1 1 1 1 J 1 1 1-2 1-2/M 2/M 2/M 2-3/M 2-3/M 2-3/M 2/M 1 1 1 1 1-2 1-2 12 12 1 i 2 1-3 2 2 1-2 2 2 2 Blood-Filled Cystic Spaces (Peliosis) Aneiosjicoina O -1 m in -4 jjs s" if PP VP PP PP PP PP VVC 0 0 0 0 0 4 4 7 2 Ail lesions arc focal unless otherwise indicated. Grading System and Abbreviations 1 = minimal in severity 2 = mild in severity lJ -- Present, nu grade 3 = moderate in seventy M- multifocal 4 = marked in severity D - diffuse 5 -- extreme in severity TABLE IV TEST MATERIAL; Vinyl Chloride (Ethylene Derived) Chronic Vapor Inhalation Study - Albino Rats DO o Cine-Year Interim Sacrifice Incidental Histopathologic Change s in Tissues Other Than Liver - Males Grean Findings Group: Number Examined: Control 10 Average incidence Grade T-I 10 Average Incidence Grade T-H 10 Average In cidence Grade T-1II 10 Average Incidence Grade Trachea Chronic tracheitis 2 2.0 3 1.0 2 1.8 3 1.7 Lung Chronic focal murine pneumonia Focal aggregates of alveolar macrophages Acute local bronchopneumonia 3 1.3 2 1.0 5 1.3 1 2.0 2 1.5 l 1.0 5 1.3 2 1.0 Heart Focal myocardial degeneration and tiLrusis 2 1.0 2 1.0 I 1.0 Kidney Bilateral focal proteinaceous tubular casts Bilateral focal tubular nephrosis Bilateral focal interstitial lymphoid infiltrations bilateral chronic focal nephritis Bilateral focal mineralized micro, oncretions in tubules Unilateral hydronephrosis bilateral hydronephrosis 3 1 2 1 1.2 1.5 1.0 2.5 2 1.0 I 1.0 1 1.5 1 2.0 2 1.8 2 1.8 1 1.0 4 1.5 3 1.7 1 1.0 VVC 0 0 0 0 0 4 4 7 3 Urinary bladder Proteinaceous plug iu lumen 2 1.0 1 1.0 3 1.0 4 1.0 Prostate gland Focal interstitial lymphoid infiltrations 1 1.0 Testes Unilateral focal testicular degeneration Unilateral focal testicular degeneration with spermatoceles, spermatic granulomas and dystrophic mineralisation ol some affected tubules 1 1.0 1 3.5 9*teuAi/ua B I O - T E S T JlsMcvuzl&u&i, Hkc. 50 V * T3 oa 41 * "a $2 <M -i M -- 0 V TJ rS.I *u * o rvj o <M a -a w 14 In c id e n ta l liiiJ u jta lljo lo y ic C hanges in T is s u e s O th e r T h a n L iv e r - M ales uj 73 '/I 35 *3 73 "3j 5 3 a '3 3 Sf o -3 < c~* 22 5u_ S 45 S oy s5 U 33 > 'J > > >> 3 "5 on >y 30 2 .s 2 *3 so] i 2 4 t sd `S '= = A J* 41 2, II ii ii H II II a e 2. w iM <*4 < in VVC 000004474 Sttdu&tMal B 1 O * T E S T Jlalxyiai&u&i. 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