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OCT 2 9 19-4 THE DOW CHEMICAL COMPANY October 25, 1974 BENNETT BUILDING 2030 DOW CENTER MIDLAND. MICHIGAN 48640 Mr. K. D. Johnson Manufacturing Chemists' Assoc. 1825 Connecticut Ave., N.W. Washington, D.C. 20009 Mr. H. L. Kusnetz Shell Oil Company Room 1574, One Shell Houston, TX 77002 Plaza Three protocols are attached. The first summarizes briefly the current study at IBT. The second summarizes the study the committee developed Sept. 18, 1974 and which it agreed should be discussed with the Rail Committee. The third is a proposal I put together since I don't like proposal #2. I see no reason to omit hamsters and I would like to delay the start of more mice until we have metabolic data. My proposal accomplishes both. I'll be calling you next week. Sincerely, ct 9. ^cdeddmd Theodore R. Torkelson Corporate Medical Department kjf end. CMA 001470 C':'I? MANUiWJTURX ASSOCIATION, INC. CHRONIC VAPOR INHALATION TOXJ.CHTi' STUDY WITH VINYL CHLORIDE IaDUSTRIAL B10-TEST L v ,0PAT0Pi2S DECATUR, ILLINOIS I. Outline of Invest i gat ion A. Typa and Lei.UthJ_ 9-month vapor inhalation ir. White Mice + S months observa-. Lon lz~nonLh vapor inhaJanion in Albino Rats + 12 months obscr'-v.-ti'jn 12-month vapor inhalation ir. Hamsters + 12 months observation. B. Number f. An in'-'' is : 800 Mice. 900 Robs. 800 H:.V.~ C. ExtJO^ u : W r-nuie : i .-.,i ii(;iivn per nv_ Five days per week. D. Tost M itcr le.J.ii: Vinyl Chloride (Ethylene doth-... : E. Organlza ~ici:: a y p] )Xe 1 F. Dose Leveinp See Table I. G. Exposures Stortap: ` ExpQ3ures~~Urrnlr\vj-d Terml r,1 Aut.oo sy : Sept, ll ?.'2 7 mice, rats 1 hams torn J ui: c 19 7 4; mi c a. Sept. 1974; rats & hamsters. March 1975; mic e *. Sept. 1975; rats & hamsters. C0II7IP3ITTIAL Subject to Protective Order In Kqs3 v. Conoco, Inc., Do. 90-4837 14th Judicial District Court Calcasieu Parish,, Louisiana . NOTE: Excess mortality made it necessary to sacrifice all TL'-IT and TE-JIl n:ce alter ii months on ey;.v;,; i.p.ent. -I- CMA 001471 itst "aterial '. .1, '.*1 Vinyl Chloride {Ethylene derived) TABLE I CHRONIC VAPOR INHALATION TOXICITY STUDY Organization of Groups Group f/.ica Males Females Number of Animals Rats Hamsters Males Females Males Female Control 100 100 100* 100* 100 100 TS-I Low Level 50 ppm too TE-II Intermediate Level - 100 200 ppm T2-III High Level 2500 ppm LOO T3-IV High Level 2500 ppm with food - 100 ICO 100 - 100* 100* 100* - 100* 100* 100* 100 100 100 100 - 100 100 100 - f'Saoh of these rat groups were reduced by sacrifice ^f 5 rats for cytogentic and histopathological 2xam ^nation at the end of-.the 12 th month of exposure. CMA 0 0 1 4 7 2 v:" ... --r.:,Trr,R cirflor m ect to Pv0^" ilo. 90-4837` Ross v.-^cnor:o_.--Court 04tb Xudxclul^^ I(0al6lana XI. Chamber Parameters Each group of animals will be exposed in a specially constructed stainless steel and plexiglass inhalation chamber having a capacity of approximately 9.3 allowing animal loading of less than 2% when the animals reach maturity. Flow rate through the chamber will be at least 3 1.53 M /min. providing a theoretical air change every six minutes. The chamber supply air will be filtered and maintained at 40% to 60% relative humidity and 70 to 75F. Food will be removed frr all animal cages during exposure except Group TE IV rats. . -T* III. Animal Parameters A. Clinics 1 Observations '....`-.S'. C--'r'Vn sti-sia.n3- 14 J' ^ a\l i'i" calc-B All animals will be observed daily for lesions and behavioral changes attributable to the test material. The time of appearance and location of all tumors that occur among both control and test animals will be recorded. Mortality records will be kept on all groups of animals. All animals which die during the study will be necropsied and their tissues processed in accordance with the methods given in the Anatomic Pathology Section. Care will be exercised to minimize loss of tissues through cannibalism or autolysis. Animals in a moribund state will be sacrificed in extremis when death is imminent. B. Body Weights Individual body weights will be recorded once before exposure and after 1, 2, 3, and 4 weeks of testing. -3- CMA 001473 Thereafter, mei:n group body 'weights v/ill be determined monthly up to the 12-month point of study. C. Clinioai Pachology Hemoglobin, hematocrit, total erylhrocyte and total leukocyte counts will be performed at 18 and 24 months on 30 (15 male and 15 female),rats of the control and each test group. Differential leukocyte counts will be performed on all animals having high total leukocyte counts. D. Anatomic Pntho1ocry 1. Methods of Sacrifice c2 uo oCh U, o ***, I c o i Kr5* r r-t o a\ p-! o 1W-1*, u- J. oo ii11 H'r*<l SEii o 9 i 't-'i Oj-p 5 j ^ Upon completion of the study, all survivors will be sacrificed by exsanguination following carbon dioxide anesthesia. 2. Gross Pathology Complete necropsies will be performed on all animals which die or are sacrificed and all macroscopic lesions will be recorded. The lungs will be inflated with formalin fixative. 3. Histopathology Representative specimens of the following organs and tissues will be taken from all animals at time of sacrifice and fixed in 10.0% neutral buffered formalin: -4- CMA 001474 Pi^r' c if oT^-ve Order in - * "J " no. 50-4337 14th Jvuiviil Calc'Siou ?ur tv; 3 jr-ict Court 'sh,, Louisiana Adrenal Glands All Gross Lesions Bone (femur, tarsal and metatarsal, including long bones of all four limbs) Bone Marrow (sternal) Brain Both Ears (external auditory canal with ceruminal (Zymbal's) glands) 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 Salivary Gland _j Gl _7!_ V ^W- O l_l_ *--< _l_ _.J Skin Small Intestine (duodenum, jejunum and ileum) Spleen Stomach Thymus Thyroid Gland Tracheo Urinary Bladder Uterus The above tissues, from animals of the control, TE-II, and TE-III will be processed by conventional methods, embedded in paraplast, sectioned (4-6 p), stained with hematoxylin and eosin, and evaluated by light microscopy. If drug-related lesions are detected in tissues from the high or intermediate dose (TE-II or TE-III) animals, affected tissues from the TE-I group animals will be processed and examined in the same manner as the above. -5- CMA 001475 Only major organa (liver, kidney, spleen, heart, lungs) ana neoplasms from animals which die and are found in an advanced state of autolysis will be processed for histopathological evaluation. IV. Reports Quarterly summaries of mortalities, clinical observations, clinicopathologic and anatomopathologic findings will be prepared. Upon completion of the study, a complete report will be prepared and issued. October 23, 1974 H CD V .H ' 1 S :1s S- o oo .of* ^ h u- dh - I o f"iAl-rti W r; Hi b -I'd o\ P* P*. o\. o o iO r if) \ wiS ^ oo # wl co sC0iV3r u a -6- CMA 001476 PROPOSED PROTOCOL A MANUFACTURING CHEMISTS' ASSOCIATION, INC. CHRONIC VAPOR INHALATION TOXICITY STUDY WITH VINYL CHLORIDE Outline of Investigation A. Type and Length*: 9-month vapor inhalation exposure in White Mice plus 9 months observation. 12-month vapor inhalation in Albino Rats plus 12 months observation. B. Number of Animals: 500 Mice 500 Rats C. Exposure Duration: Seven hours per day. Five days per week. D. Test Materials: Vinyl Chloride E. Organization: See Table I F. Dose Levels: See Table I G. Proposed Schedule: Terminal Group Exposures Start Exposures Stop Autopsy Mice: Control, I-, II, III Dec. 1974 Sept. 1975 June 1976 Rats: Control, I, II, III Dec. 1974 Dec. 1975 t Dec. 1976 1- - r;OIi?IDSlTTIAL\ _ ' v; Subject to Protective Order lit -S v. Conoco. Ino. No.; 90r4833 =55*i> Judicial District' Court CilOMlou Parish,, Louisiana CMA 001477 TABLE I CHRONIC VAPOR INHALATION TOXICITY STUDY VINYL CHLORIDE . Organization of Groups Group Control I II III Low Level 1.5 ppm Intermediate Level 5 ppm High Level 15 ppm Number of Animals Mice Rats Males Females Males Females 125 125 125 125 125 125 125 125 125 125 125 125 125 125 125 125 Total 500 500 500 500 30HPI3)EHTIAL\ to Protective Order'll! n Conoco, Ino., No. 90-482% fndiciJtl Diotrict Court eibu Pjtrish, Loulaiana 2- - 001478 __ ----srmT ,\li SiSJert so- 90'4f II. Chamber Parameters Each group of animals will be exposed in a specially constructed stainless steel and plexiglass inhalation 3 chamber having a capacity of approximately 9.3 M , allowing animal loading of less than 3% by volume when the animals reach maturity. Flow rate through the. 3 chamber will be at least 1.53 M /min. providing a theoretical air change every six minutes. The chamber supply air will be filtered and maintained at 40% to 60% relati;Te humidity and 70 to 75F. Mice are to be individually caged; rats will be caged in groups. and water will be provided ad libitum. Food III. Animal Parameters A. Clinical Observations: All animals will be observed daily for lesions and behavioral changes attributable tc the test material. The time of appearance and location of all tumors that occur among both control and test animals will be recorded. Mortality records will be kept on all groups of animals. All animals which die during the study will be necropsied and their tissues processed in accordance with the methods given in the Anatomic Pathology Section. Care will be exercised to minimize loss of tissues through cannibalism or autolysis. Animals in a moribund state will be sacrificed in extremis when death is imminent. -3- CMA 001479 B. Body Weightr. Individual body weights will be recorded once before exposure and after 1, 2, 3, and 4 weeks of testing. Thereafter, mean group body weights will be determined monthly up to the 12-month point of the study. C. Clinioal Pathology Hemoglobin, hematocrit, total erythrocyte and total leukocyte counts will be performed at 18 and 24 months on 30 (15 male and 15 female} rats of the control and each test group. Differential leukocyte counts will be performed on all animals having high total leukccycc counts. D. Anatomic Pathology 1, Methods of Sacrifice Upon completion of the study, all survivors will be sacrificed by ersanguinaticn following carbon dioxide anesthesia. Gross Pathology Complete necropsies will be performed' on all animals which die or are sacrificed and all macroscopic lesions will be recorded. The lungs will be inflated with formalin fixative. CMA 001480 Histopatho1oay ""STIAL `to Pi.utscbivs Order Int Pc53 7. Conoco , Ino. No ,i 90 t:4832 Judioisil" District Court'" Calonsisu F arist,, Louisiana; ' Representative specimens of tec following organs and tissues will be. taken from all animals at time of sacrifice and fixed in 10.05 neutral buffered formalin: Adrenal Glands All Gross Lesions Bone (femur, tarsal and metatarsal, -including long bones of all four limbs) Bone Marrow (sternal) Brain Both Eyes (external auditory canal with ceruminal (Zymbal1s} glands) Esophagus Eye Gonads (testes and ovaries) Kidneys Large Intestine (caecum and colon) Liver Lungs Lymph Nodes (tracheobronchial, cervical and tpri t? r* }- *'** r* \ Optic Nerve Pancreas Parathyroid Gland Pituitary Gland Prostate Salivary Gland Seminal Vesicles Skin Small Intestine (duodenum, Spleen Stomach Thymus Thyroid Gland Tracheo Urinary Bladder Uterus jejunum and ileum) The above tissues, from all animals, will be processed by conventional methods, embedded in paraplast, sectioned (4-6 p), stained with hematoxyline and eosin, and evaluated by light microscopy. -5- CMA. 001481 IV. Only major organs (liver, kidney, spleen, heart, lungs) and neoplasms from animals which die and are found in an advanced state of autolysis will be processed for histopathologic evaluation. Reports Quarterly summaries of mortalities, clinical observations, clinicopathologic and anatcmopathologic findings will be prepared. Upon completion of the study, a complete report will be prepared and issued. October 23, 1974 -6- h0 o ID vA> \V* O vo 2O Tij &\.- P'br"1\\.'fo. 0 o\3* |Vsu \ O C ' rtf O' o V$ o CMA 001482 PROPOSED PROTOCOL B :\ib"''" C "i;o 90-^8371 P053-V- Ccrio^^.- Court' -^th JudWli^st-10,1,alana- Calcasiau Pax--^' MANUFACTURING CHEMISTS' ASSOCIATION, INC. CHRONIC VAPOR INHALATION TOXICITY STUDY WITH VINYL CHLORIDE I. Outline of Investigation A. Type and Length*; 6-month vapor inhalation in Albino rats plus 18 months observation. 12-month vapor inhalation in Albino Rats plus 12 months observation. 12-month vapor inhalation in Hamsters plus 12 months observation. 9-month vapor inhalation in White Mice plus 9 months observation. B. Number of Animals: 1400 Rats 800 Hamsters 800 Mice C. Exposure Duration: D. Test Materials: Seven hours per day. Five days per week. Vinyl Chloride ._ E. Organization: See Table I. F. G. Group Dose Levels: Proposed Schedule: See Table I. ! Exposures Start Exposures Stop Terminal Autopsy Rats: Control, 1-6, II-6, III-6, 1-12, 11-12, III-12 Dec. 1974 Dec. 1974 June 1975 Dec. 1975 Dec. 1976 Dec. 1976 Hamsters: HI-12 1-12, 11-12, Mice: 1-9, II-9, III-9 Dec. 1974 June 1975 Dec. 1975 Mar. 1976 Dec. 1976 Dec. 1976 CMA 001483 TABLE I CHRONIC VAPOR INHALATION TOXICITY STUDY VINYL CHLORIDE Proposal B Organization of Groups X II III 1-6 II-6 III-6 Group Number of Animals Rats Hamsters Males Females Males Females Mice Males Females Control Low Level 1.5 ppm 100 100 100 100 100 100 100 100 100 100 100 100 Intermediate Level - 100 5 ppm 100 100 100 100 100 High Level 15 ppm 100 100 100 100 100 100 Low Level 1.5 ppm 100 100 Intermediate Level - 100 5 ppm 100 High Level 15 ppm 100 100 TOTAL 700 700 400 400 400 400 CMA 001484 - 0 Oli r ~ v _ Subject to Ko. 90-4837. ?oss v. Ccno2j--rr-T". _v Court Chamber Parameter Each group of animals will be exposed in a specially construe stainless steel and plexiglass inhalation chamber having a capacity of approximately 9.2 M3 , allowing animal loading of less than 2% by volume when the animals reach maturity. 3 Flow rate through the chamber will be at least 1.53 M /rain, providing a theoretical air change every six minutes. The chamber supply air will be filtered and maintained at 40% to 6C% relative humidity and 70 to 75F. Mice are to be individually caged; rats will bo caged in groups. water will be provided <_u_ libitum.- Food and All rats and hamsters will be started simultaneously. After 6 months of exposure groups TE-I-6, TE-II-6 and TE-III-6 will be removed from exposure and kept for 18 months observation. The exposures of the mice will then commence. Therefore, all terminal autopsies will take place simultaneously 24 months after the start of the experiment. Animal Parameters A. Clinical Observations: All animals will be observed daily for lesions and behavioral changes attributable to the test material. The time of appearance and location of all tumors that occur among both control and test animals will be recorded. Mortality records will be kept on all groups of animals. All animals which die during the study will be necropsied and their tissues processed in accordance with the methods given in the Anatomic Pathology Section. Care will be exercised to minimize loss of tissues through cannibalism or autolysis. Animals in a moribund state will be sacrificed in extremis when death is imminent. CMA 001485 B. Body Weights Individual body weights will bo recorded once before exposure and after 1, 2, 3, and' 4 weeks of testing. Thereafter, mean group body weights will be determined monthly up to the 12-mcnth point of the study. C. Clinica.l Pathology Hemoglobin, hematocrit, total erythrocyte and total leukocyte counts will bo performed at 18 and 24 months on 30 (15 male and 15 female), rats of the control and each test group. Differential leukocyte counts will be performed on all animals having high total leukocyte counts. D. Anatomic Pathology t- :I sCO JJ, ? t 1 2Q O O5 m * r! S2 o A"hra * 1-1 & 5 `g i'S5 -3 3 2 ^ CD tg Pi 1. Methods of Sacrifice Upon completion of the study, all survivors will be sacrificed by exsanguination following carbon dioxide anesthesia. 2. Gross Pathology Complete necropsies will be performed on all animals which die or are sacrificed and all macroscopic lesions will be recorded. The lungs will be inflated with formalin fixative. 4- - CMA 001486 Kisfcna-'holoav 1 XL SQSsusbvje.ctCotonogPpr,otIencot.!,--2Kc-O.. r'JdCe-r48in37, I4th- JudloiTrF^ i"t Court , Calctsieu Parisn, T: ouirtrrra Representative spec.-mens of the following organs and tissues will be, taken from all animals at time of sacrifice and fixed in 10.OS neutral buffered formalin: Adrenal Glands All Gross Lesions Bone (femur, tarsal arid metatarsal, including long bones of all four liuibs) Bone Marrow (sternal) Brain Both Eves (external auditory canal with ceruminal (Zymba.L's) glanus) Esophagus Eye Gonads (testes and ovaries) Kidneys Large Intestine (caecum and colon) Liver Lungs Lymph Nodes (tracheobronchial, cervical and Optic Nerve Pancreas Parathyroid Gland Pituitary Gland Prostate Salivary Gland Seminal Vesicles Skin Small Intestine (duodenum, Spleen Stomach Thymus Thyroid Gland Tracheo Urinary Bladder Uterus jejunum and ileum) The above tissues, from all animals, will be processed by conventional methods, embedded in paraplast, sectioned (4-6 p) , stained with hematoxyline and eosin, and evaluated by light microscopy. 5- CMA 001487 Only major organs (liver, kidney, spleen, heart, lungs) and neoplasms from animals which die and are found in an advanced stake of autolysis will be process ad for histopathologic evaluation. IV. Reports Quarterly summaries of mortalities, clinical observations, clinicopathologic and anatomopatbologic findings will be prepared. Upon completion of the study, a complete' report will be prepared and issued. October 23# 1974 -6- ia v $ \ * A'! 4 ft cJ CMA 001488 PROPOSED PROTOCOL A MANUFACTURING CHEMISTS' ASSOCIATION, INC. CHRONIC VAPOR INHALATION TOXICITY STUDY WITH VINYL CHLORIDE Outline of Investigation A. Type and Length*: 9-month vapor inhalation exposure in White Mice plus 9 months observation. 12-month vapor inhalation in Albino Rats plus 12 months observation. B. Number of Animals: 500 Mice 500 Rats C. Exposure Duration: Seven hours per day. Five days per week. D. Test Materials: Vinyl Chloride E. Organization: See Table I F. Dose Levels: See Table I G. Proposed Schedule: Terminal Group Exposures Start Exposures Stop Autopsy Mice: Control, t, II, III Dec. 1974 Sept. 1975 June 1976 Rats: Control, If II, III Dec. 1974 Dec. 1975 Dec. 1976 -1- ,n Court - v n . T' `' CMA 001489 TABLE I CHRONIC VAPOR INHALATION TOXICITY STUDY VINYL CHLORIDE . Organization of Groups Group Control I II III Low Level 1.5 ppm Intermediate Level 5 ppm High Level 15 ppm Number of Animals Mice Rats Males Females Males Females 125 125 125 125 125 125 125 125 125 125 125 125 125 125 125 125 T'otal 500 500 500 500 _ Q r7r?rotcctivc lOrder in Sublet 1 ire . I'0 90-403* let Court I ' l II " l, Louisiana Calcasieu Parish, -2- t. 001490 II. Chamber Parameters C""1 _ *_ '-* r r> r --- r z _ --' '-".To^dsr ;r;':=o:so-w court f'jU x.(i'J.isi^T'-a Each group of animals will bo exposed in a specially constructed stainless steel and plexiglass inhalation 3 chamber having a capacity of approximately 9.3 M , allowing animal loading of loss than ?.% by volume when the animals reach maturity. Flow rate through the 3 chamber will be at least 1.53 M /min. providing a theoretical air change every six minutes. The chamber supply air will be filtered and maintained at 40% to 60% relative humidity and 70 to 75F, Mice are to be individually caged; rats will be caged in groups. and water will be provided ad libitum. Food III. Animal Parameters A. Clinical Observations: All animals will be observed daily for lesions and behavioral changes attributable tc the test material. The time of appearance and location of all tumors that occur among both control and test animals will be recorded. Mortality records will be kept on all groups of animals. All animals which die during the study will be necropsied and their tissues processed in accordance with the methods given in the Anatomic Pathology Section. Care will be exercised to minimize loss of tissues through cannibalism or autolysis. Animals in a moribund state will be sacrificed in extremis when death is imminent. -3- CMA 001491 B. Body Weightn Individual body weights will be recorded once before exposure and after 1, 2, 3, and 4 weeks of testing. Thereafter, mean group body weights will be determined monthly up to the 12-month point of the study. C. Clinical Pathology Hemoglobin, hematocrit, total erythrocyte and total leukocyte counts will be performed at 18 and 24 months on 30 (13 male and 15 female) rats of the control and each test group. Differential leukocyte counts will be performed on all animals having high total leukccycc counts. D. Anatomic Pathology CONFIDENTIAL Subject, to rroxsotivs Order in iv Conoco, ins, , ITo. 90-4837, 1 o Methods of Sacrifice 14th Judicial District 'Court Calcasieu Parish, Louisiana Upon completion of the study, all survivors will be sacrificed by exsanguination following carbon dioxide anesthesia. * 2. Gross Pathology Complete necropsies will be performed' on all animals which die or are sacrificed and all macroscopic lesions will be recorded. The lungs will be inflated with formalin fixative. 4- - CMA 001492 Histopatholoqy COi Subject xo ^ <*\ *P <J ,, O 1 __ , j * urtr , - , ,. Ca^sio,-J- -or^ri=; Representative specimen? of the following organs and tissues will be taken from all animals at time of sacrifice and fixed in 10.0' neutral buffered formalin: Adrenal Glands All Gross Lesions Bone (femur, tarsal and metatarsal, -including long bones of all four limbs) Bone Marrow (sternal) Brain Bo til Eyes (external auditory canal with ceruminal (Zymbal1s) glands) Esophagus Eye Gonads (testes and ovaries) Kidneys Large Intestine (caecum and colon) Liver Lungs Lymph Nodes (tracheobronchial, cervical and 4rkn*'-HftUf> fwjb.. 5u* W yv r-i \ Optic Nerve Pancreas Parathyroid Gland Pituitary Gland Prostate Salivary Gland Seminal Vesicles Skin Small Intestine (duodenum, jejunum and ileum) Spleen Stomach Thymus Thyroid Gland Tracheo Urinary Bladder Uterus The above tissues, from all animals, will be processed by conventional methods, embedded in paraplast, sectioned (4-6 p), stained with hematoxyline and eosin, and evaluated by light microscopy. -5- CMA 001493 Only major organs (liver, kidney, spleen, heart, lungs) and neoplasms from animals which die and are found in an advanced state of autolysis will be processed for histopathologic evaluation. IV. Reports Quarterly summaries of mortalities, clinical observations, clinicopathologic and anatcmopathologic findings will be prepared. Upon completion of the study, a complete report vill be prepared and issued. October 23, 1974 ,* -6- / CMA 001494 PROPOSED PROTOCOL B MANUFACTURING CHEMISTS' ASSOCIATION, INC. CHRONIC VAPOR INHALATION TOXICITY STUDY WITH VINYL CHLORIDE I. Outline of Investigation A. Type and Length*; 6-month vapor inhalation in Albino rats plus 18 months observation. 12-month vapor inhalation in Albino Rats plus 12 months observation. 12-month vapor inhalation in Hamsters plus 12 months observation. 9-month vapor inhalation in White Mice plus 9 months observation. B. Number of Animals: 1400 Rats 800 Hamsters 800 Mice C. Exposure Duration: Seven hours per day Five days per week. D. Test Materials: Vinyl Chloride E. Organization: See Table I. F. Dose Levels: See Table I. G. Group Proposed Schedule: Exposures Start Exposures Stop Terminal Autopsy Rats: Control, 1-6, II-6, III-6, " 1-12, 11-12 , III-12 Hamsters: III-12 1-12, 11-12, Mice: 1-9, II-9, III-9 Dec. 1974 Dec. 1974 Dec. 1974 June 1975 June 1975 Dec. 1975 Dec. 1975 Mar. 1976 Dec. 1976 Dec. 1976 Dec. 1976 Dec. 1976 CMA 001495 TABLE I CHRONIC VAPOR INHALATION TOXICITY STUDY VINYL CHLORIDE Proposal B Organization of Groups X II III . 1-6 II-6 111-6 Group Control Low Level 1.5 ppm Number of Animals Rats Hamsters Males Females Males Females Mice Males Females 100 100 100 100 100 100 100 100 100 100 100 100 Intermediate Level - 100 5 ppm 100 100 100 100 100 High Level 15 ppm 100 100 100 100 100 100 Low Level 1.5 ppm 100 100 Intermediate Level - 100 5 ppm 100 High Level 15 ppm 100 100 - ;o N^. 'n'* 5 \. v* VVjK O\x V ^v w TOTAL 700 -2- 700 400 400 400 400 'CMA 0 0 1 4 9 6 v* ^ II. Chamber Parameters C03r?ID?*7T7 7 Hi" ***_. Cccoee t 0rder Iff *.NL4tH Jndi^TTrrr1 90'4837 '' .Calcastp? -- ' '"Tt Each group of animals will be exposed in a specially construe stainless steel and plexiglass inhalation chamber having a 3 capacity of approximately 9.2 M , allowing animal loading of less than 2% by volume when the animals reach maturity. Flow rate through the chamber will be at least 1.53 M /min. providing a theoretical air change every six minutes. The chamber supply air will be filtered and maintained at 40% to 6C% relative humidity and 70 to 75F. Mice are. to be individually caged; rats will be caged in groups. water will be provided ou_ libitum.. Food and All rats and hamsters will be started simultaneously. After 6 months of exposure groups TE-I-6, TE-II-6 and TE-III-6 will be removed from exposure and kept for 10 months observation. The exposures of the mice will then commence. Therefore, all terminal autopsies will take place simultaneously 24 months after the start of the experiment. III. Animal Parameters A. Clinica1 Observations: All animals will be observed daily for lesions and behavioral changes attributable to the test material. The time of appearance and location of all tumors that occur among both control and test animals will be recorded. Mortality records will be kept on all groups of animals. All animals which die during the study will be necropsied and their tissues processed in accordance with the methods given in the Anatomic Pathology Section. Care will be exercised to minimize loss of tissues through cannibalism or autolysis. Animals in a moribund state will be sacrificed in_ extremis when death is imminent. CMA 001497 B. Body Weight5 Individual body vreights will be recorded once before exposure and after 1, 2, 3, and-4 weeks of testing. Thereafter, mean group body weights will be determined monthly up to the 12-mcnth point of the study. C. Cl inic al Patho.log y Hemoglobin, hematocrit, total erythrocyte and total leukocybe counts will bo performed at 18 and 24 months on 30 (15 male and 15 female), rats of the control and each test group. Differential leukocyte counts will be performed on all animals having high total leukocyte counts. D. Anatomic Patholog 1. Methods of Sacrifice Upon completion of the study, all survivors will be sacrificed by exsanguination following carbon dioxide anesthesia. 2. Gross Pathology Complete necropsies will be performed on all animals which die or are sacrificed and all macroscopic lesions will be recorded. The lungs will be inflated with formalin fixative. CMA 001498 3. Histern-holoav " SuBJect to Protective Cr^er Ross V...C=noi0-^^-..^ rmtrt Representative spec." mens of the following organs and tissues will be. taken from all animals at time of sacrifice and fixed in 10.0% neutral buffered formalin: Adrenal Glands All Gross Lesions Bone (fs'mur, tarsal avid metatarsal, including long bones of all four Hubs) Bono. Marrow (sternal) Brain Both Eyes (external auditory canal with ceruminal (Zymbal's) glanus) Esophagus Eye Gonads (testes and ovaries) Kidneys Large Intestine (caecum and colon) Liver Lungs Lymph Nodes (tracheobronchial, cervical and TT'9or.t`llv' *i r*' \ Optic Nerve Pancreas Parathyroid Glanc! Pituitary Gland Prostate Salivary Gland Seminal Vesicles Skin Small Intestine (duodenum, jejunum and ileum) Spleen Stomach Thymus Thyroid Gland Tracheo Urinary Bladder Uterus The above tissues, from all animals, will be processed by conventional methods, embedded in paraplast, sectioned (4-6 p), stained with hematoxyline and eosin, and evaluated by light microscopy. 5 CMA 001499 Only major organs (liver, kidney, spleen, heart, lungs) and neoplasms from animals which die and are found in an advanced state of autolysis will be processed for histopathologic evaluation. IV. Reports Quarterly summaries of mortalities, clinical observations, clinicopathologic and anatomopathologic findings will be prepared. Upon completion of the study, a complete' report will be prepared and issued. October 23, 1974 -6- CMA 001500