Document DMeRyXmObQZMBGN8qQ0jv0o2o

0CT 'S j THE DOW CHEMICAL COMPANY October 25, 1974 BENNETT BUILDING 2030 DOW CENTER MIDLAND. MICHIGAN 4864.0 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 Plaza Houston, TX 77002 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, dot-3. Theodore R. Torkelson Corporate Medical Department kjf end. CMA102172 C't ? 7'r-OVOColj WiNUFAC7:hKT:v CHRONIC VAPOR INHALATION TOXICITY STUDY WITH VINYL CHLORIDE Ji\L>UHTHIATi BIO-TE0T L At0PATOPi.hS DECATUR, ILLINOIS I. Outline of Inver t i cat.Ion A. Type and Lc.i*gt\h_ 9-month vapor inhalation in White Mice + 9 months observe1- Ion Id-month vapor inhaJavion in A3.bino Hat..a + 12 months oh per vi 1.7-month vapor inhalation ir Hamsters + 12 months observation. B. Nunder - r. An in-" Is ! 800 Mice. 900 Re's. 800 LY.v: ''-r. C * E x uoii: vy - A r-njle i ;j?C: \ r.i 1 liC #(i`i Ot^T- (1 ri V . Five cL.ys pc-.r week. D. To s t M :;te jJ.cU._Ei_ Vinyl Chloride (Ethylene ic-r; . ) E. Organ!zazic:; i_ bee Table i .. F. Dose Leve'.u See Tabic I. G. Exposures Started : Sept. A)72; mice, rats hamsters Exposures `To rrn.',r.a1 : Juno 1974; mice. Sept. 1274; rats hamsters. Term! i, a 1 A n t o rs v: Ma r ch 19 7 5; m i c a * . Sept. 1975; rate & hamsters. * NOTE Excess mortality made it necessary to sac ifice all TE-II and TE-3I1 mica attar ii months on expert ent. -1- CMA 102173 Ast Material l.-YYlZZ 7ln.yl Chloride thylane derived) TABLC I CHRONIC VAPOR INHALATION TOXICITY STUDY Organization of Groups Group Males ieaaies Number of Animals Rats Hamsters Males Females Males Femali Control 130 ICC 100* 100* 100 100 TS-I T2-II T2-III TR-IV Low Level 50 ppm Intermediate Level 200 ppm High Level 2500 ppm High Level 25C0 ppm with food LOO LOO LOO - 100 ICO 100 - 100* 100* 100 100 100* 100* 100 100 100* 100* 100 100 - 100 - - * 5aah of these rat groups were reduced by sacrifice, of 5 rats for cytogentic and histopathological examination at the end of the 12th month of exposure. CMA 102174 ___ "A _ II. 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 M 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 75l'. Food will be removed fre all animal cages during exposure except Group TE XV rats. Ill. Animal Parr-maters A. C1 in ic " .1 Cb s cr va t i on s 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. -3CMA 102175 Thereafter, mean group body weights will be determined monthly up to the 12-month point of study. C. Clin i <:aI_Po_Lhology 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 wi.t 1 be performed on all nirkpals having high total leukocyte counts. D. Anatom' c Pa tholooy 1. Methoda of Sacrifice Upon completion of the study, a?1 survivors will be sacrificed by eusanguination 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. Histopa t.hology 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 102176 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 i tv*: -,i Skin Small Intestine Spleen Stomach Thymus Thyroid Gland Tracheo Urinary Bladder Uterus (duodenum, jejunum and ileum) 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. -5CMA 102177 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 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 -6CMA 102178 PROPOSED PROTOCOL A MANUFACTURING CHEMISTS' ASSOCIATION, INC. CHRONIC VAPOR INHALATION TOXICITY STUDY WITH VINYL CHLORIDE I 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-V II, III Dec. 1974 Sept. 1975 June 19' Rats: Control, I, II, III Dec. 1974 Dec. 1975 Dec. 19'/ 1 CMA102179 TABLE I CHRONIC.VAPOR INHALATION TOXICITY STUDY VINYL CHLORIDE Organization of Groups Group Control I Low Level 1.5 ppm II Intermediate Level 5 ppm III 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 CMA 102180 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 loss than 2% by volume when the animals reach maturity. Plow 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 60% relative humidity and 70 to 75F. ''lice arc to be individually caged; rats will be caged in groups. Food and water will be provided ad libitum. Ill, 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. -3CMA102181 B. Body Weights 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 22-month point of the study. C. Clin leal 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 Taigh total leukccy a; counts. D. Anatomic PaLhoioay 1= Methods of Sacr-lfice Upon completion of the study, all survivors will be sacrificed by exsanguinaticn 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. -4CMA102182 3 Histopathologv Representative specimens of the following organs and tissues v:iil bo taken from all animals at time of sacrifice and fixed in 10.01 neutral buffered formalin: Adrenal Glands A13 Gross Lesions Bone (femur, tarsal and metatarsal, -including long bones of all four limbs) Bono marrow (sternal) Brain Botn Eyes (external auditory canal with ceruminal ( Zymibal1 c) glands) Esophagus Eye Gonads (testes and ovaries) Kidneys Large Intestine (caecum and colon) Liver Lungs Lymph Nodes (tracheal:-X'nchial, cervical and >r\/n c* tj i- ! qr i r-'t \ Optic Nerve Pancreas Parathyroid Gland Pituitary Gland Prostate Salivary Gland Seminal Vesicles Skin Small Intestine (duodenum, Spleen Stomach Thymn s Thyroid Gland Tracheo Urinary Bladder Uterus jejunum and ileum) t 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 102183 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 suiamaries 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 102184 PROPOSED PROTOCOL B MANUFACTURING CHEMISTS' ASSOCIATION, INC. CHRONIC VAPOR INHALATION TOXICITY STUDY WITH VINYL CHLORIDE 1 * 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. 111-6," 1-12, 11-12, III-12 Dec. 1974 Dec. 1974 June 1975 Dec. 1975 Dec. 1976 Dec. 1976 Hamsters: 1-12. ir-12. in-12 Dec. 1974 Dec. 1975 Dec. 1976 Mice: 1-9. 11-9. in-9 June 1975 Mar. 1976 Dec. 1976 CMA102185 TABLE I CHRONIC VAPOR INHALATION TOXICITY STUDY VINYL CHLORIDE , Proposal B Organization of Groups TX II III 1-6 II-6 III-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 98VSOVVWO TOTAL 700 -2- 700 400 400 400 400 II. Chamber Parguotiers Each group of animals will be exposed in a specially construct 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. Flov/ rate through the chamber will be at least 1.53 M3 /mm. providing a theoretical air change every six minutes. The chamber supply sir 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. Food and water will be provided ,_u_ libitum. All rats and hamsters will be started simultaneously. After 6 months of eiposure groups TE-I-6, TE-II-6 and TE-III-6 will be removed from exposure and kept for 18 months observation. The c.posures of the mice will then commence. Therefore, all terminal autopsies will take place simultaneously 24 months after the start of the experiment. III. Animal Paramotors A. Cljnica1 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 dentil is imminent. cmm2187 B. Body Weights Individual body v;eights 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 . Clin .1 cal 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. Anatomi c Pathology_ 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 102188 3. Hi si-cn.V holoa Represcnlvtive spec'mens of the follov/ing organs and tissues vi) 1. he taken from all animals at time of sacrifice and fixed in 10.0?; neutral buffered formalin: Adrenal Glands All Gross Lesions Bone (femur, tarsal armi metatarsal, including long bones of al.1 four liube) Bono Marrow (sternal) Brain Both Eyes (external auditory canal with ceruminal (ZymbaL's) gianus) Esophagus Eye Gonads (testes and ovaries) Kidneys Largo 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 102189 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 ev^lluation. IV. Reports Quarterly summaries of mortalities, clinical observations, clinicopatholcgic and anatomopatbologic findings will be prepared. Upon completion of the study, a complete report will be prepared and issued. October 23# 1974 -6- CMA 102190 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 197 - Rats: Control, lf II, III Dec. 1974 Dec. 1975 Dec. 197 -1CMA 102191 TABLE I CHRONIC VAPOR INHALATION TOXICITY STUDY VINYL CHLORIDE Organization of Groups Group Control I Low Level 1.5 ppm II Intermediate Level 5 ppm III 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 CMA102^92 II. Chamber Parameters 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 21 by volume when the animals reach maturity. Flow rate through the chamber will be at least 1.5 3 MVmin. 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. Food and water will be provided ad libitum. Ill. 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. -3CMA 102193 B. Body Weighto Individual body ^eights 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 Pathelogy 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 Tiigh total leukccycc counts. D. Anatomic Pathology 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 102194 3. Histopsthologv Representative specimens of the afollowing organs and tissues will be taken from all animals at time of sacrifice and fixed in 10.0Z neutral buffered formalin: Adrenal Glands All Gross Lesions Bone (femur,, tarsal and metatarsal, -including long bones of all four limbs) Bone Marrow (sternal) Brain Botn 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 ivl C C j f' I* t r* \ *1* w w -w * * u w* ,,i, ^ w y Optic Nerve Pancreas Parathyroid Gland Pituitary Gland Prostate Salivary Gland Seminal Vesicles Skin Small Intestine (duodenum, jejunum and ileum) Spleen Stomach Thymus 4 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 hematoxylins and eosin, and evaluated by light microscopy. -5- CMA 102195 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 102196 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 Dec. 1974 Dec. 1974 June 1975 Dec. 1975 Dec. 1976 Dec. 1976 Hamsters: 1-12, 11-12, i"ll-12 Dec. 1974 Dec. 1975 Dec. 1976 Mice: 1-9, II-9, III-9 June 1975 Mar. 1976 Dec. 1976 CMA 102197 TABLE I CHRONIC VAPOR INHALATION TOXICITY STUDY VINYL CHLORIDE , Proposal B Organization of Groups T1. II III 1-6 II-6 III-6 Group Control Low Level 1.5 ppm Number of Animals Rats Hamsters Males Females Males Females 100 100 100 100 100 100 100 100 Mice Males Females 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 o 2- - o t-o> (> CD Chamber Parameters 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. Food and water will be provided libitum. 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 Paramaters 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 yJfcLbe recorded. Mortality records will be kept on all ^fgpgroups 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 ill extremis when death is imminent. CMA 102199 B Body weights 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 . Clin .1 ca 1 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 leukocyte counts. D. Anatomic Pathol oqy_ 1. Methodn 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 102200 Hisj-o p - holoa v Representative speoprtma of the following organs and tissues v/iJ 1 be taken from all animals at time of sacrifice and fixed in 10.0% neutral buffered formalin: Adrenal Glands All Gross Lesions Bone (ftrnur, tarsal and metatarsal, including long bones of all four liiEibw) Bone 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 mac 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- CMA102201 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 anatomopatbologic findings will be prepared. Upon completion of the study, a complete report will be prepared and issued. October 23, 1974 -6- CMa 102202