Document ga0Z5O1we6BVydgmQzND6VL5q

R&S 112715 BIO-MEDICAL.RESEARCH DOCUMENT DESCRIPTION FORM 63 Duplicate.in all cards:--> 68 69 76 tif? 1 l 00(JU3g year as-1961- File number [Right justify [Numeric only] 77 78 1 Sub-Index Code Author(s), as Last Name FS (No Punctuation) and coden for journal as JAMA preceeded by one blank space 1 Z (ft- . (L C- 20 2140 4160 61 62 t / - A' ms rsx.cs 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 22 23 f &. /U?/ d jfZ/tZ. 24 Source (Journal, Vol., Number, Pages, Date) 1 2. . 61 62 31 . 32 Brief Summary 12 10 SUMMARY: 61 62 61 62 63 64 =N EVALUATION OF THE ENVIRONMENTAL TOXICANT VINYL CHLORIDE (VC) AND VINYLIDENE CHLORIDE (VDC) 003$9 PROGRESS REPORT NO. 8 1 July through 31 December 1975 Contract No. N01-ES-2-2084 (Continuation of NIH-NIEHS-72-208^) MRI Project No. 3612-B For National Institute of Environmental Health Sciences P.0. Box 12233 Research Triangle Park, North Carolina 27709 Attn: Dr. James S. Woods ( ,-V MIDWEST RESEARCH INSTITUTE 425 VOLKER BOULEVARD. KANSAS CITY. MISSOURI 64110 316 =61-0202 ii i i i im 11 i i "1 h i1i ni pi' "i m 1 EVALUATION OF THE ENVIRONMENTAL TOXICANT VINYL CHLORIDE (VC) AND VINYLIDENE CHLORIDE (VDC) by Cheng-Chun Lee William. B. House Paul J. Peters Jagdish C. Bhandari John R. Hodgson Jack H. Hagensen Thomas W, Reddig PROGRESS REPORT 8 1 July through 31 December 1975 Contract No. N01-ES-2-2084 (Continuation of NIH-NIEHS-72-2084) MRI Project No. 3612-B 11 R&S 112717 i 1 ' 1 1 ii 'i ii ii 1 For National Institute of Environmental Health Sciences P.0. Box 12233 Research Triangle Park, North Carolina 27709 k j MIDWEST RESEARCH INSTITUTE 425 VOLKER 30ULEVARD. KANSAS CITY. MISSOURI 64110 * 816 551-0202 4 PREFACE This report was prepared at Midwest Research Institute, 425 Volker Boulevard, Kansas City, Missouri 64110, under Contract No. N01-ES-2-2084 (continuation of NIH-NIEHS-72-2084) with the National Institutes of Health, Department of Health, Education and Welfare, MRI Project No. 3612-B, "Research Capability for Environmental Toxicants." The research was sponsored by the National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709. Dr. James S. Woods is the Project Officer. The research was conducted in the Biological Sciences Division under the direction of Dr. W. B. House from 1 February 1972 through 31 December 1975. Dr. Cheng-Chun Lee, Assistant Director of Biological Sciences Division for Pharmacology and Toxicology, was the Principal Investigator. Dr. William B. House supervised the inhalation chamber operation, assisted by Dr. Paul J. Peters. Dr. Jagdish C. Bhandari, Associate Pathologist, supervised the nec ropsy and histology preparation and performed the microscopic examination. Dr. John R. Hodgson, Senior Biochemist supervised the cytogenetic analysis of chromosomes. Mr. Jack H. Hagensen. Junior Biologist, performed the analyt ical monitoring of chamber concentration and environmental air. Mr. Thomas W. Reddig (ASCP certified M.T.), Laboratory Supervisor, supervised the hemotology and clinical laboratory tests. Approved for: MIDWEST RESEARCH INSTITUTE Biological Sciences Division li R&S 112718 4 I. II. III. TABLE OF CONTENTS Page Introduction............................................... .......................................................1 Material and Methods................................................................................... 1 A. Methods of Exposure.......................................................................... 1 1. Chamber Design............................................................................... 2. Chamber Air Supply and Flow Rates.................................. 3. Generation of VC and VDC Vapor..................................... . . 4. Chamber Atmosphere Monitoring .......................................... 1 2 2 2 B. Safety................................ '...................................................................... C. Animals................................................................................................. 4 5 1. Species and Number..................................................................... 2. Animal Housing.............................................................................. 3. Animal Hygiene.............................................................................. 5 6 6 D. Experimental Protocol ................................................................ . 6 1. Experimental Design................................................................ 2. Observations and Studies....................................................... 6 7 a. General Observations andWeight Gain..................... b. Hematology andClinicalBlood Chemistry ... c. Serum Imnunoglobins....................................................... d. Serum Protein..................................................................... e. Pulmonary Macrophages ................................................... f. Cytogenetic Analysis....................................................... g. Examination of Bones....................................................... h. Necropsy and Hlstopathology ..................................... 7 7 8 8 8 9 9 9 3. Statistical Analysis................................................................. 9 Results.................................................................................................................... 10 A. Rats.............................................................................................................. 10 1. General Observations andWeight Gain................................... 10 ill R&S 112719 R&S 112720 TABLE OF CONTENTS (Concluded) Page 2. Laboratory Data....................................................................... 10 3. Cytogenetic Analysis ................................................................. 11 4. Examination of Bones................................................... 11 5. Organ Weights............................................................................ 11 6. Gross and Microscopic Examination of Tissues ... 11 B. Mice.....................................................................................................................12 1. General Observations and Weight Gain..................... 12 2. Laboratory Data........................................................................13 3. Cytogenetic Analysis....................................................................... 13 4. Examination of Bones....................................................................... 14 5. Organ Weights............................................................................ 14 6. Gross and Microscopic Examination of Tissues ... 14 IV. Discussion and Conclusion...................................................................... 16 Tables 1 - 35............................................................................. Figures 1-6................................................................................................... References................................................................................................................................... 58-59 * Appendix I - Hematology, Clinical Blood Chemistry, Urinalysis, Histopathology. Statistical Analysis, and Normal Values 52-57 iv * abstract Progress Report No. 8 We have studied the inhalation toxicity of various levels of vinyl chloride (VC) and one level of vinylidene chloride (VDC). At the end of 3 months, rats of both sexes exposed to 50, 250 or 1,000 pptn of VC or 55 ppm of VDC, 6 hours a day, 5 days a week, did not show any significant adverse effects, including effects on weight, gain, various clinical laboratory data, cytogenetic analysis, and gross or microscopic lesions. Exposure to VC or VDC caused some early deaths in mice. Histopathology revealed acute toxic hepatitis amd marked tubular necrosis of the renal cortex. The other mice"appeared to be healthy. However, bronchiolar adenoma developed after exposure to VC. Its occurrence appeared to be dose related. At the end of 2 months, bronchiolar adenoma occurred in one male at 250 ppm, and in one male and two fe males at 1,000 ppm. At the end of 3 months, it occurred in one male at 50 or 250 ppm, and in two males and one female at 1,000 ppm. At each time period, four males and four females were examined. Exposure to various levels of VC did not cause any other significant adverse effects in mice. As for rats, mice exposed to 55 ppm of VDC for up to 3 months did not show any adverse signs, any changes in laboratory data, any cytogenetic effects, or any pattern of persistent lesions. Principal Investigator Biological Sciences Division for Pharmacology and Toxicology R&S 112721 v ZZLZH sl8ti I. INTRODUCTION Under Contract No. N01-ES-2-2084 (NIH-NIEHS-72-2084) on "Research Capability for Environmental Toxicants," we have performed various studies on the toxic effects of the two commercially available dithiocarbamates, ferbam and thiramJL/ Following completion of these studies, the inhalation toxicity of various levels of vinyl chloride (VC) was chosen for study. In addition, one level of vinylidene chloride (VDC) was also included. Vinyl chloride was first prepared more than a century ago and its fire and explosion hazard are well known. Acute toxicity of VC was first reported in guinea pigs.--^ As a possible anesthetic agent in dogs, VC was found to cause incoordinated muscular activity of the extremities, cardiac arrhythmia$2/ and sensitization of the myocardium.-- Relatively high con centrations of VC (20-40Z in air) for 30 minutes produced narcosis and/or death in mice, rats, and guinea pigs ..5/ The guinea pigs were found to be more resistant. The main lesions were congestion of the lung, with pul monary edema and hemorrhages, in some animals, and congestion of the liver and kidneys. Blood coagulation was also impaired. Various laboratory animals were exposed to 50, 100, 200, or 500 ppm of VC, 7 hours/day and 5 days/weeks, for up to 6 months.-- A threshold limit value of 100 ppm, be low which no detectable changes occurred, was suggested. A recent study indicated that rats (Ar/IRE) exposed to high concentration of VC (3% V/V in air, equal to 30,000 ppm), 4 hours/day, 5 days/week for 12 months, develop ed tumors of the skin, lungs, and bones.-- Furthermore, zymbal gland car cinomas, nephroblastoma, hepatic and extrahepatlc angiosarcomas were ob served in rats, and pulmonary tumors, mammary carcinomas and liver angio sarcomas were observed in mice exposed to as low as 250 ppm of VC, 4 hours/ day and 5 days/week for 12 months.-- The growing suspicion that exposure to VC has caused a number of deaths of workers from a rare form of liver can cer, angiosarcoma, has alarmed the OSHA to set an emergency rule on April 5, 1974, that lowered the permissible worker exposure level to 50 ppm from the previous level of 500 ppm. This report summarizes our efforts on the installation of inhala tion chambers and the results of exposure to VC or VCD for 1, 2, .or 3 months. II. MATERIAL AND METHODS A. Methods of Exposure 1. Chamber Design Five stainless steel Rochester type exposure chambers of 3.5 nP (123 ft^) volume were used (Figure 1). Contaminant entered the air stream 1 rt and was Chen mixed in a plenum at Che cop of each chamber. Each chamber concained a diffusion place and two small squirrel-cage fans (100 CFM, 2.85 m^/min) mounted on opposite sides of the top cone above the diffusion plate to ensure complete mixture of the gas with air. 2. Chamber Air Supply and Flow Rates The air supply to the chambers was drawn from a stack on the roof of the building. Chamber air passed through a coarse filter and then over coils for heating, cooling, and dehumidifying. The air then passed through an absolute filter (99.97-99.999% retention of 0.3 p particles) into the plenum of the chamber; Initially, air flow rates were measured at the inlet side of the chamber with a pitot tube connected to a magnahelix guage. According to these readings, the chambers were onerated at an air flow rate giving 10.5 changes/hour (21.4 CFM, 0.6 m^/min). Other methods of measuring air flow were tried and were not considered to be satisfactory. An air flow trans ducer (Autotronics 100-sxx) has been purchased and preliminary tests have indicated, that the actual air flow is greater than the readings obtained by pitot tube measurement. The results of these studies will appear in the next progress report. The air flow rate required to make the nominal and analytical, concentrations consistent were 12 changes/hour (24.8 CFM, 0.70 min) for VC and 19 changes/hour (39 CFM, 1.1 n^/min) for VDC. We are in the process of replacing the pitot tubes with critical orifice plates on the outlet exhaust system. Regardless of the air flow, the important factor is the actual concentration of VC and VDC exposure to the animals, which is- discussed later. 3. Generation of VC and VDC Vaoor Vinyl chloride at a purity of 99.8% was obtained from Matheson Products, It is a gas at room temperature and was metered with rotameters into the chamber air supply. VDC with a purity of 99% was obtained from the Aldrich Company. Its boiling point is 32C (89.6F) and was heated to 37C (98.6F) to generate the gas. All gas lines and the- rotameter were heated to 40 C (104F) to prevent condensation. 4. Chamber Atmosphere Monitoring Chamber concentrations were monitored using a gas chromatograph (Varian-2700) with a flame ionization detector. A 6 ft x 1/8 in. stainless steel column packed with 0.4% Carbowax 1500 on Carbopak A was used with a nitrogen carrier flow rate of 80 ml/min. The injection, column, and detec tor temperatures were 135C, 65C,and 170C respectively. 2 ZZLZW t Standards: VC standards at dilutions of 10, 50 and 1,000 ppm were obtained in lecture bottles from Supelco Inc., Bellefonte, Pennsyl vania, VDC standards were prepared by a serial dilution (weight/volume) of VDC in carbon tetrachloride. The desired final concentrations in a 4 ill injection were determined by the following procedure: mg/1 (pg/ml) pom x Hf 24450 With a molecular weight for VDC of 96.94, the final concentrations of 10 ppm, 50 ppm, and 100 ppm, the amount of VDC in a 1-ml sample from the chamber would be 0.04, 0.20, or 0.40 pg respectively. For 1 pi of standard, these same amounts would be obtained from solutions of 40, 200, and 400 mg/ liter; but since 4 pi of standard were injected, the final dilutions of VDC in carbon tetrachloride were 10, 50, and 100 mg/liter. All liquids were injected at a volume of 4 pi and all gase3 at 1 ml. Standards at each point on the calibration curve were injected in triplicate. Chamber Sampling: Each chamber was fitted with 10 sampling ports on two sides of the chamber. During preliminary studies a series of dis tribution studies were made to determine the uniformity of the test material in the chamber. These tests were made with three sampling lines of poly ethylene capillary tubing, of varying lengths, passed through each sampling port. It was determined that a valid measure of chamber contaminant dis tribution could be obtained by sampling periodically through three positions and occasionally through nine positions (Figure 2). Samples were withdrawn from the chamber with the same syringe used to introduce the sample to the gas chromatograph. Tests indicated that a valid sample could be withdrawn by pumping the syringe three times on the short sampling lines, and five times on the longest line. All sampling was done in triplicate. During the first 3 months, distribution studies were compared with a reference point in the center of the chamber. The restilts showed that average chanter concentrations were t 37. of the desired concentration; the reference point averages were 93.47. to 100.4% of these chamber averages. Chambers were routinely sampled from the reference point 3 or 4 times a day. The average weekly concentrations and the range of all samples for VC are shown in Figure 3. The average weekly variations in the 1,000 ppm and 250 ppm chambers did not vary more than 57, except during the third week. The variation in the 50 ppm chanter was greater with may!.mum devta- R&S 112724 3 >* tions for the chamber average of 47, 53, and 54 ppm during the second, 7th and 8th weeks (Figure 3). All of these values are within + 10% of the de sired average concentration. The ranges about the'averages, shown in Figure 3, represent the maximum and minimum values chat occurred in any week. Since the chambers were sampled a minimum of three times a day, the exposure to a maxisum deviation from the norm would be no store than 3 hours. At the beginning of testing with VDC it was planned that the ex posure level would be 50 ppm. Due to technical problesis with the standards, the objective of 50 ppm for VDC was not achieved. Hiese problems were associated with the use of chloroform as the solvent during the first 4 weeks, which was subsequently changed to carbon tetrachloride, and a non linear calibration curve during the 5th through the 10th weeks. By the 11th week, these problesis had been solved and the slightly higher dosage of 55 ppm was obtained. The average weekly concentrations of VDC are shown in Figure 4, as well as the daily nw-g-fmum and minimum values from the 11th week on. Because of the problems discussed above, the analytical concentration during the first 10 weeks is not precisely known. Since the air flow rates have been constant throughout, we can calculate the nominal concentration, for this time period, by assuming 19 air changes per hour (based on the 16-19th week data) and using the amount of VDC used per day. This has been done (Figure 4) for the 4th through the 10th week. However, the material balance also was not recorded during the first 3 weeks. Therefore, we have estimated the average concentration during the first 3 weeks by replicating all magnahelix and rotameter settings, which were known, and measuring the chamber concentration. Thus, we estimated the average concentration during the first 3 weeks to be 30, 80, and 95 ppm consecutively. B. Safety All chambers were operated with a slight negative pressure (0.1 0.2 in. water) to prevent escape of contaminant through leaks into the room. Before opening, chambers were flushed with clean air for a minimum of 30 minutes. Sampling from the chambers and from the doorway upon opening al ways showed less than 1 ppm of contaminant. In addition, after operating for 30 minutes without contaminant, on one occasion, the air supply and exhaust were shut off to the 1,000 ppm chamber for 10 minutes. Thus, the animals sat in a static environment and any off-gassing would be allowed to accumulate. Samples taken from the chamber after the 10-minute period showed less than 1 ppm of VC in the air. Additional samples from doorways and numerous other places in the area were collected on charcoal tubes and by grab sampling with a syringe. All samples showed less than 1 ppm of VC. Boom monitoring for VC and VDC 4 R&S 112725 R&S 112726 was performed on a routine basis by grab samples with a syringe and analysis with the gas chromatograph used for chamber monitoring. During*all initial work, personnel wore impervious coveralls, gloves, and a demand type face mask when the chambers were opened for trans fer of animals to holding racks. This practice was discontinued only after extensive sampling had shown that exposure to more than 1 ppm did not occur. All air from the chambers travels under negative pressure to an incinerator outside the building. Integrated bag samples of stack gas were collected on January 12 and 13. These samples were collected accord ing to. Method 106, "Determination of Vinyl Chloride from Stationary Sources," Federal Register, Vol. 40, No. 248, December 24, 1975. This method in volves pulling a sample of stack gas into, a leak proof Tedlar bag with a vacuum pump set at a flow rate of 500 cc/min for a 1-hour period. The sample site was determined by cutting a hole in the stack wall at two duct diameters downstream from the flame. Two tests were conducted, on January 12, while three chambers operated at a combined level of 1^00 ppm of VC; and one chamber was oper ated at 55 ppm of VDC. An additional three chambers operated with only clean air. Test one, analyzed at the inhalation facility, showed 39 to 40 ppm of VC. Test two showed 38.5 to 39 ppm of VC. To check these values, a sample was drawn by syringe directly from the stack. This sample con tained 29 ppm of VC. VDC was never detected. On January 13, a third test was conducted and showed 38 ppm of VC. Samples from the third test were taken to the Environmental Measure ments Section at MRI. After setting overnight, the average of 6 peaks equalled 29 ppm of VC. This lower value could be due to loss of VC through absorption in the Tedlar bag, or due to loss through the used septum. During all three tests, the burner of the incinerator operated at 1700 to 1800F. Subsequent tests at higher or lower temperatures did not lower the- VC. C. Animals 1. Species and timber Albino CD rats and albino Swiss Mice (Charles River Breeding Laboratory) about 2 months old at the start were used in these studies. They were acclimated in our holding quarters for 1 week before the experi ment began. For each species, a total of 360 animals were divided into five groups, each consisting of 36 males and 36 females. 5 R&S 112727 2. Animal Housing All animals lived In Che same stainless steel cages during ex posure and outside of the chambers. Bats were housed 2/cage and mice 6/cage. Powdered laboratory chow (Wayne Manufacturing Company) was provided at all times except during the 6-hour period of exposure each day. Water was available ad libitum. The temperature In the chamber ani In the room averaged 24 + 1.3C (75 + 5F). The relative humidity in the chambers ranged from 25-60%; In the ro m it also ranged from 25 to 60%, but was regulated at 45 + 5% after the 6th month. A 12-hour light cycle was maintained at all times. When out of the chambers, the animals were placed In holding racks similar to those in Figure 1. A continuous supply of room air was drawn over the cages while in the holding racks. Provisions were made for covering the frpnt of each compartment in the holding rack In the event of significant off-gassing from the animals. This was found not to be necessary with VC or VDC. 3. Animal Hygiene All cages and feeders were washed, and sanitized each week. The floors of the room, the holding racks, and the interior of the chambers were frequently washed with disinfectant. Animal caretakers wore gloves and surgical masks when handling animals. It was found that when the cages were loaded into the chambers without drop-pans (so :not to impair air flow) the animals on the bottom shelves were frequently drenched with urine and water. In order to allevi ate this problem, the cages were loaded into the chambers with clean drop pans in place. Sampling studies showed that the vapor distribution was actually improved slightly. Thus, from the 6th mouth on, drop pans were washed daily and a clean drop pan without bedding was inserted into each cage before loading into the chamber. D. Experimental Protocol 1. Experimental Design A total of five chambers were used. Each chamber holds one group of 72 rats and one group of 72 mice. Animals in the chambers were exposed at 6 hours/day and 5 days/week to the following: Chamber 1: Uncontaminated air Chamber 2: 50 ppm of VC 6 R&S 112728 < Chamber 3: Chamber 4: Chamber 5: 250 ppm of VC 1,000 ppm of VC 55 ppm of VDC Four animals of each species, sex and exposure level were ter minated for various studies, necropsy, and histopathology at the end of 1, 2 and 3 months. The remaining animals will be terminated at the end of 6, 9 and 12 months. All terminated animals are replaced in the respective chandlers. The replacement animals are again exposed for 1, 2, 3, 6, 9 or 12 months, respectively. They are then held 1 year for observation. At the end of the observation period, all animals will be terminated for necropsy and histopathology. 2. Observations and Studies a. General Observations and Weight Gain All animals were observed throughout the study for behavioral changes and any unusual or toxic signs. Food consumption was recorded weekly and body weights biweekly at a uniform time of the day. Any unusual changes in weight were investigated. An attempt was made to determine the cause when any unscheduled death occurred. b. Hematology and Clinical Blood Chemistry Aortic blood from four males and four females of each group of each species was collected under anesthesia (sodium pentobarbital for rats and ether for mice) at 1, 2 and 3 months. Hematology (various blood counts, hematocrit, hemoglobin, methemoglobin and Heinz bodies) and clinical blood chemistry (fasting blood glucose, SGOT, SGPT,1 alkaline phosphatase, BUN, creatinine and/or LDH) were performed on all samples. The procedures for these tests and the normal values for rats are given in Appendix I. In addition, the following tests were also performed for rats. Prothrombin time: The prothrombin time was measured by the one-step method of QuickCusing the Fibro System (BBL) with activated thromboplastin (Dade). Standardized Normal Plasma (Dade) was used as the control for each assay. Bilirubin: Total and direct bilirubin was measured using the Billi-Strate Kit (General Diagnostics) which is based on a modified Jendrassik-Grof method.-ifi/ Moni-Trol I, Versatol (General Diagnostics), and Calibrate I (General Diagnostics) was used as the reference for each assay. 7 R&S 112729 c. Serum Irnmunoglobins Serum Imminogloblns were measured in four male and four female rata from the control, 1,000 ppm of VC, or SS ppm of VDC groups. The radial Immunodiffusion technique of Mancini et al.^ was used to assay Inmunoglobin groups IgA; IgG, subgroups A and B; and IgM. A replicate serum sample from each rat was placed In wells In an Immunodiffusion chamber along with a standard, and held until' the precipitin ring reached equilibrium. Hie square root of the diameter of the preclpltan ring Is directly proportional to the concentration of antibody. d. Serum Protein ,?"byroteinr Total protein was measured by the biuret method of Kingsley.-- Standard Human Protein (Dade) was used as the stan- dard for each assay. Albumln: Albumin was measured by the Albu-Strate Kit (Gen eral Diagnostics) which Is based on the bromeresol green method of Doumas et al.,H/ Dab-Trol (Dade) and Calibrate I was used as the reference for each assay. Globulin: Globulin was calculated by the difference of total protein and albumin. e. Pulmonary Macrophages Four males and four females of each species from the control, 1,000 ppm of VC, or 55 ppm of VDC groups were used for the pulmonary macro phage count. ( At: the time of necropsy, the left lung was removed at the bifurcation of the trachea; a small needle was inserted Into the bronchus; and 5 ml of sterile saline was gently flushed into the lungs of rats and 0.5 ml in the lungs of mice. The fluid was withdrawn and flushed again several times. This fluid was then transferred from the syringe to a glass tube and stained. The number of cells in the preparation was counted under a microscope with a differentiation made between macrophages and leukocytes. If changes were observed in lungs from animals exposed to 1,000 ppm of VC, then animals from the groups exposed to 250 or 50 ppm were also examined. 8 tR&S 12730 f. Cytogenetic Analysis Bone marrow samples were collected from four males and four females of each species from the control, 1,000 ppm of VC or 55 ppm of VDC groups. Short term bone marrow cultures were made according to the methods of Tjlo and Wang.--^ After allowing approximately 5 hours for sufficient members of cells to be arrested in metaphase, the cells were collected, swollen in hypotonic saline, and processed for spreading on glass slides according to the method of Moorhead and Norwell.--'^ Slides were then stained with giesma and scanned under low power optics. Those slides showing a nrtTVftmim scattering of cells in metaphase were selected for chromosomal analysis using oil immersion optics. Estimation of cell polyploidy was obtained by rough chromosomal estimates of at least 200 cells. Exact chromosomal counts and chromosomal aberration estimates were determined for at least 50 metaphase spreads. Special attention was given to chromosomal damage such as breaks, gaps, and translocations. g. Examination of Bones At the time of necropsy, the limbs of both species were excised and fixed in formalin. After fixation the limbs were wrapped and frozen for storage. Arrangements have been made to use the x-ray facilities of the Radiology Department, University of Kansas Medical School, Kansas City, Kansas. After the 12-month terminations, limbs from animals with the longest exposures will be examined for any evidence of osteolysis or other changes. If abnormalities are present, the bones from animals ex posed for progressively shorter periods will be examined. h. Necroosv and Histopathology When moribund or at the end of 1, 2, and 3 months, four males and four females from all groups were euthanized for necropsy after the collection of blood. Gross examination of all tissues, especially for any appearance of abnormal growth or other lesions, were carefully per formed. The brain, liver, kidney, spleen and gonads were removed and weighed. Portions of these and other tissues were fixed, processed, sectioned and stained for microscopic examination. The procedures are given in Appendix I. 3. Statistical Analysis The results of the various parameters were compared with the control groups at the respective time intervals, whenever applicable, according to the Dunnett's Multiple Comparison Procedure.!!' 9 R&S 112731 t. XXX. RESULTS A. Rats 1. General Observations and Weight Gain The rats terminated after 1, 2, or 3 months were exposed for a total of 22, 41 and 61 days, respectively, at 6 hours/day and 5 days/week. All groups gained weight and appeared in good health. The body weights of the control rats and rats treated with the high level of VC (1,000 ppm) or with VDC (55 ppm) are shown in Figure 5. The weight gains of the fe male rats exposed to VC or VDC became less than that of the control rats after the 5th week. ; However, the differences were not statistically significant. The weight gains of the rats exposed to the middle and low levels of VC (250 or 50 ppm) were comparable to that of the control rats and their body weights were not Included on the graph. 2. Laboratory Data The results of hematology, clinical blood chemistry, serum immuno globulins, serum proteins, and macrophage counts in the lungs of male and female rats after exposure to VC or VDC for 1* 2, or 3 months are summarized in Tables 1 through 6. At the end of 1 month, the peripheral blood elements and clinical chemistry values of various groups (Tables 1 and 2) were within normal ranges for rats observed, in our laboratory as shown in Appendix I. When compared with control rats, there were a few occasional differences not related to the compounds. These differences included an increase in direct bilirubin concentration, of male rats exposed to 250 ppm of VC or 55 ppm of VDC, and increases in the leukocyte count and creatinine concentration of female rats exposed to VDC. Serum levels of lmmunoglobins were not apparently altered after exposure to high levels of VC. The total protein level of female rats exposed to VDC was statistically higher than that of the control rats; however, the increase was not clinically significant. The concentrations of serum albumin and globulin were not statistically different. The macrophage count in the lung washings was not apparently affected after exposure to high levels of VC or to VDC. At the end of 2 months, the peripheral blood elements and clinical chemistry values (Tables 5 and 6) were also not apparently altered after exposure to VC or VDC. However, a few differences were observed in these rats. These differences were mild and included a decrease in prothrombin 10 1 time in male rats at high doses of VC, and decreases in hemoglobin concen tration, MCV, and MCHB in female rats at 55 ppm of VDC. Serum levels of immuneglobins were not affected in rats after exposure to VC. There were mild decreases in imminoglobin A and Subgroup A of immunoglobin G in male rats and immunoglobin A in female rats after exposure to VDC. Serum, levels of proteins were not altered after exposure to VC or VDC. Macro phage counts in the lung washings were not affected in the male rats and were slightly lowered in female rats at the higfc level of VC and at 55 ppm VDC. 3. Cytogenetic Analysis The results on numerical distribution and morphological aberr ation of chromosomes are shown in Tables 7 and 8, respectively. Bone marrow preparations from rats exposed to the high level of VC or 55 ppm of VDC for up to 3 months did not show any changes in the chromosome frequency distribution or number of tetraploids, or any changes in the frequency of chromatid breaks, gaps, or translocations. 4. Examination of Bones Hind llmb3 of the control rats and rats exposed to VC or VDC were removed at the end of 1, 2, and 3 months, fixed, frozen and stored. They will be available for examination at a later date by x-ray for pos sible osteolysis or other abnormalities. 5. Organ Weights The organ weights of male and female rats after exposure to VC or VDC for 1, 2, or 3 months are summarized in Tables 9 through 14, The absolute organ weights and their relative weights based on the brain weight were not apparently altered by VC or VDC when compared with that of the control rats. Although the absolute brain weight of female rats ex posed to 55 ppm of VDC for 2 months was statistically smaller than that of the controls, the difference was small and was not seen in rats after ex posure for 3 months. 6. Gross and Microscopic Examination of Tissues The rats exposed to various levels of VC or VDC were in good nutritional condition at the end of 1, 2 or 3 months. Microscopic ex amination of tissues revealed a number of lesions in the control rats and rats exposed to the high level of VC or 55 ppm of VDC. 11 R&S 112732 AC the- end of 1 month, all control rats, and rats exposed to VC or VDC, had lesions in the lung, liver, and/or kidney (Table 15). The lesions included perivasculitis; peribronchiolitis (associated with chronic murine pneumonia); microfoci of mononuclear cells and/or vascuolization of hepatocytes; and chronic lnterstital nephritis and/or microcalculi in the kidney. Other occasional lesions were chronic tracheitis in five rats and myocarditis in one rat. These lesions in the control rats and rats ex posed to VC or VDC were mild and focal; they were considered as spontaneous lesions. The myeloid erythrold (M/E) ratios in the bone marrow smears of these rats were normal. At the end of 2 months, spontaneous lesions also occnrred in the lung, liver, kidney, and/or heart of the control rats and rats exposed to VC or VDC (Table 16), In addition, one rat had niFositis of the skeletal muscles and one rat had pinworms in the cecum. The M/E ratios in the bone marrow smears of these rats were normal. At the end of 3 months, spontaneous lesions similar to those seen at i or 2 months also occurred in the lung, liver, kidney, and/or heart of these rats (Table 17). In addition, a few lesions occasionally occurred in other tissues, including a focal necrosis in the liver of one control rat. Other lesions included retinal rosettes of the eye in one control rat and one. rat exposed to VDC; pinworms in the cecum of one rat exposed to VDC; chronic pancreatitis in two rats exposed to VDC; tubular degeneration of the testis in one rat exposed to the high level of VC; chronic prostatitis in one control rat; and cystic ovary in another con trol rat. All of these alterations were mild, focal and were also con sidered as spontaneous or incidental lesions. The M/E ratios in the bone marrow smears of these rats were normal. B. Mice 1. General Observations and Weight Gain The mice terminated after. 1, 2 or 3 months were exposed for a total of 19, 39 and 59 days, respectively, 6 hours/day, 5 days/week. Due to scheduling changes at the end of the year, the termination for mice at 3 months was actually during the 11th week. The weight gains of all groups of male and female mice were comparable to those of the con trols. The body weights of the control mice and mice treated with the high level of VC (1,000 ppm) or VDC (55 ppm) are summarized in Figure 6. 12 R&S 112733 4 All mice appeared to be in good health. However, there were fiv unscheduled deaths. Two males and one female exposed to the high level of VC were found dead between the third and 9th exposure days; two males exposed to VDC were found dead on the 13th exposure day. Their deaths were apparently due to the acute effect of these compounds. 2. laboratory Data The results of hematology, clinical blood chemistry, and macro phage counts in the lung of male and female mice after exposure to VC or VDC for 1, 2 or 3 months are summarized in Tables IS through 23. At the end of 1 month, the peripheral blood elements and clinical chemistry values were not apparently altered after exposure to VC or VDC (Tables 18 and 19). When compared with those of* the control mice, the MCV index of male mice at the low level of VC, the MCHB index of male mice at the middle level of VC, and the SGFT level of male mice at 55 ppm of VDC were elevated. The differences were small and/or not seen at the end of 2 or 3 months. The macrophage count in the lung washings was not apparent ly affected by exposure to VC or VDC. At the end of 2 months, the peripheral blood elements, clinical chemistry values, and the macrophage count of both male and female rats (Tables 20 and 21) were not apparently affected by exposure to VC or VDC. When compared with those of the respective control mice, the percentage of eosinophils of male' mice at the low level of VC, the reticulocyte count and the BUN levels of female mice at 55 ppm of VDC were higher, and the BUN levels of male mice at all levels of VC were lower. The differences were mild and were not seen at the end of 3 months. The macrophage count in the lung washings was not apparently altered by exposure to VC or yDC. At the end of 3 months, the peripheral blood elements, clinical chemistry values, and the macrophage count in the lung washings of both the male and female mice exposed to VC or VDC (Tables 22 and 23) were not statistically different from those of the control mice. 3. Cytogenetic Analysis The results on numerical distribution and morphological aberr ation of chromosomes are shown in Tables 24 and 25. As for the rats, the bone marrow preparations from mice exposed to the high level of VC or 55 ppm of VDC for up to 3 months did not show any changes in the chromosome frequency distribution or number of tetraploids, or any changes in chromatid breaks, gaps, or translocations. 13 R&S 112734 R&S 112735 4. Examination of Bones As for the rats at the end of 1, 2, and 3 months, hind limbs of the mice were removed, fixed, frozen and stored for possible future examin ation by x-ray for osteolysis or other abnormalities. 5. Organ Weights The organ weights of male and female mice after exposure to VC or VDC for 1, 2 or 3 months are summarized in Tables 26 through 31. The absolute brain weights of male mice after exposure to various levels of VC or VDC for 1 month were statistically smaller than that of the controls (Table 26). This difference was not seen after exposure for 2 or 3 months. Based on the brain weight, the relative spleen weight of female mice ex posed to 250 ppm of VC for 2 months (Table 29) and the relative spleen and testis weights of male mice exposed to 50 ppm of VC for 3 months (Table 30) were statistically larger than those of the control mice. The signifi cantly enlarged spleen and testis are unexplained and will be examined in the remaining mice. The absolute and relative weights of other organs of the treated mice were not significantly different from those of the control mice. 6. Gross and Microscopic Examination of Tissues Microscopic examination of the five- unscheduled deaths (two males and one female exposed to 1,000 ppm of VC for 3 to 9 days, and two males exposed to 55 ppm of TOC for 13 days) revealed a number of lesions. These included an acute toxic hepatitis, characterized by focal to marked congestion, and marked diffuse coagulation type necrosis of hepatocytes beginning in the ,centrilobular area of the liver. Marked tubular necrosis characterized by pyknosis and eosinophilic granulation of the cytoplasma in the renal cortex was also observed. In addition, one mouse had R-E cells and lymphoid hyperplasia and a large number of megakaryocytes in the spleen* The mice exposed to various levels of VC or VDC for 1, 2 or 3 months were' in good nutritional condition. Microscopic examination of tissues revealed a number of lesions in the control mice and mice exposed to the high level of VC or 55 ppm of VDC. At the end of 1 month, chronic interstitial nephritis, tubular casts, tubular basophilia and/or microcalculi occurred in the kidneys of all four male control mice, one of four male mice exposed to the high level of VC and all four male mice exposed to 55 ppm of VDC (Table 32). These lesions were mild to moderate and were not considered related to the compounds. Other occasional lesions were present in three females exposed to VDC. One mouse had focal degeneration and Inflammation of the 14 R&S 112736 liver, microfoci of mononuclear cells in the salivary gland, and myositis of the skeletal muscles; and another mouse had myocarditis, bile duct hyperplasia, and cholelethiosis. These lesions were considered as spon taneous or incidental. The M/E ratio in the bone marrow smears of these mice were normal. At the end of 2 months, one male and two females exposed to 1,000 ppm of VC had bronchiolar adenoma characterized by focal areas of acinar or papillary growth forming small solitary nodules which were well demarcated but not encapsulated (Table 33). There were a number of other lesions not caused by VC or VDC. The renal lesions, seen at the end of 1 month, also occurred in these control mice and mice exposed to VC or VDC at 2 months. In addition, a number of lesions occasionally occurred in other tissues. In the control group; one mouse had microfoci of Inflammatory cells in the liver;. and another mouse had a marked suppurative inflammation and moderate hyperplasia of the perianal gland; and three mice had an acute metritis. Some female mice exposed to 1,000 ppm of VC had myocarditis; microfoci of inflammatory cells or focal mineralization in the liver; acute metritis; and/or myositis of the skeletal muscle. The mice exposed to 55 ppm of VDC had microfoci of Inflammatory cells in the liver or focal mild fatty change in the brain. Hie M/E ratio in the bone marrow smears of all these mice were normal. .At the end of 3 months, two males and one female exposed to 1,000 ppm of VC had bronchiolar adenoma or focal acinar epitheial pro liferation In lungs, as seen at the end of 2 months (Table 34). A number of die control mice and mice exposed to VC or VDC had renal lesions, as seen at the end of 1 and 2 months and. In addition, hepatic lesions including microfoci of mononuclear cells and intranuclear inclusions of the hepatocytes.' The liver of one male souse exposed to VDC also had focal necrosis. Furthermore, two mice exposed to VC had focal chronic inflammation in the salivary gland and four mice exposed to VDC had focal myocardial degener ation. These mild occasional lesions were not caused by the compounds. The M/E ratio in the bone marrow smears of these mice were normal. Due to the occurrence of bronchiolar adenoma in the lung of mice exposed to the high level of VC, the lungs of mice exposed to the low or middle levels were also examined. One male exposed to 250 ppm at the end of 2 months, and one male exposed to 250 ppm and one male exposed to 50 ppm at the end of 3 months also had bronchiolar adenoma. The com posite incidence of bronchiolar adenoma are summarized in Table 35. The occurrence of bronchiolar adenoma in these mice exposed to VC appeared to be dose-related. 15 R&S 112737 IV. DISCUSSION AND CONCIDSION Exposure of rats of both sexes at 6 hours/day and 5 days/week for up to 3 months to 50, 250, or 1,000 ppm of VC or 55 ppm of VDC did not cause any significant adverse effects. The weight gains of female rats exposed to 1,000 ppm of VC or 55 ppm of VDC became less than that of the control rats after the 5th week. However, the differences were not statistically significant. The laboratory results, including hematology, clinical blood chemistry, serum immuneglobins, serum proteins, and macro phage count In the lung, were not apparently affected* Exposure to VC or VDC did not cause any cytogenetic effects as measured by numerical dis tribution or morphological aberration of chromosomes In the bone marrow preparations. Various organ weights at termination after 1, 2 or 3 months were not apparently altered when compared to those of the control rats. Although a number of spontaneous lesions occurred in various tissues, ex posure to VC or VDC did not present any pattern of lesions in rats. Exposure to VC or VDC caused some early deaths In mice. Among a total of 36 mice of each sex, two males and one female exposed to 1,000 ppm of VC died between the third and 9th exposure days, and tvo males ex posed to 55 ppm of VDC died on the 13th exposure day. Hlstopathology re vealed acute toxic hepatitis and marked tubular necrosis of the renal cor tex. The other mice appeared to be healthy. However, bronchiolar adenomas developed after exposure to VC. Its occurrence appeared to be dose-related. At the end of 2 months, bronchiolar adenoma occurred in one male at 250 ppm, and in one male and two females at 1,000 ppm. At the end of 3 months, it occurred in one male each at 50 and 250 ppm, and in two males and one fe male at 1,000 ppm. At each time period, four males and four females were examined. Exposure to various levels of VC did not cause any other signifi cant adverse effects in mice. As for rats, mice exposed to 55 ppm of VDC for up to 3 months did not show any adverse signs, ary changes in laboratory data, any cytogenetic effects, or any pattern of persistent lesions. 16 R&S 112738 TABLE 1 LABORATORY DATA OF MALE RATS AFTER EXPOSURE TO VC OR VT)C FOR 1 MONTH a1 erythrocytes mo /mm 1 RETICULOCYTES. s MCNATOCRIT, VOL. 1 HEMKL09IN* GM. I . MCVa CUGIC MICRONS MOW* MICRO NICROOMS. MCMNC. GH * S3 PLATELETS <X10 /MM ) 93 LEUKOCYTES (210 /MM 1 NEUTROPHILS. S %lymphocytes, AMOS* % EOSINOPHILS. t BASOPHILS. 1 NOMOCYTES. ATYPICAL, S NUCLEATED MC. % PROTHROMBIN TIME, SEC. SCOT, IU/L SOFT, IU/L ALX. AMOS., IU/L BILIRUBIN, TOTAL m % BILIRUBIN, DIRECT NO S SUN. MO S CREATININE,.NO s LON. IU/L IMMUNOGLOBULINS A, IU/hl 0. A FACTOR. IU/ML 0. FACTOR. IU/ML N. IU/ML TOTAL PROTEIN. CM ALBUMIN. On s SLONULIN. ON t 3 MACROPHAGES ino./nh 1 Control o.oo * .94 (3) so * .23 (3) AR.a m. 1S.1 0 49.9 0 .6 *2 4.5 (3) (3) (3) 22.* * 1.4 (3) 34*2 .a (3) T.4 * tt.T 14.3 * .7 4 1*4 <33 (33 43.9 * 2.4 o.s * .9 * o.a .3 0.0 a.o l *4 .4 a.* M* 0.0 4.4 0.0 12.2 .3 43.3 * 4*3 30.3 * *3 0 1.+ .1 .1 4.4 * 0.0 i'A.o 1*1 .7 * .1 913 * 30T VC to tt* 7*05 * .13 1.34 * *32 4**3 *. 4 15.0 * .2 42.4 * 1*0 21.2 * 33.5 2 *2 4*2 * .4 11.4 * 1*3 14.3 * 3*2 3.0 * 4.4 0.0 * 0.0 1.4 I 1.2 0.0 * 0*4 1.0 * .7 0.0 * 0.0 0.0 o.a 11.* * 4 45.0 * 3.5 32.5 125 * 1.3 i/ * J * 1 .1 * 17.0 *0 .5 4 i 0.0 235 * 104 4.2 * .4 393 * 2 79 47 0 22 2 9.3 0 *3 3.3 0 *2 1.9 0 144 0 .2 43 00 3*4 * 3*5 1.4 1 0 .2 *1 .2 0 vc 130 nm 7.44 * 1**4 . 45.0 * .04 .47 4 13.2 40.3 0 1 .7 20.4 . 33.0 .3 *2 0.2 5.0 0 *0 ,5 5.3 * 1.4 05.4 * 1.4 0.0 * .5 0 0.0 0 3 0 0.0 0 0.0 0 ii.o 0 *3,5 0 0.0 3 0.0 .3 0,0 0,0 ,1 2.5 33.3 1.4 03 * 9 .3 * .2 * .1 .1 2/ 13.4 . 3 4 . 0.0 337 * 143 00 3,4 0 3.7 l 2.0 i 0 .1 .2 .1 0 VC 1,000 ,<> A.ON * . 44 2.90 . l.U *2.9 * .0 l*.* * .3 Tl.T . 4.2 2*.2 . 33.9 . 1.0 .4 0.7 . Ul 11.3 . l.l 10.4 * s* 00.3 0.0 . 1.0 m 5.7 0.4 .4 0.0 0.4 .9 # 0.0 0 .3 0.4 0.0 0 0*0 12.1 * .3 A*. 9 . 1.9 3*. a . 1.2 102 * 5 .3 . .1 .1 *a .0 19.3 .4 0 4 0.0 302 * 104 3.3 * 344 * .1 20 107 24 30 . l 3.6 * .1 3,3 . .3 2.4 .3 430 03 lacrlai in lua - 5,2. at k r*ci atcapc 11 oaead 1> 2/ SlislElcaacly dllfirac trom ciu CmctbL uni, p < 0.02, OiHMt'i aalcipla caaiailHi proeaNma. YSC Si jp- 7.01 . 1*17 * *2.3 0 14.3 0 40*3 . 20.4 0 .0* .10 .9 .2 .9 .3 33*0 1 9.2 .6 9*0 - .6 L*.0 2.7 03.3 2#* 0.0 0.0 1.0 0.0 0.0 i 0.0 1.4 . 3 0.0 * 0.0 0.0 . 12*1 *9 03.0 0.4 30.3 i.* 143 .3 -- .2 . .1 .14/ 14.4 . i.t .7 * .i 340 . 194 00 3.4 * --J.* .1 .1 2.9 .1 203 136 17 TABLE 2 LABORATORY DATA OF FEMALE RATS AFTER EXPOSURE TO VC OR VDC FOR 1 MONTH ai ERYTHROCYTES (XIt /mm I RETICULOCYTES. t HEMATOCRIT, vot_. MEMOOL09IN. dm, % MCV, CUBIC MICRONS MCMO, MICRO HtCROGMS. MCMBC* GM t 53 PLATELETS Uto /MM ) 33 leukocytes <xi /mm i NEUTROPHILS. LYMPHOCYTES. iAMOS. EOSINOPHILS. BASOPHILJ. * MONOCYTES* % ATYPICAL* S nucleated <*ec. t PROTHROMBIN TIME* SEC. SOOT. 1U/L MPT. IU/L ALX. PMOS*. tU/L bilirubin. total n bilirubin. direct mo \ SUM. ho CREATININE. HO * LOM. TU/L immunoglobulins A. TU/ML 0, A PACTOR, tU/HL 0. B PACTOR, (U/ml n* TU/"L TOTAL PBOTEIM* 9M 1 ALBUMIN* om OLOBULIN. CM * 3 MACROPHAGES (MO./MM I (uni **19 X 1*57 * .3* .AT + 1*5 * .5 l+,5 X .2 *7.* X 4.7 23. 7 X 1.7 34*9 * .1 *.5 * .3 7.3 X .A 17,3 t 3.1 1*1 X 3.1 0.0 * 0.9 i * .5 o.o * o.o *3 * .2 0*0 * 0.0 0*0 * 0.0 12*1 i .3 (2) T+*3 * A.A 33*5 * 7+ * 2.* 7 .1 * .1 ,1 * .1 15*3 * l.A .4 * 0.0 391 * 21A YC JO A.A9 21 1.34 * .53 43.0 * 0.0 l*.7 * ,2 44.0 * 2.0 zz.i * 34.2 * *0 + 3.9 * * + 7.3 * *9 10.0 * 2.0 BO.9 * 3.3 0.0 * 0*0 1.0 * * + 0.0 * 0*0 .9 * .3 0.0 X 0.0 0.0 * 0*0 11.9 * .2 A9.0 10*7 Z7.0 2.1 A9 * 0 .2 .1 .1 * *1 13.0 1.2 * * 0*0 +9+ 23* 7,3 * 393 71 +9 5*3 * 3.+ 2*1 * 210 4 .0 34 tz 3 .1 .1 .1 TZ s.r * 3.7 * 2.0 * .1 *2 *1 vc ua (* 4*22 * 1.17 * .19 .35 41*0 * ,4 1**2 * 47.2 * .3 1.3 22*9 34*1 * 7.3 * .2 .3 .4 7.7 * .5 13*5 4.A 05*3 * 4.1 0*0 o.o .0 * 0*0 .9 o.o 5 * 0*0 * .3 0.0 0*0 * 0.0 11*4 * .2 71.3 * A.A 27.4 * 1.4 77 * 10 .2 3 .1 .1 X 14*0 .1 .7 .7 * .1 *4* * 1ST 5.4 * 3*4 X 2.0 X .2 .2 .1 VC 1.000 pfM 9.07 SI l.TA *77 40.0 * 14,1 * 4 .1 70.0 * 24.A 7.4 2*4 39.2 * .3 7.9 3.9 X' .5 14.3 3.3 43.0 3.4 0.0 . 0.4 .3 3 0.0 0*4 .5 *3 0.0 * 4.0 o.o * 0*0 11.7 '* 4 41.0 * 2,4 29.3 * 2*1 (0 * 2 2 * .0 * 1 ,4 13.0 * *9 *4 * 72 . 4.0 4 7.1 4 4 304 *1 BO * u 44 2 5.7 . 3.5 * .1 .2 2.2 X .2 243 x 43 vtc JJ PPM 6*73 .9* * +* I + .0 * .14 .11 .7 .2 59.5 .0 24*4 * .2 34*9 .2 5*5 * 4 Ll*9 * 1.0 A 12.4 * 3.0 14.0 - 2.7 0*4 i 0.0 4 - . 1 4.0 - 0.0 .5 - .1 0.4 - o.o 0*4 * a.a 11*2 * 73.3 1 ( 0-3 { 3+.0 * 1.7 < *3 * 3( .1 ( 2 - 4 1 17*3 - 1-7 < 1*0 - .1 ( a-' 329 - (4 ( k.l .1 3*9 X 1*4 X .3 .3 249 X 45 EatriM r wia i S.E. of 4 rtci xe?c o ooc4 la fOfriichaoao. i Sltolflcoaclr 4lfrac ixom c4 Control froa** p < 0.03, DuBaoct'i aaltipl* pro<o4t*r. R&S 112739 18 TABLE 3 LABORATORY DATA OF MALE RATS AFTER EXPOSURE TO VC OR VPC AFTER 2 HO *3 ERTTMMOCYTES IXIA turn 1 RETICULOCYTES. * hENATOCRIT. VOL. * HCM04L00IM* So. MCV. CIMIC MICRONS nCMU. MICRO MlCROAMS. MCNUC. CM V 33 PLATELET* (MO /NM ) 33 LEUKOCYTE* (MO /MM ) neutrophil*. * LYMPHOCYTES. SAMOS. * EOSINOPHILS. * BASOPHIL*. * MONOCYTES. * atypical. * NUCLEATED roc. * PROTHROMBIN time. SEC. SOOT. tU/L SORT. tU/L ILK. PhO*.. IU/L BILIRUOIM* TOTAL MO BILIRUBIN. OIRECT mo * SUM. MO , CREATININE, mo * ldm. iu/l ALRMA-MSOH. iu/l IMMUMOOLOSULINS A. IU/ML 0* A FACTOR. IU/ML 0. a FACTOR. IU/ML M. IU/ML TOTAL PROTEIN. OM * ALBUMIN. OM s SLOauLIM. SM 1 3 AC*0PA4CS tHOm/m 1 Cam* ipl 4,94 * .10 1,01 .12 44,5 * .3 14,5 * .2 *4,1 * .A 2loJ .3 13,3 * .A 3.7 .0 9,4 *0 12.0 , 1.2 04,3 2.1 0,0 0.0 1,0 t.A 0,0 , o.o 0,0 , 0.0 0,0 , 0.0 0,0 * 0.0 11,4 * 10Q * (3) T 43,0 * 3.3 73 , 3 .0 * .0 0,0 * 0.0 13*0 * .3 *4 * 0.0 IDEA * IA3 A3A * 4A 10.3 * 2.1 325 , 1* AB 3 7* 20 3.7 * .1 3.3 , .1 * 2.A * .2 206 , *0 so 9m 7.03 * 1*42 43,0 . 14,0 .12 ,00 0 *0 42,3 * 21*2 * 34.1 5,0 . 12.4 10*0 . 1*7 .3 *4 . ,3 ,7' 4.1 70.3 * 4,0 . 4*0 4*0 *5 * 0*0 . 1.3 * 0.0 * .3 0*0 4 0*0 0,0 * 0*0 lo.a , 74 * a 4*: 34,0 * 2*4 40 * 4 a a 0,0 * 0,0 14.3 . ,4 ,4 0*0 344 * 34 U 22T * 23 -- 0a 5*7 . 3.4 * 2.3 1. 0 *1 *1 .2 4 2*0 PYM 4,02 * .12 1*34 * 10 43*0 .7 14.7 .2 42*2 * 1.3 21.2 * 34,1 * .A .A 4,7 .7 0.3 * l.l 14,4 * 2.7 2,0 * 2.4 o.o * a.a .5 * .5 0,0 * a.a *3 * 0.0 * .3 a.a 4.0 * a.a 10,4 * 44 . 32,5 * A a A 2.9 17 ' 73 9 a* 0.0 * .1 a.a 14.5 * .4 i.a a.a 311 *129 * 73 if 2B * 0A 5.4 * 3.3 * 2.1 * 0 .1 .A .1 A 1,000 Of* 4.03 . `.A~2 1.32 * .18 42.0 * .3 14*1 * .2 42.4 .5 20*4 * *2* 32.9 * * 5.9 * .5 9.2 * .7 9,0 1.1 94.3 * .9 0.4 0.0 .3 * .3 o.o * 0.0 .5 . .3 0.0 * o.o 0.0 Mr 0,0 10.4 TO * .3 3* 34.4 * 60 , 1.* 2 a , .A 0.0 A.A 13*4 .3 4 A.A 501 * 207 . AA if 2. * 10.2 A** 294 . 47 2.* 3ft f 47 * 17 5.5 * .2 3.5 * .A 2.0 * .2 29* * 13A Saerlat ar **aa s 3.1. of 4 net a aocod la piroatbu**. j/ SifaifLemcly dlffome fvoa e& Caacrol {roup, p < 0.03, Qumact'i sulclpl* eo^ortaon procttfcsra, * S3 9m A.ST * USB * *2.3 . .20 .03^ lA.a - * AA.S 1.9 22.2 3A.S . * .6 S.T * .2 i.s . .5 11.3 - 1.3 S7. - 1.4 A.A 1 0.0 l.A . .7 A.A * A.A * A.A . 0,0 0.0 0.0 A.A * 0*0 11.3 * 7A * 2B.5 SB * 0 1.t7 5 .2 .1 * 19.S , a a .6 .A 0.0 A*B - 143 3A2 47 12.2 * 1 .A 310 - 12 I* - 5 1A3 24 A.A * .2 J.S 2.* 2IS L AI 46 R&S 112740 19 TABLE 4 LABORATORY DATA OF FEMALE RATS AFTER EXPOSURE TO VC OR VDC FOR 2 MONTHS R&S 112741 43 ERYTHROCYTES (XIO /MM ) RETICULOCYTES* ft *M4T0CR1T, VOL* % HEMOGLOBIN* Gm. ft HCV* CUBIC MICRONS hChB* MICRO MICROHMS- HOH9C. 0** ft 53 PLATELETS 1*10 /MM > 33 LEUKOCYTES (X14 /MM 1 NEUTROPHILS. * LtmmmOCtTES* 1 BANOS* % EOSINOPHILS* ft BASOPHILS* t MONOCYTES, ft atypical* ft NUCLEATED ROC* ft PROTHROMBIN TIHC* SEC* cmnl 0.50 . 1*06 . .12 *13 *2.3 *S 1*.5 * 4**3 * 22.0 , *2 U* .* 3**2 . 6.0 * .* ** T.A L .* tl.a . 3.1 07.S * 3.3 3 1.3 * .3 .5 0.0 * 0.0 4.0 * 0.0 . 0.0 0.0 0.0 * 0.0 10.4 1 30 PP* 4.41 * 23 l.*B L *23 *1.* 1*.2 * .4 .2 44.3 * 21.4 * 2,1 .4 3**1 * 4.3 * .9 7.S L *9 11.S , 2.* or, 0 * 2.4 0*0 0.0 1.0 . 0.0 0.0 .5 # 0.0 * 0.0 *9 0*0 0.0 * 0.0 10.4 * .3 230 ppm 4.14 L. 1.44 m .21 ,2*' *1.* * .4 l*.l .2 47.7 2. * 22.* .7 33.7 *2 9.* * .7 t.l # l.i 1*,3 . s.o 0*.3 * S.A 0.0 0.0 1.3 .9 0.0 . a.o 0.0 0.0 0.0 . 0,0 0.0 . 0,0 10.9 .2 SOOT* 1U/L SGPT* EU/L ALK pnos~* IU/L BILIRUBIN, TOTAL m 0 BILIRUBIN. DIRECT MO m HUN. MO ft CREATININE* MO ft LOH* IU/L ALPHA *H0OH* IU/L tMMUNOOLOOULlNS A. tU/ML G. A FACTOR* tU/ML G* B FACTOR. Iu/hL 4* IU/"L TOTAL PROTEIN* on 1 ALBUMIN* oh 1 globulin, oh % 3 MACROPHAGES (NO./MM t 7S.G * 10.3 32.5 m 2.* SO . 3 0 a 0,0 % o.a 1S.0 L .* .0 . .1 370 * 4*~ ISO * ll.R AS* * TO * SI *2 3.S 19 16 IS t.o . 3.3 * 2.* * 200 . .2 .2 *2 *3 4*.* * 4.2 35.3 . *.r 9* . to *1 . *1 ,0 *4 14.* . 1.3 .4 . .1 22* , 4* 4* . 34 00 4.2 * 3.* . 2,* a .1 .4 .1 0 47,0 * 4.1 33.0 . 3.1 *3 L 4 ,1 L Q 0.0 0.9 l*.a . .4 .7 * .1 2*0 * "9 121 i, *7 00 4.0 . 3.4 * 2.* . a ,2 *1 ,2 0 Cacrlii an mb x LL I A nu UK17C ia e*d 1a NnRC&MN* g/ Slfatll4*aely AlfftrMC from th Cmc1 froa? , p < 0*03. Dmiwcc1! Milclpl* pro<*4r. 1,000 PR* *.70 * .20 l.*S . ,19 # 4 l*.l .1 *0.1 *1.1 1.3 ,3 33.* .3 *.t * .2 7.2 * .4 l*.S , 7,3 12.9 * 7.2 . . 0.0 ,* * .5 0.0 * o.a .3 . .3 0.0 * 4.0 0.0 0,0 10.4 * .2 fl*,0 7,7 34,3 . A.l 40 . 4 1 * 1 0 0 IS,3 * *4 . 4 *1 S21 . 20* . PI u 11.1 * AS* . 2,9 33 *3 . 12 S3 * 13 4*1 *2 3.S 2,3 .1 ,1 190 * to S3 PP 4.39 . 29 1.2* * 2* *0.5 . 13.4 # *4 .2 43.4 * 1.2 22.0 * .* 3*.5 . .3 7.2 * .7 4,0 . .4 14.3 * 2.* 41.3 2.1 0,0 . 0.0 2.0 . 0.0 . 1.1 0.0 .J * 3 0.0 . 0.0 0.0 * 0.0 10.2 . .3 73,3 . A* J 22*4 . 2.1 34 . 2 ,1 *0 . .1 .0 17.0 . *4 . 34* * t *2 O.ffS 43 2*3 32 11./ 1 343 -- 1*7 AA 43 - M 2* . 3 4.0 . 3.4 2.3 * .2 *0 .2 W3 . to R&S 112742 TABLE 5 LABORATORY DATA OF MALE RATS AFTER EXPOSURE TO VC OR VDC FOR 3 MONTHS 63 ERYTHROCYTES <*U />** 1 RETICULOCYTES. 1 HEMATOCRIT. VOC. t \ HCMQOLORlN. ON, - *CV. CU91C HICHONS HCH9, MICRO MICROSMS. hcmsc. nt* t 93 PLATELETS (XU /MH % 33 LEUKOCYTES (XU /MN ) NEUTROPHILS. 9 LThPHOCYTES. * 9ANOS. 9 EOSINOPHILS, % 9A30PHIL5. 9 HOXOCYTES, 9 ATYPICAL. 9 NUCLEATED RBC. 9 prothrohrin tine. SEC. soar, ju/l SflPT. IU/L ACK. PHOS., IU/L 9ILIRU9IN. total ho * OILIRUOIN, DIRECT HO 9 SUN, ho 9 creatinine. ho t LOH, IU/L ALPHA-NROH. tU/L IHHuhOOLOSULINS A, IU/HL a, a xactor. iu/"L 9, 9 XACTON, IU/"L H, IU/HL total PROTEIN, OH ALSUMIN, OH 9 OLOSULIN, OH 9 J MACBOPHAGC5 (NO./W* > Casual 9,93 *. 1.39 * Ai.a * .39 .90 1.3 i*.* .9 91.9 * 1.9 21.2 * ,0 39.3 . 3 5.9 * .5 9.3 * 1.1 11.5 *.9 99.3 * S.l .3 .3 2.3 , 9.0 .9 9.9 .3 * .5 9.0 . 9.9 9.0 . 0.9 11.9 . .2 112 21 So.9 19.9 97 * .9 . .9 9.9 9.9 19.9 . 1.1 .9 * 9.9 999 222 379 * 91 9.9 * .3 A19 hi IS 9 193 * s' 9.9 * 3.3 * Z.* * 190 * .1 .9 .1 90 VC 9.73 * 1.31 A3.9 # t*.7 * 03.9 * .07 .1* .7 .3 .7 21.9 3A.2 .3 0.9 * 4 9.9 .7 19.3 * 3.1 92.9 * 3.4 9.9 . 0.4 1.9 . * 9.9 * 0.4 .9 . .3 9.9 * 0*4 9,9 * 0.0 11.4 * .0 79 * 7 31.9 1.2 74 * .1 .0 9.9 . a.a 13.3 * 9 .9 .2 594 * 1*4 293 . 41 94 9.0 * 3.2 * 2.4 9 .1 .4 .4 0 VC 130 99m 9.99 .07 1.43 * 41.3 * 14.2 . *31 3 3 92.0 . .4 21.3 .4 34.4 * *4 T.7 . .7 .* a.. 1.2 11.3 *.5 97.3 1 2.1 9,9 0.4 1.3 - .4 9.9 - 0.4 .3 .3 9.9 - 0.0 9.9 0.4 11.3 *2 79 34,9 . t.7 49 * . .1 . *4 9.9 A* 0.0 12.3 * .3 .4 ** .2 393 137 232 94 90 3.7 . 3.1 * 2.4 * 9 .1 .0 .1 0 SatrtM iTf *ma 1,1. 4 rt* rce u dsctd la si iiilcABClf diffamc tttm cha Control frmip, p < 0*05* Ooaaoce** atietpU eoaporlooa ?toc*dr*. vc 1,000 9?a 9.47 .i* 1.91 .23 *4.9 .* 13.9 .3 43.2 1.1 21.4 .* 14.2 .3 4.3 4^ 19.4 2.1 22.9 6.4 77.3 6,0 9.9 4*0 9.9 0.0 9.9 0.4 .3 .0 9.9 0,4 9.9 11.9 *2 0.4 .3 4/ 4 33.9 3*5 71 7 .1 9,9 14,5 .0 0*0 .4 .5 2 777 156 335 70 4.5 .5 4t3 4 44 27 *4 r 5.4 .3 3.1 2 2.5 *2 2*4 - 55 voc 33 Pf 7.32 .11 121 1.05 *0? (2> 42,4 1.0 (21 14.6 57.3 20.0 *3 (2 *5 (21 *1 (21 a*,* 2 (2) 5.2 *3 (21 4*4 1*7 (2) 10.5 *5 (21 86.5 1*5 (21 4.0 0*4 U> *3 *3 (21 0.4 4.4 W *3 *3 U) 4.3 a.a at 0*0 0*0 U) 103 u 40.4 4.4 ijr 51 4 3) * l .1 fil 0.0 3*0 O 15*0 *6 (31 .6 0.0 (31 442 183 (3) *32 44 (31 5.1 4.4 in i' 2** 87 (3) 17 40 24 Ol 124 - 4 < 3) 6.4 2 (3) 3*3 *2 (31 2*7 .1 (3) 246 133 21 TABLE 6 LABORATORY DATA OF FEMALE RATS AFTER EXPOSURE TO VC OR VDC FOR 3 MONTHS 43 RYTM*oCrrE$ CHO /pm i reticulocytes. % HEMATOCRIT* YOU* * HEMOGLOBIN. 4m. 5 MCV* Ct/fKC *CCRONS MCMd* MICRO H1CROUPS- MCHBC. $ * 53 PLATELETS (Xio /MM I 33 leucocytes <no /mm i nUTCPNILS. 1 LYMPHOCYTES* % SAMOS* * COSIMOMMILS* % SaSOPnILS* % MONOCYTES* 1 ATYPICAL* S nucleated roc. % PROTHROMBIN TlMCt SEC. SOOT* IU/L SORT* IU/L *l*. pmos*. tun. AtURUGtN. TOTAL h % SILIRUBIN* DIRECT "0 A SUN* MO % CREATININE* MO 1 LOH. IU/L alpma-hrom. iu/l iMuNOGt.oeui.tNS A IU/ML 0* A FACTOR* IU/ML S. 0 FACTOR* IU/ml n* IU/ml total protein* GM % ALBUMIN, gm 1 GLOBULIN* ON * j* MACRORhaOCS (NO./mm 1 Conceal 6*37 - 14 1.2. X .3* *2.8 X *0 1*.S X *2 *4.2 & 1.9 22.8 , A 3*.8 6.2 * .2 .9 9.6 - 3 14.J 3.A *.3 3*G 0.8 % 0,0 1.3 2. .A 8.0 X 0.0 .3 -- .3 8.8 0.4 0.0 0.0 11.8 & *2 HO.3 * A.I 34.3 X 9*1 39 4 .1 . 80 8.8 & 0,0 13.3 X ,4 .4 & 0*0 488 75 242 33 18.4 - .'6 624 i *7 74 X 5 74 X 10 4.3 X .1 3.4 X .0 2.7 X *0 129 13 VC* 30 nm 4,54 * IS U26 * .20 41.3 x 1.3 1*,2 * .2 42.1 * 1*4 21*7 *3 3*.5 * .5 9.7 i 4.4 i t .4 10.5 * 4*4 74,3 *, 4.7 0.0 X 0.0 *1 * 0*0 * ,3 0.0 .9 * *3 0.0 * 0.0 0*0 * 0.0 10*7 * *2 03*3 * 4*4 34*3 X 1,4 34 * 3 1 X .1 0.0 * 0*0 1**3 X .4 4 * 0.0 TOO 140 314 * 02 00 YC 130 TP* 4*49 . .U 1.44 * .14 42,3 * .9 14*4 X 49.5 22*7 * .1 * *3 34.7 * 5.4 * 4*9 * 4,4 . .7 * .7 1-4 44.S * 1.7 0,0 . 0.0 *5 . 0*0 * .3 . 3 0.0 .3 0.0 * 0,0 * 0.0 0,0 11.0 * 41,0 * .3 9,1 33.0 * 50 * .1 . 0*0 * 15.0 * 3.3 4 .0 0.0 .7 .4 X 0.0 421 X 54 275 * 14 00 4*1 * .1 3.4 * .1 2*9 ,1 0 '0 9.4 . 3*3 * 2.7 . 0 .1 .1 .0 0 TO l.ooo w4.53 * .19 1.7* * .06 41,5 * .4 14,3 * .2 43.4 .8 21*4 . .2 34.* * .* 4.2 .9 4*1 * .4 14,3 * 2.4 3.5 * 2.7 0,0 . 0,8 1.4 * .5 0.0 * 8.8 .3 . .2 0*0 . 8.a 0.0 * a,a 11.1 * .1 44,3 * 10.9 32*3 * 6,4 *4 . 7 .1 - 0 0.0 * 8.8 19*3 .4 ,3 . .1 444 * 1*6 304 * *6 4.2 .* *53 . 104 4 * 79 40 * U 5.0 * 3.4 m .3 .0 2.4 * A* * .3 7 ,J totrl r iw : j.l. of 4 nu oaoft u 14 1/ Jltslfleisclr dlffotosc fro* cho Control troop, ? < 0.051, moet'o oulclplt co(xcl*oo ;n<in. VOC 95 pv> 4*51 - .1* .04 X ,07 34,3 * *4 13*5 * 40.3 . .2 1.2 20*7 X .5 34.3 X 5.5 X 5.0 # .3 .3 .5 12*0 * 07,0 . 0,0 , .3 2*4 2.7 0.0 .3 0.0 X a.o 0,0 X 0*9 0*0 X 0.0 4*0 X 0,0 11,0 X ,* 40,3 X *,4 31.0 X 1,2 21 X 1 l X , 0.0 * 1**3 * 0.4 A *4 0*0 *43 * 112 224 . 4* .5.2 X .0 345 X 22 9* 10 100 X 10 4.4 X 3.* 3*a - *>1 x .1 .1 .1 4 R&S 112743 TABLE 7 aniiM;TCAL DTSTRIBOTTON OF CHROMOS.OMES_IN BONE MABROW EXPOSED TO VC OR.VDC Treatment Control VC (1,000 ppm) VDC (55 ppm) Control VC (1,000 ppm) VDC (55 ppm) No. of Chromosome Frequency Rats ^40 41 42 43 a44 Tetraplolds (4N) per 100 cells After One Month 6 3^/ 4 42 . 1 0 0.250.11-/ 6 4 4 41 l 0 6 3 3 43 1 0 0.33+0.17 0.4210.08 After Three Months 5 6 7 34 2 1 0.00.0 5 9 7 30 3 1 0.0+0.0 3 5 5 39 1 0 0.0+0.0 a/ Mean b/ Mean + S.E. R&S 112744 23 Treatment Control VC (It000 ppm) VDC (55 ppm) Control VC (If000 ppm) VDC (55 ppm) a/ Mean S.E. TABLE 8 MORPHOLOGICAL ABERRATIONS OF CHROMOSOMES IN BONE MARROW EXPOSED TO VC OR VDC Ho. of Rata Chromatid saps per 50 cella Chromatid breaka per 50 cella Tranalocationa per 50 cella Total Aberrations per 50 cells After One ^ontl] 6 Q.67Q.2l&f 0,330.33 6 0.83+0.31 0.500.22 6 G,330.21 0.870.48 0.33+0.21 0.33+0.21 0.17+0.17 1.330.21 1.67+0.56 1.37+0.61 5 0.80+0,40 5 0.14-1-0.14 3 0,33+0,33 After Three Montha 0.0+0.0 0.0+0.0 0.53+0.53 0,250.25 0.20+0.20 0.0+0.0 1.05+0.32 0.34+0.21 0.86+0.47 wLZii s'sa 9pizu ssy Terminal Body Weight Brain Liver Kidney Spleen Tea tea Liver Kidney Spleen Te8 tea TABLE 9 ORGAN WEIGHTS OF HALE RATS AFTER EXPOSURE TO VC OR VDC FOR 1 HONTH Control VC 50 _EE VC 250 ppm VC 1.000 _REB VDC 55 ppm Absolute Weight feirrt 350 45^ 2.11 0.03 10.4 0.9 2.91 0.13 0.75 0.06 3.35 0.05 4.9 0.4 1.4 0.1 0.36 0.03 1.59 0.02 371 27 1.82 0.25 11.4 0.9 3.36 0.40 0.73 0.05 2.98 0.14 367 11 1.98 0.02 11.2 0.5 2.87 0.08 0.64 0.02 3.07 0.00 384 26 2.04 0.04 12.4 0.7 3.20 0.13 0.80 0.02 3.18 0.11 Relative to Brain Weight fgm/ftral 6.6 1.0 2.1 0.7 0.43 0.08 1.77 0.34 5.7 0.3 1.5 0.1 0.33 0.01 1.55 0.02 6.1 0.2 1.6 0.0 0.39 0.00 1.56 0.05 363 3 1.87 0. 04 11.0 0. 5 2.98 0. 03 0.64 0. 04 3.12 0. 10 5.9 0. 3 1.6 0. 0 0.34 0. 02 1.66 0. 03 a/ Mean S.E. of four rata. TABLE 10 ORGAN WEIGHTS OF FEMALE RATS AFTER EXPOSURE TO VC OR VDG FOR 1 MOtJTtj Control Terminal Body Weight Brain Liver Kidney Spleen Ovaries 235 153/ 1.91 0.06 6.9 0.2 1.82 0.09 0.46 0.02 0.22 0.06 Liver Kidney Spleen Ovaries 3.63 0.05 0.96 0.06 0.24 * 0.01 0.12 0.04 VC 50 ppm VC 250 ppm Absolute Welsht VC 1.000 .RES 235 8 1.95 0.04 7.3 0.2 1.92 0.06 0.51 0.03 0.13 0.01 230 8 1.88 0.04 7.5 0.2 1.88 0.02 0.48 0.02 0.14 * 0.01 210 8 1.81 0.07 7.5 0.3 1.77 0.10 0.48 0.04 0.17 0.03 Relative to Brain Weight Cgm/gnO 3.72 0.09 0.98 0.02 0.26 0.02 0.07 0.01 4.01 0.15 1.00 0.03 0.26 0.01 0.07 0.01 4.19. 0.28 0.98 0.06 0.27 0.02 0.09 0.02 VDC 55 -EES 230 i 7 1.74 0. 05 7.0 0. 2 1.55 d: 0. 23 0.50 0. 04 0.12 d: 0. 02 4.05 0. 22 0.90 0. 14 0.29 0. 03 0.07 0. 01 a/ Mean S.E. of four rata. 8PLZU SSU Terminal Body Height Brain Liver Kidney Spleen Testes Liver Kidney Spleen Tea tea TABLE 11 ORGAN HEIGHTS OF HALE RATS AFTER EXPOSURE TO VC OR VDC FOR 2 MONTHS Control VC 50 ppm VC 250 ppm VC 1.000 ppm VDC 55 ppm Absolute Weight (Rnp 477 9sJ 2.14 0.05 12.1 0.4 3.32 0.17 0.81 0.05 2.83 0.27 5.7 + 0.3 1.55 0.09 0.38 0.02 1.3 0.1 475 11 2.09 0.04 12.4 0.7 3.25 0.08 0.74 0.05 3.27 0.16 451 16 2.19 0.02 13.0 0.8 3.30 0.08 0.76 0.08 3.53 0.17 488 15 2.21 0.01 14.6 0.7 3.65 0.13 0.83 0.08 3.32 0.10 Relative to Brain Weight fcm/gnO 6.0 0.4 1.55 0.03 0.35 0.03 1.6 0.1 6.0 0.4 1.51 0.03 0.35 0.03 1.6 + 0.1 6.6 0.3 1.65 0.06 0.38 0.01 1.5 0.1 479 11 1.70 0.28 12.7 0.6 3.13 0.11 0.79 0.05 3.08 0.06 8.4 1.9 2.07 0.46 0.51 0.09 2.0 0.5 a/ Mean S.E. of four rata. TABLE 12 ORGAN WEIGHTS OP FEMALE RATS AFTER EXPOSURE TO VC OR VDC FOR 2 MOUTHS Control VC 50 ppm VC 250 ppm VC 1.000 ppm VDC 55 ppm Absolute Weight fgnO Terminal Body Weight 271 gS/ 274 9 273 12 265 3 270 11 Brain 2.08 0.04 2.16 * 0.09 2.03 0.03 1.98 0.05 1.84 X 0.03^ Liver 7 .4 0.3 8.0 * 0.8 8.1 0.2 7.9 0.1 6.9 X 0.3 Kidney 1.97 0.06 2.13 0.07 2.02 0.03 1.95 0.05 1.59 * 0.18 Spleen N> 00 Ovaries 0.59 0.05 0.14 0.02 0.54 0.05 0.18 0.02 0.58 0.04 0.15 0.00 0.50 0.02 0.13 X 0.01 0.48 O: 0.01 0.11 0.00 Liver Kidney Spleen Ovaries 3.57 0.16 0.95 0.01 0.28 0.03 0.07 0.01 Relative to Brain Weight fgm/gml 3.66 0.20 0.99 0.03 0.25 0.03 0.08 0.01 4.02 0.09 1.00 0.02 0.29 0.02 0.07 0.00 3.98 0.06 0.99 0.04 0.25 X 0.01 0.06 X 0.00 3.75 0.20 0.86 0.11 0.26 X 0.01 0.06 0.00 a/ Mean * S.E. of four rata, Ji/ Significantly different from control, p < 0.05, Dunnett*a Multiple Comparison procedure. WLZll SSH osizn ssa TABLE 13 ORGAN HEIGHTS OF HALE RATS AFTER EXPOSURE TO VC OR VDC FOR 3 MONTHS Control VC 50 ppm VC . 250 ppm VC 1,000 ppm VDC 55_ES Absolute Weight fgm) Terminal Body Weight Brain Liver Kidney Spleen Testes 554 2i*y 2.22 0.05 15.0 0.4 3.65 + 0.19 0.76 + 0.03 3.4 + 0.1 Liver Kidney Spleen Testes 6.8 0.3 1.65 0.12 0.34 t 0.02 1.53 0.06 519 35 2.18 0.05 13.8 0.5 3.28 0.18 0.80 0.12 3.5 0.1 516 37 500 12 2.22 0.07 2.23 0.04 12.8 1.3(3) 14.2 0.6 3.42 0.10(3) 3.30 0.21 0.78 0.07(3) 0.83 0.07 3.4 0.0 2.9 0.5 Relative to Brain Weight (gm/grirt 6.3 0.1 1.50 0.06 0.36 0.05 1.61 0.04 5.6 0.6(3) 1.49 0.03(3) 0.34 0.03(3) 1.54 0.06 6.4 0.3 1.48 0.10 0.38 0.03 1.29 0.21 520 17 2.36 0.22 13.9 0.3 3.43 0.33 0.77 0.07 3.4 0.2 6.0 0.4 1.51 0.24 0.33 0.02 1.50 0.17 a/ Mean S.E. of four rata except as noted in parentheses TABLE 14 ORGAN WEIGHTS OF FEMALE RATS AFTER EXPOSURE TO VC OR VDC FOR 3 MONTHS Control VC 50 ppm VC 250 ppm VC 1.000 ppm VDC 55 ppm Absolute Weight fgnp Terminal Body' Weight 322 10*/ 309 6 284 2k/ 305 10 278 9k/ Brain Liver 2.06 0.08 8.6 0.4 2.13 0.03 8.4 Of 2 2.05 0.01 8.4 0.3 2.02 0.02 8.7 0.5 1.91 0.03 7.1 0.3 Kidney wo Spleen 2.08 0.06 0.55 0.04 2.26 0.05 0.63 0.06 2.22 0.07 0.58 0.03 2.13 0.08 0.57 0.02 1.98 0.04 0.48 0.02 Ovaries 0.14 0.01 0.17 0.02 0.16 0.01 0.15 0.01 0.17 0.02 Relative to Drain Weight (gra/gm) Liver Kidney Spleen Ovaries 4.16 0.16 1.01 0.03 0.27 0.02 0.07 0.00 3.97 0.09 1.06 0.01 0.29 0.03 0.08 0.01 4.08 0.16 1.09 0.04 0.29 0.02 0.08 0.01 4.29 0.27 1.06 0.04 0.28 0.01 0.08 0.01 3.74 0.16 1.04 0.02 0.25 0.02 0.09 0.01 a/ Mean S.E. of four rats. b/ Significantly different from control, p < 0,05, Dunnett's Multiple Comparison procedure. ISIZU S9U TABLE 15 ZSIZU ssu SUMMARY OF TISSUE LESIONS AND M/E RATIOS IN RATS AFTER EXPOSURE TO VC OR VDC FOR 1 MONTH 4/ Severity of leeloni 1 - lid, 1 " Moderate, 3 - marked, 4 ~ eavere, f - questionable, ll/A * not available. TABLE 16 SUMMARY OF TISSUES LESIONS AND H/E RATIOS IH RATS AFTER EXPOSURE TO VC OR VDC FOR 2 MONTHS &l Severity of lealont 1 ~ ulU, 2 - noderate, 3 - narked, A - nevera, - queetlonable. eszzu s*a TABLE 17 tszzii ssa SUMMARY OP TISSUE LESIONS AND M/E RATIOS IN RATS AFfER EXPOSUBE TO VC OR VDC FOR 3 MOUTHS Lee Ionaa/ Sex Rat Ho. Hata. 111 11 Control 12 45 Female 16 42 VC (1,000 ppm) Y!>g 155 CEgl -Haig. -feasts ..Hale. Female Afl 225 224 122 228 241 242 142 244 297 298 299 300 333 334 335 336 Eye Retinal roaattaa Heart Focal, chronic myocarditis____ _l _I________________ ____ ______ I 1l1 Lung FocaL chronic murine^ gmoumonla^ _ 1 ^____l I1111I1 I |. Ivor Hlcrofocl of mononuclear cell* Focal vact uoltratlon Focal necroele Uu) Cecum Ptnworam in lumen 112 1 1111 1111 1 Il1 11 11 11 l 2I Pancreae Focal, chronic pancreat1tla Kidney Focal, chronic lntaratltlal nephritis Hit at ton of tlie pelvis Hlcrocelcnl1 Tea tie Tubular degeneration 11I 2 Proaiate Focal, chronic prostatitis Ovary Cyatlc fitttia Harrow H/K ratio l.S 1.5 1.4 1.6 1.5 1.5 1.4 1.5 1.7 1.6 1.5 1.6 1.5 l.S 1.6 1.7 1.6 1.4 1.6 1.7 1.6 1.7 1.6 1.5 g/ Suva of leeluim; 1 - *lld, 2 - moderate, 3 - marked, 4 - severe, ]uuatlonable. 18 LABORATORY DATA OF MALE MICE AFTER EXPOSURE TO VC OR VDC FOR 1 MONTH 63 ERYTHROCYTES 1X10 /HH 1 RETICULOCYTES. % MEHATOCR1T. VOL. % HEHUGLOHIN. GH. % HCV. CUIIIC MICRONS HCUII, HICHU H1CR00HS. HCN13C. 6H % S3 PLATELETS (X10 /MM 33 LEUKOCYTES (XIO /HH l NEUTROPHILS. * u>i LYMPHOCYTES. HANDS. \ EOSINOPHILS. * (JASOPHILS * MONOCYTES. * ATYPICAL, s NUCLEATED IHIC. * SOP!, Itl/L HUN. HU % 1J MACROPHAGES 1N0./HH ) Control 0.17 * 1.0 .6b .09 45.5 1 13.4 4 .6 .3 56.0 1 4.5 16.7 1 .2 29,4 1 .5 3.6 1 1.0 4.2 i S 18.0 4.3 80.0 i 5.1 0.0 4. 0.0 1.0 .6 0.0 * 0.0 1 .0 i .6 0.0 i 0.0 0.0 I 0.0 70 X 5 (2) 21.5 X 2 20 X 3 <2) (2) VC SO p|ta 6.33 1 .80 (3) l.OS t 44.3 .28 O) 2.7 (3) 13.0 1 71.3 1 1.2 (3) oW 4.9 20.7 L 1.0 (3) 29.2 1.0 (3) 6.1 L .9 (3) 5.2 1 .6 (3) 27.0 t 4.8 71.3 4.2 0.0 0.0 1.3 .5 0.0 i 0.0 .5 .5 0.0 0.0 .3 170 X 28.5 X .3 59 (2) 9 (2) VC ISO ppm 6.99 4 .48 1.63 1 211 45.8 2.8 15.1 .4 65.5 1 1.7 21 .A 4_ *1.5^ 33.4 2.6 4.7. 1 6.3 I.l .8 15.8 i 2.3 83.3 2.0 0.0 0.0 .3 .3 0.0 1 0.0 .A 0.0 .5 0.0 0.0 1 0.0 119 X 5 (2) Cntrlea are Mean x S.B. of 4 Mice except noted In parentheses. .!/ Significantly different from the Control group, p * 0.05, Ihmuett'e antUlple coMp.rlaon procedure. VC 1,000 pp 7.27 * .43 .74 1 47.3 .23 2.1 14.0 .6 65.2 4. 1.7 19.3 1 29.6 .5 .2 4.6 1 .9 6.4 1 1.2 18.8 1 4.5 79.3 4.8 0.0 0.0 .8 .5 0.0 0.0 1.0 1.0 0.0 0.0 0.0 0.0 95 i 2 (2) 17.5 X 1 (2) 33 B (3) VDC 55 pp 6.83 .44 1.27 4b.5 4 .21 1.2 14.0 .6 68.6 4 2.9 20.7 3U.1 4 1.0 .7 5.4 .8 6.8 4_ 1.1 9.5 4 2.1 89.8 4 1.8 0.0 4 .5 * 0.0 .3 U.0 _ 0.0 .3 4 .3 0.0 4 0.0 0.0 0.0 282 X 19 (2) 19.0 X 3 (2) 72 4 24 SSLZVV S'SU 9SLZVY SSH TABLE 19 LABORATORY BATA OP FEMALE MICE AFTER EXPOSURE TO VC OR VDG FOR 1 MONTH EIIYTIIHOCYItS 1X10 /HH ) RET 1 CULGCYTES * * HEHATOCRIT. VOL. * HEMOGLOBIN, GM. X MCV* CUII 1C H]CHONS HCHit, HICRO HICROGHS. HCtlllC* GH X &J PLATELETS 1X10 /HH ) 33 LEUKOCYTES IXID /HH I NEUTHOPIMLS. X LYHPHOCYTES, * HANDS % EOSINOPHILS. X IIASOPHILS. X HONOCYTES. X ATYPICAL. X NUCLEATED HOC. X SOP T. IU/L DUN. HU X .1 HACHUPHAGE S INO./HH 1 7.47 .14 .96 3l 49.U *10 .9 14.5 .4 65.6 1 1.0 19.5 .5 29.6 1 5.5 * .6 .6 9.0 2.1 12.3 04.0 2.0 2.a 0.0 1.5 0.0 .b 0.0 0.0 1.5 .6 0.0 i 0.0 0,0 t 0.0 171 i 47 (2) 22.0 X l (2) 71 27 (1) vc 6.9(1 66 1.19 4_ .31 47.5 4_ 2.7 14.6 * .11 60.9 1 21.2 3.2 1.0 3u.b i .2 6.3 JL .7 9.1 2.0 15.3 2.3 fll .3 1 3.4 0.0 1, 0.0 3.3 1.4 (1.0 0.0 .3 .3 0.0 4 0.0 Q.O 0.0 197 8 (2) 21 .5 x 0.5 (2) vc 7.21 4 .29 l.bO i .34 40.3 2.4 14.5 .7 66.9 1 20.2 * 30.1 1.0 .4 .3 S.U i 1.3 O) 0.9 2.3 13. H _ 3.1 114.0 i 4.0 0.0 i 0.0 2.0 .11 0.0 * 0.0 .5 .3 0.0 4 0.0 0.0 4_ 0.0 143 * 64 (2) VC 7.16 .33 O) 1.0b la .14 (3) 44.7 2.2 (3) |2.9 i .7 (3) 62.4 t 2.0 {3) 17.9 . .2 (3) 20, a .9 (3) 4.1 la .9 (3) 3.6 t .3 (3) 24.7 4 5.5 (3) 73.0 t 5.0 (3) 0.0 1 1.3 * 0.0 (3) .0 (3) 0.0 Q.O (3> .5 la .5 (3) 0.0 4 0.0 0.0 (3) 0.0 (3) 201 (1) 19.0 20 X 5 m (2) VUG 7.40 .56(3) 1.71 t .50 41.0 7.4 15.1) 4 .6 (3) 66.6 2.3 (3) 20.4 4^ 30.7 4 .0 (3) . 1 (3) 3.7 X 5.3 4 .4 (2> 1.4 (3) 15.3 4 4.0 H3.3 4 5.5 0.0 Jt 0.0 1.0 .6 Q.O 0.0 .5 4 .3 0.0 4 0.0 0.0 4_ 0.0 311 X 48 (2) 24.0 X 0.0 (2) 20 X 5 (2) Entries art) alum A. H.B. of 4 wlce except as nutetl In pareiiLlmaas. a/ SJ^fcglcantly different fron the Control ftroup, p 0.05, Uminett'a Xlple coiaparlaon procedure. .BLK 20 LABORATORY DATA OF MALE MICE AFTER EXPOSURE TO VC OR VDC FOR 2 MONTHS Control VC SO ppm VC 250 ppm VC 1,000 ppm 61 ERYTHROCYTES (XIO /HH 1 RE 11 CULOCYTt'S, % HEMATOCRIT. VOL, * HEHOIU.OR IN OH, * HCV, CliniC HI CHONS MCHII. HICltO HICHORUS. MCH11C, C.H % 53 PLAIELffS 1X10 /HH 1 33 LEUKOCYTES 1X10 /HH I NEUTROPHILS, f LYMPHOCYTES, * HANDS EOSINOPHILS. X IUSOPHUS. X MONOCYTES. * ATYPICAL. X NUCLEATED HOC. * SOP I, Ml/L HUN, HP % 3 HACIIOPHAGFS (NO./HH 1 6.AO 1.10 17 .14 40.o i 14.2 1.3 .5 75,0 22.1 4 1.2 .4 29.5 i 5.0 _ .0 5.9 1 .0 40.0 11.B 57.5 1 11.9 0.0 (1.0 .1 4 .3 0.0 1.5 4 0.0 I .0 0.0 4 0.0 0.0 4^ 0.0 129 4__ 40 35.5 4^ 2.6 ?3 4^ 13 0.00 4_ .22 1.70 4_ .07 45.n 4_ 1.7 13.6 4^ .1 76.3 4 7 22.7 .4 29. fl 4 .3 6.3 4_ .7 4.4 4 .5 4 3,11 4_ 7.9 54.3 4_ 7.3 0.0 1.0 4 0.0 ` .6i/ 0.0 4 0.0 .3 4^ ,3 0.0 4_ 0.0 0.0 *_ 0.0 7 4_ 24.0 4_ 1? 3.4^ 6.70 .08 4 50.0 4 .26 (3) .13 13) 1.0 13) 14.8 4 73.9 4 .5 13) 2.0 13) 21.0 4 .1 13) 29.5 i 5.9 4 .7 (3) .7 6.8 4 .7 30.3 4 13.6 (3) 60.5 4 14.2 .3 .3 .5 4 .3 0.0 4_ 0.0 .5 4_ .1 0.0 0.0 .3 4 .3 184 4 14 23.0 1 . l-f 6.59 4^ 1.10 4^ .19 .30 40.8 4. .6 U.3 1 74.0 4^ .2 1.3 21.7 4 .3 29.3 1 .1 6.0 i 1.0 6,1 1 .9 39.0 4^ 11.1 59.5 4 10.7 0.0 0.0 1.0 4 0.0 * .4 0.0 .5 4 .5 0,0 0.0 0.0 4 0.0 163 1 22.5 35 1.2i/ 162 4 82 (3) Entrfoa ira mean * 3.8. of 4 mlcq except * noteJ In parentliaaea. i/ Slpnt f (cant ly different from the Control firoup, p < 0.05, (hmnott'e multiple comparlaon procure. vu: SS ppm 6.37 .19 1.31 1 45.3 4 .11 .5 13.5 .1 71.2 4_ 1.8 21.3 4 .6 29.9 1 4.0 4 .2 .4 4.4 4 1.4 31.0 4 4.4 68.5 4_ 4.3 0.0 4. 0.0 0.0 0.0 0.0 4 0.0 .5 4 .5 0.0 i 0.0 4_ 0.0 0.0 236 24 35.8 1.9 87 4 50 O) zsz? 11 s'sa TABLE 21 IABORATORY DATA OF FEMALE MICE AFTER EXPOSURE TO VC OR VDC FOR 2 MONTHS 8SIZll S'SU 6 .1 EHYTHIlOCYIt'S (X 1 a /HH 1 RETICULOCYTES. * HEHATOCHIT. VOL. * HEHUUL0H1N. HH. X HCVi CUIlIC H1CR0NS HCim, HI Clio HICH06HS. Hcimc. oh % &3 PLATELETS (XIO /tm ) 33 leukocytes mo /hh t NEUTROPHILS. % LYMPHOCYTES. * HANDSi % EOSINOPHILS. X IIASUPHILSi % HOHOCYIES. % AlYPICAL. 1 NUCLEATED IlllCt X SGPI. IU/L IIIIN, HG X A MACHOPHAGtS (NO./HH 1 Control 5.94 . 3U 78 .07 46.a 3.3 13.A 4 1.0 78.7 1 1.8 23.2 1 .5 29.5 1 5.1 .2 .6 5.0 .3 29,3 i 6.8 67.5 t 5.9 0.0 0.0 2.5 1 .9 0.0 4 0.0 .8 .5 0.0 1 0.0 0.0 0.0 119 111 23.3 .9 213 * 123 (3) SO p|H* 7.09 .24 1.13 4 .17 54.0 1.7 15.9 .5 76.2 1 22.5 * .5 .1 29.5 1 .1 4.1 1 5.3 .6 .3 26. A 1 7.3 71.0 _ 7.3 0.0 1 1.0 0.0 .6 0. A 1 1.3 0.0 .9 0.0 0.0 0.0 t 0.0 200 1 25 24.3 1 .3 250 ppia 6.52 .38 1 .04 i 50.5 .23 1.2 15.1 1 78.0 .3 2.0 23.4 1.0 30.0 t 4.5 1 5.0 .6 .3 .5 22.5 t A.6 76.5 1 0.0 A.6 0.0 .0 * .5 0.0 _ 0.0 .3 0.0 .3 0.0 0.0 4 0.0 296 f 7? 27.5 2.3 1,000 ppw 6.66 4 .30 1 .26 4 .24 49.5 * 1.7 14.6 1 .5 74.5 i 2.1 21.9 I .7 29.5 l .7 5.7 i 5.1 * .3 .5 29.0 4 12.0 60.8 11.3 0.0 0.0 1.8 i 1.1 Q.O t 0.0 .5 .3 0.0 0.0 0.0 0.0 222 40 24.8 1.0 1A 1 6 Kill ties are wean t a,B. of 4 alee except aa noted (n pnrcntlinaaa. a/ Significantly different trow Ilia Control group, p < 0.05, Dnnnolt'a iwiltipta cowparlaon procedure. 55 ppa 6.90 1 1.51! 1 .41 .07*' 50.11 * 1 .9 15.2 .5 73.8 1.7 22.1 .6 29.9 i 4.9 4.8 * .2 .5 .2 22.8 6.2 75.8 ,5.6 0.0 0.0 .5 .5 0.11 0.0 1.0 * .6 0.0 * 0.0 .3 * .3 217 4 50 33.3 i. i-' 218 1 ua (2) 1 TABLE 22 LABORATORY DATA OF MALE MICE AFTER EXPOSURE TO VC OR VDC FOR 3 MOHTHS 6J EH Y ItlHOC Y T E S U1U /Hrt I lltTICULOCr [ES, IIEHAIOCHIf. VOL. * HEHUGLOlIlh, CM. * HCVt cutiic Hicito^s HCIIII, HlCHO MCHUGHS. HCMIIC. Oh % &a PLAlELtTS 1X10 /HM 1 3a LEUKOCYTES (X|{| XHH ) NEIJ THlIPH 1L S, % L YHPHUCYTE b< % HANDS, * tOSINlIHUll.S, i HASIlPHILS, * HONOCYTtS, * AlYPlCAL. * NUCLEATED HHC, % Slil`1, 1U/L UIIN, HG % j HAL.dlPlIAGLS INO./HH 1 Control 7.52 I .46 (3) 1.11 ,33 (3) a.a .0 (3) 15,5 4 .4 (3) 64.6 L 20.7 3.4 . (3) (3) 32.1 .7 (3) 7.0 1.2 (3) 6.2 1.0 (3) 22.3 77.0 3.3 2.1 0.0 0.0 .5 4 .3 0.0 4_ 0.0 .3 .3 0.0 4 0.0 u.a 0.0 55.0 I 1. 1 20.0 l .3 mo 121. 30 ppm H. 15 * .2 1.23 * .2 (3) (I) 49.0 lb. 3 .4 5H.lt 19.9 1 .1 34.) (l> (2) 7D (2) (1) 6.2 7.3 1 .0 0) (2) 25.ll i 12.6 73.H i 12.7 0.0 * 0.0 .3 t .3 U.O i_ 0.0 .6 L *3 0.0 i 0.0 0.0 i 0.0 96.7 t 21 .5 (3) 19.0 i 1.2 (3) Kit trie* nee Muan S.K. except aa notej In parenthese*. 250 ppn H.01 i .5 <2) 1.63 4 .4 (2) 46.0 12 .0 (2) 15.3 4 .7 (2) 57.5 41 (2) 19.1 * .3 (I) 33.3 t .1 (2) 3.9 11 .0 (2) 30.5 i II.5 67.11 ft.9 1.0 - 0.0 t 1.0 0.0 0.0 1 0.0 . 1 .ft 0.0 t 0.0 2.0 2.0 72.0 i 6.9 24.11 t 3. / 1,000 ppw 6.13 ,6ft 1.53 4 .25 46.3 1 13.3 4^ 6fl. 1 1 21.9 4 1.2 1.3 .9 .7 (3) (3) 31.3 4 6.ft 1 3.0 t .& (3) .9 (3) .7 15.3 1 B4.3 * 2.1 2.1 0,0 4 0.0 0.0 4^ 0.0 0.0 0.0 .5 1 .5 0.0 4 0.0 0.0 0.0 06.0 1 9.2 <3> 21.0 53 2.1 (3) In 55 PLH* 7.14 4 1.24 4_ 44.0 *_ .22 .20 .9 14.5 .4 61.7 1.2 20.3 .1 33.0 .5 7.1 t .7 6.1 4 1.2 m 19.0 4^ H. 6 HO .U 4 0.5 0.0 4 0.0 0.0 * 0.0 0.0 0.0 .3 4 .3 0.0 0.0 0.0 4 0.0 112*5 4 5.9 27.0 * 2.4 135 4. 99 6SLZU SSH 09LZVl S^y TABLE 23 LABORATORY DATA OF FEMALE MICE AFTER EXPOSURE TO VC OR VDC FOR 3 MOMTIIS 6J ehy iiihocyits mu /hh t iilticulocytes. * iifHAiocmr. vm.. % ttEHOGL Dll IN* (i 14. t MCV, CUIt 1C HlCriUNS HCIID. H [CriO MfCHOGKS. HCIIHC. GH X 53 PLATELETS (XIO /MM 1 L4 VO J 3 leukocytes mu /mh i UtUniOHtllLS, * LYMPHOCYTES, i HANDS, * EOSINOPHILS, % UASGPHlLS, * HONOCY TES* * AIYI'ICAL, * NUClEA1tO HUC, * SGI* f , 1 U/L 1UJN, MG * i MACHIIPUaOES 1N0./HH 1 Control 6.73 * 1.0* 4 **.3 4 14.1 4~ 65.4 20.9 * .17 (3) .07 (3> 1.2 (3) .5 (3) .3 (3) .2 (3) 31 .H 5,9 .2 (3) .S (2) 3.2 i 12.0 4 H7.3 .*i 3.2 3.6 (3) 0.0 0.0 . 0.0 .5 0.0 0.0 Q.O 0.0 1 0.0 4, 0.0 0.0 1 1 6 30 19. U 1 24 4_ 1.4 H VC SO ppta 7.72 1 .22 4* .47 .23 4U.fl 1.2 15.9 .H 62.6 1 2.2 20.7 1 33.1 4^ .3 .it 6.4 O) 5,2 * .4 tt.5 4_ 2.1 90.3 1 0.0 4_ 2.6 0.0 .It 4_ .S 0.0 0.0 .5 .1 0.0 * 0.0 .3 t 64 4* 22. tt 4" .7 11 3.1 VC 250 I'P* 7.12 1.15 .94 (3) .21 (3) 44.7 3.0 14.9 . 64.0 4.11 (3) (3) (3) 21.5 * 2.1 (3) 33.4 4 .11 (3) 4.3 * .5 (2> 6.1 1 .5 (3) 10.3 4 1. r 110, ft 4 2.0 o.n 0.0 .5 4 .5 0.0 * 0.0 .5 .6 0.0 * 0.0 O.U 1 0.0 127 4^ 13 26. n 3.0 VC 1. 000 ppai 6.24 .77 (3) 1 .54 4 42.7 4_ 14.0 4_ .30 (3) 3.5 (3) 1.1 (3) 69,2 4_ 3.5 (3) 22.7 1 1.4 (3) 32.0 6.0 t .5 (3) .5 (2) 4.2 4. .7 (3) 0.0 3.3 91.0 1 3.5 0.0 1 0.0 0.0 * 0.0 0.0 t 0.0 .3 4 .3 0.0 4 0.0 0.0 4_ 0.0 66 4 6 20.11 _* 165 4* 1.0 (19 VDC 55 ppai 5.A3 4 1 .411 4 .54 (3) .25 (3) 39.3 4 3,2 13) 12.4 4 67.7 1.0 1.6 (3) O) 21.3 4^ .3 {3) 31.5 4 .6 (3) 3.1 JL ,1 12.3 *_ 5.1 (16.3 4.9 0.0 0.0 .5 4 .5 O.b 4 0.0 1.0 4 .4 0.0 4 0.0 4^ 105 4 0.0 0.0 16 29.11 4 116 4 2.4 lift 73) CiiCrlca aiu mean t S.E. except aa noted In parentlieaca. R&S 112761 TABLE 24 NUMERICAL DISTRIBUTION OF CHROMOSOMES IN BONE MARROW OF MICE EXPOSED TO VC OR VDC Treatment Control VC (1,000 ppm) VDC (55 ppm) Control VC (1,000 ppm) VDC (55 ppm) No. of Mice Chromosome Frequency * 38 39 40 41 a 42 Tetraploids (4N) per 100 cells After One Month 6 sa/ 5 36 3 1 0.42 2s 0.20^/ 6 6 4 39 1 0 0.25 0.17 6 2 5 40 2 1 0.25 0.17 After Three Months 4 5 7 37 1 0 0.0 = 0.0 4 6 6 37 1 0 0.0 = 0.0 4 6 6 35 3 0 0.15 - 0.15 a/ Mean b/ Mean = S.E. 40 Z9LZIV s'sa TABLE 25 MORPHOLOGICAL ABERRATIONS OF CHROMOSOMES IN BONE MARROW OP MICE EXPOSED TO VC OR VDC Treatment No. of Mice Chromatid gaps per 50 cells Chromatid Translocations breaks per 50 cells per 50 cells After One Month Total Aberrations per 50 cells Control VC (1,000 ppm) VDC (55 ppm) 6 6 6 0,67 0.33^ 0.52 0.23 0.97 0.26 0.0 0.0 0.17 a. 0.17 0.33 0.21 0.20 d 0.20 0.17 d 0.17 0.17 d 0.17 0.B6 d- 0.31 0.86 * 0.13 1.A7 0.A3 After Three Months Control VC (1,000 ppm) VDC (55 ppm) A A A 0.25 0.25 0.0 0.0 0.50 0.50 0.0 0.0 0.25 0.25 0.25 0.25 0.0 0.0 0.0 d 0.0 0.0 0.0 0.25 0.25 0.25 i 0.25 0.75 0.A8 a/ Mean S.E TABLE 26 ORGAN WEIGHTS OF MALE MICE AFTER EXPOSURE TO VC OR VDC FOR 1 MONTH Terminal Body Weight Brain Liver Kidney Spleen Tes tea Liver Kidney Spleen Testes jCantrol 33.0 1.0^ 0.51 0.02 1.77 0.24 0.54 0.03 0.17 0.02 0.26 0.04 3.5 0.5 1.06 0.08 0.34 0.05 0.52 0.08 VC 50 ppm VC 250 ppm VC 1.000 ppm VDC 55 ppm Absolute Weight (gnO 31.0 1.2 0.47 0.01^/ 1.34 0.13 0.51 0.04 0.13 0.02 0.23 0.01 32.0 0.7 0.47 0,01^/ 1.35 0.12 0.51 0.02 0.14 0.01 0.24 0.01 32.5 0,6 0.46 0.01-^' 1.31 0.02 0.54 0.01 0.13 0.01 0.22 0.02 29.8 0.8 0.46 0.00^' 1.35 0.03 0.46 0.02 0.11 0.01-^ 0.24 0.01 Relative to Brain Weight fgm/gnO 2.9 0.3 1.09 0.08 0.27 0.04 0.49 0.03 2.9 0.3 1.11 0.06 0.30 0.02 0.52 0.02 2.8 0.0 1.17 0.04 0.27 0.02 0.49 0.04 2.9 0.1 1.01 0.05 0.24 0.02 0.52 0.03 5/ Mean S.E. of four mice, b/ Significantly different fron control, p<0,05, Dunnett's Multiple Comparison procedure. 09LZVi SSH P91ZU SSU TABLE 27 ORGAN WEIGHTS OF FEMALE MICE AFTER EXPOSURE TO VC OR VDC FOR 1 MONTH Terminal Body Height Brain Liver Kidney Spleen Ovaries Liver Kidney Spleen Ovaries Control VC 5Q_BBS VC 250 onm Absolute Weight fern! VC 1.000 jem 1.3 1.1V 48 0.01 13 0.08 33 0.03 ,10 0.01 ,02 0.01 27.8 0.47 0.01 1.15 0.07 . 0.38 0.02 0.15 0.02 0.01 0.00 26.5 1.5 0.47 0.01 1.12 0.02 0.37 0.02 0.13 0.02 0.02 0.00 24.8 0.9 0.49 0.01 1.03 0.06 0.36 0.01 0.14 0.03 0.02 0.00 Relative to Brain Height {gm/gnrt ,36 0.18 ,69 0.05 ,21 0.02 .05 0.16 2.43 0.12 0.81 0.03 0.31 0.04 0.03 0.01 2.40 0.06 0.79 0.04 0.27 0.03 0.03 0.01 2.13 0.07 0.74 0.01 0.30 0.08 0.05 0.01 VDC 55--BBS 26.5 0.9 0.49 0.01 1.31 i 0*06 0.39 0.01 0.13 0.01 0.02 0.00 2.69 0.08 0.80 0.00 0.27 0.02 0.03 0.01 _g/ Mean S.E. of four mice. b/ Significantly different from control, p < 0.05, Dunnett'a Multiple Comparison procedure. TABLE 28 ORGAN WEIGHTS OF MALE MICE AFTER EXPOSURE TO VC OR VDC FOR 2 MOUTHS Terminal Body Weight Brain Liver Kidney Spleen Testes Liver Kidney Spleen Testes Control 33.5 O.S^ 0.50 0.01 1.72 0.13 0.69 0.04 0.11 0.04 0.27 0.02 3.4 0.3 1.37 0.08 0.23 0.08 0.54 0.03 VC 50 ppm VC 250 ppm VC 1,000 ppm Absolute Weight (gm) 33.8 1.9 0.47 0.04 1.50 0.08 0.70 0.08 0.15 0.03 0.26 0.01 34.3 1.7 0.51 0.01 1.50 0.07 0.66 0.03 0.15 0.01 0.28 0.02 36.8 dh 1.4 0.48 0.03 1.57 0.09 0.69 0.02 0.16 0.00 0.26 0.02 Relative to Brain Weight (gm/gm) 3.3 0.4 1.56 i 0.29 0.33 0.05 0.58 0.06 2.9 0.1 1.29 0.05 0.30 0.02 0.55 0.05 3.3 0.2 1.44 0.10 0.34 0.03 0.55 0.07 VDC 55 ppm 32.0 0. 6 0.49 0. 02 1.60 0. 04 0.52 0. 04 0.11 0. 02 0.26 0. 02 3.3 0. 1 1.06 0. 04 0.22 0. 03 0.53 0. 02 a/ Mean S.E. of four mice. SSlZll 99Z2U S9H TABLE 29 ORGAN WEIGHTS OF FEMALE MICE AFTER EXPOSURE TO VC OR VDC FOR 2 MONTHS Terminal Body Weight Brain Liver Kidney Spleen Ovaries Control 20.8 0.6^ 0.53 0.02 1.28 0.10 0.43 0.02 0.14 0.01 0.03 0.00 VC 50 ppm VC 250 ppm Abaolute Weight (gml 29.0 1.9 0.53 0.02 1.29 0.07 0.42 0.01 0.13 0.01 0.04 0.01 29.3 0.6 0.47 0.01 1.25 0.04 0.39 0.01 0.17 0.01 0.03 0.00 VC 1.000 ppm 29.5 1.2 0.54 0.02 1.2a 0.07 0.44 0.02 0.14 0.01 0.03 0.00 VDC 55 ppm 28.0 0.4 0.51 0.01 1.32 0.08 0.42 0.01 0.12 0.01 0.04 0.01 4> Ui Liver Kidney Spleen Ovaries 2.44 0.16 0.81 0.03 0.26 0.01 0.06 0.01 Relative to Brain Weight fgro/gm) 2.44 0.09 0.79 0.03 0.24 0.02 0.07 0.01 ` 2.66 0.11 0.84 0.03 , 0.36 0.03-^ 0.07 0.00 2.40 0.12 0.82 * 0.05 0.26 0.02 0.06 0.00 2.60 0.17 0.82 0.01 0.23 0.02 0.07 0.00 a/ ' Mean + S.E. of four mice. b/ Significantly different from Control, p< 0.05, Dunnett'a Multiple Comparison procedure. TABLE 30 ORGAN WEIGHTS OF MALE MICE AFTER EXPOSURE TO VC OR VPC FOR 3 MONTHS Terminal Body Height Brain Liver Kidney Spleen Tea tea Liver Kidney Spleen Testes Control 34.9 l.t*' 0.52 0.02 1.64 0.09 0.63 0.03 0.12 0.01 0.22 0.02 3.19 0.14 1.23 0.04 0.23 0.01 0.43 0.03 VC 50 ppm VC 250 ppm VC 1.000 ppm Absolute Height (gup 34.8 1.1 0.49 0.01 1.54 0.08 0.67 0.06 0.16 0.02 0.30 0.03 32.8 1.2 0.51 0.02 1.46 0.06 0.64 0.05 0.14 0.01 0.28 0.01 34,4 1.2 0.52 0.01 1.62 0.08 0.68 0.03 0.12 0.01 ` 0.27 0.02 Relative To Brain Weight (mn/gmT 3.16 0.13 1.37 O.iO 0.33 0.04-^ 0.62 0.06^/ 2.88 0.19 1.25 0.07 0.27 0.02 0.54 0.02 3.13 0.16 1.31 0.07 0.24 0.01 0.52 0.04 VDC 55 ppm 28.3 1.8^ 0.51 0.01 1.50 0.07 0.50 0.01 0.12 0.01 0.27 0.02 2.97 0.18 0.99 0.04 0.23 0.02 0.55 0.05 / Mean S.E. of four mice. b/ Significantly different from control, p < 0.05, Dunnett's Multiple Comparison procedure. 89LZIV S'Sy TABLE 31 ORGAN WEIGHTS OF FEMALE MICE AFTER EXPOSURE TO VC OR VDC FOR 3 MONTHS Terminal Body Weighf Brain Liver Kidney Spleen Ovaries Control 25.9 t 1.5^ 0.49 0.01 1.18 0.10 0.50 0.14 0.12 0.01 0.04 0.01 VC 50 ppm VC 250 ppm Absolute Weight (uml VC 1.000 ppm 29.4 1.2 0.51 0.02 1.27 0.06 0.41 0.02 0.11 0.01 0.04 0.01 26.0 1.1 0.55 0.01 1.12 0.07 0.38 0.04 0.14 0.02 0.04 0.01 26.9 0.7 0.51 0.03 1.21 0.07 0.45 0.07 0.14 0.01 0.04 0.01 VDC 55 ppm 25.9 0.B 0.47 0.03 1.32 0.07 0.41 0.01 0.13 0.00 0.04 0.01 Relative to Brain Weight fgm/gml Liver Kidney Spleen Ovaries 2.38 0.17 1.01 0.27 0.24 0.01 0.07 0.01 2.49 0.04 0.81 0.03 0.22 0.01 0.09 0.01 2.03 0.10 0.70 0.05 0.24 0.03 0.08 0.01 2.40 0.21 0.89 0.11 0.27 0.02 0.08 0.03 2.81 0.11 0.87 0.05 0.28 0.02 0.10 0.02 a/ Mean S.E. of four mice. TABLE 32 SUMMARY OF TISSUE LESIONS AHD H/E RATIOS IN MICE AFTER EXPOSURE TO VC OR VDC FOR 1 MONTH Lasisir^ Heart Hyocardltla Control* VC ft.OOP ppiO Sex: .Halas Fenglea Halo* Females gflL-Hsi _1____J____L JL 1] M 22 AS 112113 219 220 253 254 255 256 -I'ES-Ill-Etig). Hales Fowaloe 2B9 290 291 292 325 326 327 3 Liver Focal degeneration and tnfUaaatlon Bile duct hyperplasia and cholalathloals 11 Salivary Claud Hlcrofocl of aunonuclear cells Kidney Chronic Interstitial nephritis Co Tubular cast* Tubular nosophilia Hicrocatcull Skeletal Muscle Myositis Bona Harrow H/E ratio 1 2 12 2 2 21 11 I 1 1l11 11 1l 11 1 1*2 1*3 1*3 1*2 1*3 1*4 1*2 1*2 1*1 1*3 1*3 1*2 1*3 1*5 1*3 1*4 1*6 1*5 1*3 1*4 1*3 1*2 1*2 1 if Severity of lesion: 1 - mild, 2 - moderate, 3 - sarked, 4 - severe, j. - questionable. 69Z2U SSH TABLE 33 0LLZVV SSU SUMMARY OF TISSUE LESIONS AND M/E RATIOS IH MICE AFTER EXPOSURE TO VC OR VDC FOR 2 MONTHS Lsilsu^ Seat Hat.Mai Controls ..VS fl.figfl caaL win* Irfrf ki*hi ,HftlSi . kl k ..Fsaaleii- JlaiaiL faMlgg. Ha lea. Fesaka hi hi hi hk 211 m 211 Hk 211 21k 212 2M> 221 21k 211 23k 329 33ft m 332 Heart Myucardltta Lung lironchlolar adenoma 11 Liver Hicrofocl of inflammatory cell* Focal mineralization Puneroue Pancreatitis and degeneration 22 Perianal Gland Suppurative influiuauU ion _ jlyfejplesio________________ Kidney CUronlc iuterstltai nephritis Tubular casta tllcrocalculi Tutiular basophilia _ v_l"________________ Uterus Acute metritis Brain Mild, focal fatty change Skeletal Untiele Hyua|tie Hone Harrow M/K ratio 1*3 1*3 1-4 1>3 1-2 1.3 1.4 1.5 1*4 1*3 1*4 1*2 1*4 1*5 1*5 1*4 1*3 1*3 1*4 1*5 1*5 1*2 1*4 1*3 it/ Severlesion: l - mild, 2 - moderate, 3 - marked, 4 - severe, - questionable, + - present Lesion5t Sex Fat Ho. TA 34 SUMMARY OF TISSUE LESIONS AND M/E RATIOS IN MICE AFTER EXPOSURE TO VC OR VDC FOR 3 MONTHS Control vc g.ooo pt.> VDC 155 pp0 Hale .EsaaU .lla la.. -Haig. .fssalt. 1 LO 11 i! 45 46 il is 11% 236 237 228 261 262 363 264 297 298 299 300 333 334 33S 336 Heart _Focal ayocardlat degeneration Lung Uronchiolar adenona _Focal acinar epithelial jiroliferatlon 1I l.lver Microfoci of BKinanuclaar cella Intranuclear inclusions Focal necroala Soltvary eland lOa Focal chronic Inf tarnation Kidney Focal, chronic Interatltlal nephrltla 1 lubular caata 1 Hlcrocalculi 1 1I + .+ + 1 Done Harrow H/t ratio Severity of lea Iona; 1.6 1.6 1.7 1.4 1.7 l.S 1.6 1.4 l_. 5_ J._6_ l_.7, J -J _ l,,. 5_ J J _ b 1 J -J l - lld, 2 - nodarale, 3 - Barked, 4 - aavera, ^ - questionable, + - present. L:L J J_ L-L Js*_ LL bl JJ ULZIY ssa I ZLLZVY SSH TABLE 35 SUMMARY OF BRONCHIOLAR ADENOMAS IN MICE AFTER EXPOSURE TO VC OR VDC Exposure Time 1 month 2 months 3 months Control <f ? Or* 0 00 00 50 ppm VC rf 00 00 10 250 ppm VC <f 00 10 10 1,000 ppm VC <s ? 00 12 21 55 ppm VDC tf ? 00 00 00 a/ Number of adenomas in four mice of each sex. * Figure l - Inhalation Chambers and Holding Racks. ZLLZIY SSH . mute 1 \,lnea. 0g Sawp1^5 th. Volition. ** *-1'* o a VJ1 f- J------ 1------- 1-------- 1------- 1III 2 4 6 8 10 12 14 WEEK 16 I1 18 20 sllz 11 s^u Figure 3 - Average Meekly Analytical Concentrations and the Range of All Samples for VC. 9LLZIV S3H 140 130 120 110 100 90 80 I 70 New Standard 60 50 40 - / 30 _ I t.--A 20 Estimated 10 0 J_____L .1_____L J____ I____ I____ I____ I____ I 8 10 12 14 16 18 20 WEEKS Figure 4 - Average Weekly Analyticln iConcentrations and the Range of All Samples for VC Figure 5 - Body Weighta of Rata Exposed to VC or VDC. LLLZW S3H BODY WEIGHT (gm ) 15 I----- 1----- 1-------1----- 1IIIIIIill 0 I 2 3 4 5 6 7 8 9 10 11 12 13 WEEK OF TREATMENT R&S 112778 Figure 6 - Body Weights of Mice Exposed to VC or VDC. R&S 112779 REFERENCES 1. Lee, C. C., et al.: Toxicological Evaluation of Ferric Dimethyldithiocarfaamate (Ferbam) and Dithiocarbamate (Thiram) with Acute Toxicity of Manganese and 21nc Ethylenebisdithioearbamates (Maneb and Zineb). NTS Contract No. N01-ES-2-2084 (Continuation of NIH-NIEHS-72-2084). pp. 1-356, 1975. 2. Fatty, F. A., W. P, Yant, and C. W. Waite: Acute Response of Guinea Pigs to Vapors of Some New Commercial Organic Compounds V: Vinyl Chloride. Pub. Health Rept., 45: 1963-1971, 1930. 3. Oster, R. H., C. J. Carr, J. C. Krantz, and M. J. Sauerwald: Anesthesia XXVII, Narcosis with Vinyl Chloride. Anesthesiology, 8: 359-361, 1947. 4. Carr, J., R. M. Burgison, J. F. Vitcha, and J. C. Krantz, Jr.: Anesthesia 30CIV,. Chemical Constitution of Hydrocarbons and Cardiac Automaticity. J. Pharmacol., 2Z? 1949. 5. Mastromatteo, E., A. M. Fisher, H. Christi, and 0. Danziger: Acute Inhalation Toxicity of Vinyl Chloride to Laboratory Animals. Am. Ind. Hyg. Asso. J., 21: 391-397, I960. 6. Torkerson, T. R., F. Oyeri, and V. K. Rowe: The Toxicity of Vinyl Chloride as Determined by Repeated Exposure of Laboratory Animals. Amer. Ind. Hyg. Assn. J., 22_: 354-361, 1961. Viola, P. L., A. Bigotti, and A. Caputo: Oncogenic Response of Rat Skin, Lungs, and Bones to Vinyl Chloride. Cancer Research, 31: 516-522, 1971. ~~ 8. Maltoni, C. and G. Lefemlne: The Potential of Experimental Stages of the Prediction of the Ambient Oncogenic Risks. An example: Vinyl Chloride* Lincel-Rend. Sc. fs. mat. enat. 56:1, 1974. 9. Quick, A. J.: A Study of the Coagulation Defect in Hemophilia and in Jaundice. Am. J. Med. Sci., 190: 501, 1935. 10. Jendrassik, L., and P. Grof: Simplified Photometric Method for the Determination of the Blood Bilirubin. Biochem, Z., 297: 81, 1938. 58 Mancini, G., A. 0. Carbonara, and J, F. Heremans: Inntunochemical _ Quantitation of Antigens by Single Radial Immunodiffusion. Iamuno* chemistry, Zi 235-254, 1964. 12. Kingsley, G. R.: The Direct Biuret Method for the Determination of Serum Protein as Applied to Photoelectric and Visual Colorimetry. J. Lab. Clin. Med., 27: 840, 1942. 13. Deumas, B. T., W. A. Watson, and H. G. Biggs: Albumin Standards and the Measurement of Serum Albumin With Bromeresol Green. Clin. Chim. Acta, 31: 87, 1971. 14. Tjio, J. H. and J. Wang: Direct Chromosome Preparations of Bone Marrow Cells. In: H<imj,n Chromosome Methodology. J. J. Tunis (ed.). Academic Press, New York, 1965. 15. Moorhead, P. S. and P. C. Norweil: Chromosome Cynology. In: Methods in Medical Research, H. N. Eisen (ed.). Tear Book Medical Pub1., Inc., Chicago, 10: 310, 1964. 16. Dunnett, C. W.: A Multiple Comparison Procedure for Comparing Several Treatments with a Control. J. Am. Stat. Assoc., 50: 1096-1121, 1955. 59 R&S 112780 R&S 112781 APPENDIX I MANUAL FOR HEMATOLOGY. CLINICAL BLOOD CHEMISTRY. URINALYSIS. HISTOPATHOLOGY. STATISTICAL ANALYSIS. AND NORMAL VALUES Cheng-Chua Lee Loren D. Kintrier Thomas W. Reddig John J. Kowalski Midwest Research Institute May 1975 TABLE OF CONTENTS R&S 112782 I. Hematology, Clinical Blood Chemistry, and Urinalysis ............................ 1 A. Hematology........................................................................................................................ 1 B. Clinical Blood Chemistry ................................................................................... 2 C. Urinalysis........................................................................................................................ 3 D. Precision of Hematology and Clinical Blood Chemistry Tests . . A 1. Reproducibility Among Test Days.............................................................3 2. Reproducibility Within a Test Day........................................................ 4 3. Proficiency Test Service...............................................................................^ II. His topathology......................................................................................................... * * 5 A. Necropsy and Gross Examination.......................................................................... 5 B. Organ Weights.................................................. 5 C. Tissues for Microscopic Examination................................................................. 3 D. Fixation and Staining of Tissues ................................................................ 5 HI. Statistical Analysis....................................................... ......................................... 6 17. Normal Values.......................................... " A. Hematology, Clinical Blood Chemistry and Bone Marrow...................6 B. Absolute and Relative Organ Weights............................................................6 C. Presence of Various Substances In the Urine................................................ 7 V. References.................................................................................................................................. ? Tables A - N . . 9 - 22 A H3STOPATHOLOGY. NORMAL VALDES. AND STATISTICAL ANALYSIS I. HEMATOLOGY. CLINICAL BLOOD CHEMISTRY. AND URINALYSIS The usual blood sample from dogs Is 8 ml, from monkeys 4 ml, and from rats 0.3 ml for hematology and about 8 ml for full analysis at termin ation. A. Hematology The following hematological analyses are performed on all blood samples from rats, dogs, and monkeys. 1. Erythrocyte and leukocyte counts: A Coulter Electronic Particle Counter with 100 p aperture is used.-i^ Particle-free diluents (Isoton for RBC, Zap-Oglobin in Isoton for WBG, Coulter Electronics, Inc.) are counted to establish the background. Each blood sample is cotinted in duplicate. For each test day, two control blood samples (Diagnostic Technology, Inc.) are counted separately in duplicate. 2. Hematocrit: Hematocrit is determined in capillary tubes using a microcapillary centrifuge (International Equipment Company, Model MB). Two control blood samples (Diagnostic Technology, Inc.) are measured separately in duplicate. 3. Hemoglobin: Hemoglobin is measured as cyanomethemoglobin.^ Each blood sample is measured in duplicate. Cyanomethemoglobin (Coulter Electronics, Inc.) is used as the standard. For each assay, two levels of the standard are used and two control blood samples (Diagnostic Technology, Inc.) are measured in duplicate. 4. Methemoglobin (Met-Hb): Met-Hb is measured by the method of Dubowski.,2/ A positive control is made by adding potassium ferricyanide to control blood. 5. Heinz bodies: Heinz bodies are stained with methyl violet and the percent of Heinz bodies is calculated. 6. Mean corpuscular volume (MCV): MCV is calculated as follows: MCV (n3) Hematocrit r 10 Erythrocytes in millions/nnnJ 7. Mean corpuscular hemoglobin (MCHb): MCHb is calculated as follows; MCHb (jipg) Hemoglobin fgn 7.1 x 10 Erythrocytes in millions/nm^ 8. Mean corpuscular hemoglobin concentration (MCHbC): MCHbC is calculated as follows; MCHbC (gm %) - Hemoglobin (m X) x 100 Hematocric 9. Differential leukocyte counts; Wright's stain is used to stain the leukocytes for examination. 10. Reticulocyte count: Reticulocytes are counted by the methy lene blue method using the Miller disc.^Z 11. Platelet count; A Coulter Electronic Particle Counter with 70-ia aperture is used. $/ Particle-free Isoton is used as diluent and counted to establish the background. At weekly intervals, platelets are also visually counted in a hesaocytometer with a phase microscope for comparison JU .12. Clotting time (dog and monkey): Clotting time is determined by the capillary tube procedure using two capillary tubes ,U The time elapsed from the appearance of the blood from the animal and coagulation in either tube is measured. R&S 112784 B. Clinical Blood Chemistry The following clinical blood chemistry tests are performed on `all blood samples from dogs and monkeys and on blood samples from rats at termination. 1. Blood glucose; Fasting blood glucose is determined by Stein1 s hexokinase method.il/ Standard glucose solution (Dade) is used to establish a standard curve. For each assay, one level of the standard, and three controls (Mdni-Trol I, Dade; Reference Serum, Worthington; and Versatol, General Diagnostics) are measured. 2. Serum glutanH c-oxaloacetic transaminase (SGOT): SG0T is measured by the method of Amador and Wacker.iL/ Moni-Trol I, Enza-Trol, (Dade), and Reference Serum are used as the enzyme reference for each assay. 3. Serum gluram-Lc-pyruvic transaminase (SGFT); SGPT is measured by the method nf Hwrry a1..1_P7 Moni-Trol I, Enza-Trol, and Reference Serum are used as the enzyme reference for each assay. 4. Alkaline phosphatase; Alkaline phosphatase is measured by the method of Bowers and McCornb.TM/ Moni-Trol I, Enza-Trol, and Reference Serum are used as the enzyme reference for each assay. 2 5. BUN: BUN is measured using the BUN Strate Kit (General Diag nostic) which is based on the urease method.-i^ Three levels of Calibrate (General Diagnostics) are used to establish a standard curve. For each assay, three controls (Calibrate X, Moni-Trol X, and Versatol) are used as the reference. 6. Creatinine: Creatinine is measured by a modified kinetic alkaline picrate procedure..!^ Creatinine Standard Solutions (Sigma Chemical Company) are used to establish a standard curve. For each assay, one level of the standard and two controls (Moni-Trol X and Versatol) are used as reference. ' 7. Lactate dehydrogenase (LDH): LDH is measured by the method of Wacker et a 1.2^_ Precinorm E and Frecipath E (Boehringer, Maimhalm Corpora tion) are used as the enzyme controls for each assay. 8. tf-Hydroxybutvrate Dehydrogenase fg-HBDH): a HBDH is measured by the method of Rosalki and Wilkins on. Precinorm E and Precipath E are used as the enzyme controls for each assay. 9. Creatine Phosohokinase (CPK): CPK is measured by the improved procedure of Rosalki--^ based on the methods of Oliver.HJ Precinorm E and Precipath E are used as the enzyme controls for each assay. R&S 112785 C. Urinalysis Urine samples are collected from animals before and during treat ment as are the blood samples. The urine from rats is collected by slight manipulation of their body, and'samples within each group are pooled. The monkeys and dogs are placed individually in metabolism cages, and urine is collected in the stainless steel pan. The urine from each dog and the pooled urine from rats are tested and examined for the following: 1. Protein: Urinary protein is determined with Labstix (Ames Company, Elkhart, Indiana). ^ 2. SupflT; Urinary glucose and reducing substance are determined with Labstix (Ames Company). 3. Microscopic exa^ nation: Urine samples are centrifuged and the supernatant discarded. The residue is resuspended and examined micro scopically for the presence of erythrocytes, leukocytes, epithelial cells, and crystals under high power field and for casts under low power field. A positive urine control prepared with known amounts of protein and glucose in saline adjusted to pH 6.0 is run with each assay to check the reliability of the Labstix. 3 D. Precision, of Hematology and Clinical Blood Chemistry Tests X. Reproducibility For erythrocyte and leukocyte counts, hematocrit, hemoglobin, and the various clinical blood chemistry tests, the same control blood samples or control standards are used for day-to-day assays. The replication of results are excellent and are summarized in Table A. The determination of differential leukocyte counts and reticulocyte counts are performed by experienced personnel. At weekly intervals, a blood sample is counted by two or more personnel to confirm the accuracy of the counting. Also at weekly intervals, the platelet counts obtained from a Coulter Electronic Particle Counter are compared with the direct visual counts in a hemocytometer .using a phase microscope. 2. Reproducibility Within a Test Dav At monthly intervals, a blood sample is taken from a control dog and six or more determinations for erythrocyte, leukocyte, reticulocyte, and platelet counts, hemoglobin, and various clinical blood chemistry tests are performed to establish the reproducibility within an assay. The results are summarized in Table B. 3. Proficiency Test Service We subscribe to the Proficiency Test Service of the Institute for Clinical Science, Hahnemann Medical College, Philadelphia, Pennsylvania (F. Wm. Sunderman, M. D., Director). On the first day of each month, this Service sends two samples containing two different sera or solutions to all subscribers for measurements of one or more of the parameters usually analyzed in clinical laboratories. Participants report their results on a form fur nished by the Service. On the 15th day of the month, each participant receives a report from the Service which Includes: the results of a statis tical analysis of the values reported by all the participating laboratories; a current review of pertinent methodology; a comprehensive bibliography; and validation of the results which the participating laboratory reported. This Service enables each participating laboratory to obtain an unbiased and critical assessment of its proficiency in relation to that of 1,000 or so other clinical laboratories throughout the country. The Service has been in continuous operation since 1949 and was given endorsement by the American Society of Clinical Pathologists in 1952 and by the Association of Clinical Scientists in 1957 and 1968. Our results have been found to be satisfac tory and are summarized in Table C. 4 R&S 112786 H. HISTOPATHOLOGY A. Necropsy and Gross Examination At termination or prior to imminent death, rata are killed with ether, and dogs and monkeys with an overdose of sodium pentobarbital. Animals that die on tests are kept refrigerated but not frozen until necropsy. The general physical condition and nutritional status of each animal at the time of death or termination are observed and recorded. Necropsy is performed as soon as possible after death. Gross changes ofall tissues are carefully ex amined and recorded. B. Organ Weights The liver, spleen, kidneys, adrenals, thyroids and gonads are trimmed free from surrounding tissues and weighed. The organ weight to body weight ratios are then calculated. C. Tissues for Microscopic Examination Tissues to be examined Include the heart, lung, liver, gall blad der (except rats), spleen, pancreas, fundic and pyloric stomach, duodenum, jejunum, ileum, colon, kidneys, adrenals, urinary bladder, gonads, and ac cessory organs, thyroids, submaxillary salivary gland, thymus, prescapular (except rats) and mesenteric lymph nodes, diaphragm and gracilis muscle, rib bone with bone marrow, eyes, anterior pituitary, brain (sagittal section for rats; coronal sections of cerebral cortex, cerebellum, and brain stem for dog and monkey), spinal cord (lumbosacral plexus, dog and monkey), and any other structures not mentioned which show abnormal gross changes. D. Fixation and Staining of Tissues All tissues are cut not to exceed 1 cm in thickness for fixation. For most tissues, neutral buffered 10% formalin is used. Sufficient volume of fixing solution is used and the tissues are changed to a fresh solution after 24 hours. The fixed tissues are processed in an Autotechnicon for dehydration, clearing, and infiltration and then embedded in paraffin. Rou tine H & E staining is used to stain the sectioned tissues for microscopic examination. Supplementary tissue fixatives and staining techniques may be employed for more positive identification of special lesions such as calci fication, pigments, fat deposition and other abnormal changes. 5 R&S 112787 III. STATISTICAL ANALYSIS Data are analyzed statistically using the Dunnett's multiple com parison procedure following an analysis of variance,--^ or our modification of this procedure for uneven numbers among groups. The chosen criterion significance is p <0.05. The means of each group at various intervals during treatment are compared with pretreatment levels. For most experi ments in beagles, three baseline (pretreafment) levels are obtained. The baseline levels for each animal are averaged and the mean is used in the analysis. In addition, the means of the various treated groups are compared with that of the control group at the respective time Intervals. IV. NORMAL VALUES A. Hematology. Clinical Blood Chemistry and Bone Marrow Since June 1971, we have used about 80 rhesus monkeys (Woodard Research Corporation, Herndon, Virginia and Primate Imports, Port Washington, New York) for various studies. The peripheral blood elements and clinical blood chemistry values of these monkeys before treatment and the myeloid/ erythroid (M/C) ratio of the bone marrow of the monkeys used as normal con trols varied among individual animals. The mean r S.D. and the range of the various parameters for the males and females are summarized in Tables D and E, respectively. Since September 1971, we have used about 410, 5 to 9 months old, beagles dogs (ARC registered, Hazelton Research Animals, Inc.). The peripheral blood elements, clinical blood chemistry values and the M/E ratio of the bone marrow varied considerably among individual dogs. The mean S.D. and the ranges of the various parameters for the males and females are summarized in Tables H and I, respectively. During the same period, we have used about 350, 7 to 10 weeks old, male albino rats (CD^ Strain, Charles River Breeding Laboratories). As for the dogs, the individual variations of the peripheral blood elements, clinical blood chemistry values and the M/E ratio of the bone marrow were large. The mean S.D. and the ranges of the various parameters for these male rats are summarized in Table L. B. Absolute and Relative Organ Weights Organ weights, both absolute and relative to body weight, of rhesus monkeys, beagle dogs, and albino rats are summarized In Tables F and G, J and K, and M, respectively. These were control animals used between June 1971 and December 1974. 6 R&S 112788 r &S 112789 C. Presence of Various Substances In the Urine Various substances occasionally occurred in the urine of dogs and rats. The results are summarized in Table N. Protein, erythrocytes, leukocytes, and epithelium were present in 9.4-12.97., 19.1-24.97., 22.2-24.6%, or 18.9-23.6%, respectively, of the samples from dogs collected for analysis. Glucose, crystals, and casts occurred in 1-2% of these samples. Some dogs had been bled and returned to the metabolism cages before the urine was removed for analysis. The high incidence of some of these substances in the urine of these dogs might be due to contamination with the fecal material and traces of blood dropped in the cage. Special care to avoid contamination has been undertaken. A number Of urine samples from rats contained protein, erythro cytes, leukocytes, epithelium, or crystals. These urines were pooled samples of four to six animals. V. REFERENCES 1. Brecher, G., M. Schneiderman, and C. Z. William: Evaluation of the Electronic Red Cell Counter. Am. J. Clin. Path., 26: 1439, 1956. 2. Selegson, D.: Standard Methods of Clinical Chemistry. Academic Press, Inc., New York, Vol. 2, p. 52, 1958, 3. Dubowski, K. M. : Measurement of Hemoglobin Derivatives in Hemoglobin, Its Precusors and Metabolites. (F. W. Sunderman and F. W. Sunderman, Jr.,eds.), J. B. Lippincott Company, Philadelphia, p. 49, 1964. 4. Brecher, G., and M. Schneiderman: A Time-Saving Device for the Count ing of Reticulocytes. Am. J. Clin. Path., 20: 1079, 1950. 5. Bull, B. S., M. A. Schneiderman, and G. Brecher: Platelet Counts With the Coulter Counter. Am. J. Clin. Path., 44: 678-688, 1965. 6. Brecher, G., M. Schneiderman, and E. P. Cronklte; The Reproducibility and Constancy of the Platelet Count. Am. J. Clin. Path., 23: 15, 1953. 7. Hepler, 0. E,: Manual of Clinical Laboratory Methods, p. 83, Charles C. Thomas, Springfield, Illinois, 1935. 8. Slein, M. W.; Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), p. 117, Academic Press, New York, 1963. 7 R&S 112790 9. Amador, E., and W. E. C. Wacker: Serum Glutamic-Oxaloacetic Transamin ase Activity: A New Modification and an Analytical Assessment of Current Assay Technics, Clin. Chem., 8: 343, 1962. 10. Henry, R. J., N. Chiamori, 0. J. Golub, and S. Berkman: Revised Spectrophotometric Methods for the Determination of Glutamic- t Oxaloacetic Transaminase, Glutamic-Pyruvic Transaminase, and Lactic Dehydrogenase. Am. J. Clin. Path., 34: 381, 1960. 11. Bowers, G. N., Jr., and R. B. McComh: A Continuous Spectrophotometric Method for Measuring the Activity of Serum Alkaline Phosphatase. Clin. Chem,, 12: 70, 1966. 12. Chaney, A. L., and E, P. Manback; Modified Reagents for Determination of Urea and Ammonia. Clin. Chem., 8: 130, 1962. 13. Lustgarten, J. A.: A Simple, Rapid, Kinetic Method for Creatinine Concentration. Clin. Chem., IB: 1419, 1972. 14. Wacker, W. E. C., D. D. Ulmer, and B. L. Vallee: Metalloenzymes and Myocardial Infarction. IX. Malic and Lactic Dehydrogenase Activities and Zinc Concentrations- in Serum. New Eng. J. Med., 225, 449, 1956. 15. Rosalki, S. 3., and J. H. Wilkinson: Reduction of a-Ketobutyrate by Human Serum. Nature (London), 188, 1110, 1960. 16. Rosalki, J. B.: An Improved Procedure for Serum Creatine Phosphokinase Determination. J. Lab. Clin. Med., 69, 696, 1967. 17. Oliver, I. T.: A Spectrophotometric Method for the Determination of Creatine Phosphokinase and Myokinase. Biochem. J., 61, 116, 1955. 18. Dunnett, C. W.: A Multiple Comparison Procedure for Comparing Several Treatments with a Control. J. Am. Stat. Assoc., 50: 1096-1121, 1955. 8 R&S 112791 TABLE A REPRODUCIBILITY AMONG TEST DATS ON THE SAME CONTROL SAMPT.ES OR STANDARDS^ No. of Determina tions Mean S.D. Erythrocytes (x 10/mm^) Normal level Abnormal level Hematocrit (vol Z) Normal level Abnormal level Hemoglobin (gm 7.) Normal level Abnormal level Leukocyte Counts (x lO^/mnt^) Normal level Abnormal level 17 4.69 i 0.11 17 2.24 0.03 17 42.8 i 0.4 17 19.6 0.4 17 13.8 0.5 17 6.8 0.1 17 8.8 0.3 17 18.4 0.4 Fasting Blood Glucose (mg Z) SGOT (IU/i) SGPT (IU/i) Creatinine (mg Z) BUN (mg 7.) Bilirubin (mg Z) Alkaline Phosphatase (IU/l) 29 136.4 4.4 33 66.6 3.5 31 31.4 * 3.2 13 1.9 0.3 14 11.1 0.3 6 1.1 t 0.1 24 107,3 5.7 a/ Performed in November/December 1974. Range 4.40 -.5.21 2.19 - 2.28 42 - 44 19 - 20 13.6 - 14.4 6.6 - 6.9 8.1 - 9.1 17.7 - 19.1 131 - 149 62 - 74 28 - 37 1.5 - 2.4 10.7 - 11.9 0.9 - 1.2 94 - 115 9 R&S 112792 TABLE B REPRODUCIBILITY WITHIN A TEST DAY ON THE SAME SPECIMEN^? Erythrocytes (x lO^/mm^) Reticulocytes (7.) Hematocrit (vol X) Hemoglobin (gm 7.) Platelets (x lOVmmS) Leukocytes (x IQ^/msn?) Bands (%) Neutrophils (71) Lymphocytes (7t) Eosinophils (X) Basophils (71) Monocytes (71) Atypical (%) Nucleated BBC (%) Pasting Glucose (mg X) SGOT (XU/i) SGPT (lU/i) Creatinine (mg %) BUN (mg 7.) Alkaline Phosphatase (IU/i) Mean S.D.^ 6.30 0.10 0.53 0.08 44.8 0.4 15.5 0.1 2.24 0.08 9.0 0.2 00 53.7 + 2.1 39.5 3.7 6.2 Jm 1.9 0 -f- 0 0.7 0.8 0+ 0 00 102.6 -fc. 0.6 21.0 0.0 18.5 + 1.2 0.7 0.0 12.8 0.4 38.0 0.6 Ranee 6.15 - 6.40 0.44 - 0.65 44 - 45 15.4 - 15.6 2.15 - 2.37 8.7 - 9.3 0 -0 50 - 56 35 - 45 4- 9 0 -0 0 -2 0 -0 0 -0 102 - 103 21 - 21 18 - 21 0.7 - 0.7 12 - 13 37 - 39 a/ performed in October 1974. b/ Six determinations from an adult beagle blood sample. 10 \ R&S 112793 TABLE C PROFICIENCY TEST SERVICE (PTS) REPORTS (1973-1974W Unknowns MRI Results PTS Results Participating Laboratories (10-90 Percentiles) Median Mean Acceptable Performance^/ Hemoglobin 12.9 gm Z 8.5 gm Z 12.8 ' 8.6 . 12.8 8.6 12.75 8.6 12.4 - 13.1 8.2 - 8.9 Serum Protein 7.2 mg Z 7.1 7.'19 7.16 6,8 - 7.5 5.4 mg Z 5.3 5.25 5.27 4.9 - 5.6 Fasting Glucose 145.0 mg % 380.0 mg 7. 143.0 378.0 142.6 379.7 142.5 378.4 129 - 156 342 - 409 BUN 13.0 mg Z 12.5 12.7 12.7 11.0 - 14.2 36.0 mg Z 37.3 37.0 36.7 32.0 - 40.0 Creatinine 4.1 mg Z 4.8 4.8 1.4 mg Z 1.6 1.6 4.77 1.58 4.1 - 5.2 1.3 - 1.9 Bilirubin 0.1 mg Z 0.3 0.2 0.25 0.1 - 0.6 3.5 mg Z 3.9 3.92 3.92 3.4 - 4.5 Ca 9.2 mg Z 9.4 9.3 9.3 8.9 - 9.9 13.9 mg Z 14.4 14.5 14.5 13.8 - 15.1 Na 162.0 meq/2 162.0 161.0 161.0 158-164 136.0 meq/A 135.0 135.0 134.8 132 - 137 K 7.1 meq/jt 7.0 7.0 7.0 6.8 - 7.2 7.4 meq/A 7.1 7.2 7.1 6.9 - 7.3 Cl 84.1 meq/A 83 32.6 82.7 80 - 86 102.2 meq/jt 103 103.5 103.6 100 - 107 Mg 1.6 meq/A 1.7 1,70 1.71 1.5 - 1.9 1.6 meq/A 1.7 1.7 1.7 1.5 - 1.9 a/ Up to date. we received unknowns for uric acid , cholesterol, total protein serum iron, and inorganic phosphorus. We do not routinely perform these determinations. b/ Based on values submitted by participants by 10th of month. 11 176LZW S'Sb TAULE D HEMATOLOGY. CUHICAI. BlitOD CIIHH1STHY VALUES . AND BONE HAH HIW (HYELOIP/EKYTIIKOIDI RATIOS OF HALE HUES IS HUHKEYsI? _____ Hale Wicaiia Monkeys Humber Body Uelght (kg) Studied Haan 1 S.D, Erythrocyte* (x l06/ma3) Reticulocyte* (X) ' Hematocrit (vol X) Hemoglobin (gm X) HCV (ii3) HCIIb (Wig) MCHbC (mg X) Platelet* (x 103/im31 Leukocyte* (x 103/nnr) Neutrophil* I (X) Neutrophil* H (X) Lymphocytes (X) Eotlnophll* (X) HonopLil* (X) Baiophll* (X) Atypical cell* (X) Nucleated RBC (X) Fa*ting Clucoae (mg X) SCOT (IU/1) SOFT (ID/i) Alkaline Phoaphataae (1U/J) BUN (mg X) Froth. Tima (aac) Serum Creat, (mg X) Bilirubin Total (mg X) Direct (mg X) BSP 15 min (X ret.) Ha (mEi)/ i) K (mEq/1) Cl (Eq/i) Ca (m<|/ l) Mg (K<j/ 1) Bone Harrow Hyelold/arythrold ratio 52 52 52 52 52 52 52 44 52 52 52 52 52 52 52 52 52 44 44 44 44 44 28 44 28 28 28 28 28 28 28 28 6 3.63 0.48 3.63 0.48 3.63 t 0.48 3.63 0.48 3.63 0.48 3.63 0.48 3.63 1 0.48 3.66 * 0.50 3.63 1 0.48 3.63 0.48 3.63 1 0.48 3.63 0.48 3.63 1 0.48 3.63 1 0.48 3.63 0.48 3.63 0.48 3.63 t 0.48 3.66 t 0.50 3.66 0.50 3.66 1 0.50 3.66 t 0.50 3.66 0.50 3.58 0.51 3.66 0.50 3.51 0.50 3.51 t 0.50 3.51 0.50 3.51 0.50 3.51 i 0.50 3.51 1 0.50 3.51 0.50 3.51 0.50 3.77 0.67 a/ Data collected between June 1971 and December 1974. Obaerved Result* Haan JLUL. Range 5.4B t 0.54 0.95 t 0.52 42.7 2.9 13.2 t 0.9 79.1 9.4 24.3 i 2.3 3.1 1 0.2 3.34 1 1.35 11.4 5.8 0.19 t 0.44 40.10 18.72 55.44 18.47 2.13 t 2.79 1.77 1.86 0.06 0.31 0.00 t Q.0Q 0.00 0.00 92.7 16.2 37.2 10.1 29.8 7.7 308.8 99.6 21.9 9.3 10.4 0.7 1.1 0.3 3.75 - 6.61 0.07 - 2.41 37,0 - 50.0 10.8 - 15.4 69.6 - 117.3 21.0 - 33.6 2.7 - 3.4 0.80 - 7.10 3.8 - 29.8 0 -2 10 - 83 13 - 84 0 - 13 0-7 0 -2 0 -0 0 -0 59 - 127 20 - 60 IS - 46 143 - 501 12 - 65 9.3 - 11.9 0.6 - 1.8 0.1 i 0.2 0.0 0.0 17.7 t 8.2 156.9 7.0 4.7 0.5 106.0 6.3 5.2 0.5 1.6 t 0.1 0.0 - 0.8 0.0 - 0.0 2 - 34 144 - 179 3.9 - 5.7 93 - 118 4.2 - 6.3 1.2 - 1.8 1.48 t 0.32 1.3 - 2.1 TABLE E HEHATOlOCTf. CUBICAL BIjOOP CKEHI STBY VALUES, AHfl BOHE HARROW ftWELOTD/EEVTHEOXP^ RATIOS OF FEMALE RHESUS HOTKEYS*/ Female Khleaua Monkeva Humber Body Weight (kg) Studied Haan t s.p. II Obeerved Remit! Z S.D. list*, Erythrocyte! (x 10^ l*n?) 26 3.37 0.54 5.20 x 0.45 4.25 - 6.03 Reticulocyte! (X) 26 3.37 A 0.S4 1.16 z 0.64 0.35 - 3.31 Hematocrit (vol X) Hemoglobin (mg X) 26 3.37 A 0.54 40.B A 3.7 30.0 - 45.0 26 3.37 A 0.54 12.7 A 1.4 7.9 - 14.1 HCV CM3) 26 3.37 A 0.54 78.5 a 7.6 66.5 - 95.2 HCHb (ut>g) 26 3.37 A 0.54 24.5 A 2.6 17.6 - 29.7 KQtbC (mg X) 26 3.37 A 0.54 3.1 A 0.2 2.6 - 3.4 Platelet! (x 10S/m3l Leukocyte! (x 10-^/ea^) 26 3.37 A 0.54 3.84 A 1.57 1.85 - 7.90 26 3.37 A 0.54 10.3 A 5.2 3.2 - 24.8 Heutrophlla I (X) Neutrophil! H (X) 26 26 3.37 A 0.54 3.37 A 0.54 0.15 A 0.61 32.54 A 10.83 0-3 13 56 Lymphocyte! (X) 26 3.37 *0.54 63.08 A 10.02 41 - 79 c Eoatnophil! (X) 26 3.37 *0.54 3.12 a 4.35 0 18 Honophlla (X) 26 3.37 * 0.54 1.00 A 1.39 0 -4 Baaophtla (X) 26 3.37 *0.54 0.08 * 0.27 0-1 Atypical celta (X) Nucleated RBC (X) 26 3.37 *0.54 0.00 A 0.00 19 3.43 *0.55 0.00 A 0.00 0 .0 0 .0 Faatlng Glucoae (mg X) SCOT (lU/i) 6CPT (IU/i) Alkaline Phoaphataae (IU/j) BUN (mg X) 26 26 26 26 26 3.37 3.37 3.37 3.37 3.37 *0.54 *0.54 1 0.54 *0.54 a 0.54 88.1 a 16.7 1134.1 * 11.2 25.4 7.2 319.9 109.8 20.1 * 4.5 57 . 116 20 - 65 12 - 39 148 - 572 13 - 29 Proth. Time (tec) Serum Great, (mg X) 126 3.37 *0.54 10.8 * 0.7 9.7 - 12.3 26 3.37' 1 0.54 1.2 0.3 0.6 - 1.7 Bilirubin Total (mg X) Direct (mg X) BSP IS min (X ret.) Na (Eq/|) K (mEq/A) Cl (mEq/i) Ca (mEq/i) Mg (mEq/1) Bone Harrow 26 3.37 x 0.54 0.1 A 0.2 0.0 - 0.8 26 3.37 A 0.54 0.0 A 0.0 0.0 - 0.0 26 3.37 Z 0.54 15.9 1 8.5 5 - 34 26 3.37 Z 0.54 158.7 A 6.6 147 - 174 26 3.37 Z 0.54 4.9 A 0.6 3.9 - 6.2 26 3.37 Z 0.54 106.0 1 5.7 95 - 113 26 26 3.37 A 0.54 3.37 A 0.54 5.3 A 0.5 1.6 A 0.2 4.3 ` 6.3 1.3 2.0 Hyelold/erythrold ratio 2 3.60 Z 0.57 1.05 a 0.07 1.0 - l.l a/ Data collected between June 1971 and December 1974. S6LZVV S3H i'AliLb tf ABSOLUTE ORfiAH HEIGHTS OF HALE AHD FEMALE RHESUS MONKEYS^ 96IZU s^U Monkey Ho. _ Terminal Body Height fkgl ______________________________ ________Absolute Organ Weights Liver Spleen Kidneys Adrenals Thyroids (firo) _ fRm) ffiml (r0 fgml Testes feml Ovaries 12 3.4 71 4.7 9.1 0.81 0.50 1.35 41 3.0 71 6.0 11.0 0.80 0.75 0.53 55 3.8 67 2.6 11.5 0.45 0.42 1.20 67 3.7 86 6.4 15.6 0.86 0.81 0.94 72 4.8 122 5.2 21.0 1.05 0.61 1.60 73 4.3 102 4.0 22.0 0.82 0.37 2.01 74 3.9 83 9.3 17.0 0.61 0.49 1.80 75 2.8 85 3.2 18.0 0.71 0.40 0.86 3.7 0.7b/ 86 18 5.3 2.1 14.7 * 4.8 0.76 0.18 0.54 0.16 1.29 0.50 40 3.2 106 5.4 20.0 0.75 1.11 54 3.8 73 3.1 13.7 0.64 0.37 3.5 o.4k/ 90 23 4.3 1.6 16.9 2.5 0.70 0.08 0.74 0.52 MS 0.45 0.25 0.35 0.14 a/ Data collected between June 1971 and December 1974. b/ Mean * S.D. TABLE G RELATIVE ORGAN WEIGHTS OF HALE AMD FEMALE RHESUS MONKEYSli/ Monkey No. Terminal Body Height (kg) ___________________________ Organ Weights Per kg Body Weight Liver Spleen Kidneys Adrenals Thyroids (Rm) (fim) (Rm) (mg) (mg) Testes (gm) Ovaries (mg) 12 3.4 21.0 1.39 2.68 240 148 0.40 41 3.0 23.7 2.00 3.67 266 250 0.18 -- 55 3.8 19.1 0.74 3.03 129 120 0.34 -- 67 3.7 23.2 1.73 4.22 232 219 0.25 -- 72 4.8 25.4 1.08 4.38 219 127 0.33 -- 73 4.3 23.7 1.12 5.12 191 86 0.47 -- 74 3.9 21.3 2.38 4.36 156 126 0.46 -- 75 2.8 30.4 1.14 6.43 254 143 0.31 -- 3.7 0.7^ 23.5 3.4 1.45 0.55 4.24 1,18 211 48 152 55 0.34 0.10 40 3.2 33.1 1.68 6.25 234 346 140 54 3.8 19.2 0.82 3.61 168 97 -- 66 3.5 * 0.4b/ 26.2 * 9.8 1.25 0.61 4.93 1.86 201 47 222 176 103 x 37 a/ Data collected between June 1971 and December 1974. b/ Mean S.D. L6LZU SSH B6LZU S?U TABLE 11 HEMATOLOGY. CLINICAL BLOOD CHEMISTRY VALUES. AND BONE MARROW (MYELOIP/ERYTHROID) RATIOS OF MALE BEAGLE DOGSS7 Number S tudled Male Beagle Dogs Age Body Height (kg) (months) Mean S.D. Erythrocytes (x lO^/mra3) Re ticulocytea (7.) Hematocrit (vol 7.) Hemoglobin (gm 7.) MCV (n3) MCHb (jtpg) MCHbC (mg 7.) Platelets (x 10-*/mnr*) Leukocytes (x 103/mm3) Neutrophils I (X) Neutrophils M (%) Lymphocytes (%) Eosinophils (7>) Mono phi la (/i) Basophils (7i) Atypical cells (%) Nucleated RBC (7) Fasting Glucose (mg 7.) SCOT (IU/4) SGPT (IU/1) Alkaline Phosphatase (IU//) BUN (mg %) Bone Marrow Mycloid/erythroid ratio 196 204 196 196 196 196 196 170 204 204 204 204 204 204 206 204 204 196 196 196 196 204 24 4-7 4 -7 4 -7 4-7 4-7 4 -7 4 -7 4-7 4 -7 4-7 4 -7 4 -7 4.- 7 4 -7 4-7 4-7 4 -7 4-7 4-7 4-7 4-7 4-7 5 -9 8.2 1.7 8.2 1.7 8.2 1.7 8.2 1.7 8.2 1.7 8.2 1.7 8.2 1.7 8.4 1.7 8.2 1.7 8,2 1.7 8.2 1.7 8.2 1.7 8.2 1.7 8.2 1.7 8.2 1.7 8.2 1.7 8.2 1.7 8.2 1.7 8.2 1.7 8.2 1.7 8.2 1.7 8.2 1.7 9.3 1.6 a/ Data collected between September 1971 and December 1974 Observed Results Mean S.D. Range 5.40 0.73 0.72 0.49 41.4 3.4 13.2 1.3 77.2 8.8 24.8 3.4 32.1.1 1.4 3.18 1.04 11.1 3.6 0.91 3.24 56.79 9.83 37.70 9.59 2.63 2.80 1.96 1.97 0.00 0.00 0.11 0.37 0.03 0.18 100.8 14.1 23.9 7.9 25.7 7.9 73.2 16.9 12.2 3.5 3.62 - 7.60 0.04 - 4.35 31 - 50 10.0 - 16.9 56.7 - 127. 17.1 - 41.7 28.1 - 40.3 0.95 - 6.35 4.6 - 24.6 0 -6 22 - 80 13 - 71 0 - 16 0 - 10 0 -0 0 -2 0 -1 66 - 134 11 - 59 8 - 46 30 - 128 4 - 23 1.6 0.5 1.1 - 3.0 TABLE I HEMATOLOGY. CLINICAL BLOOD CHEMISTRY VALUES. AND BONE MARROW (MYELOID/ERYTHROID) RATIOS OF FEMALE BEAGLE DOGSSV Number S tudled Female Beagle Dors Age Body Weight (kg) (months) Mean S.D. Erythrocytea (x lQ^/mra3) Reticulocytes (%) Hematocrit (vol %) Hemoglobin (gm %) MCV (p3) MCHb (ppg) MCHbG (mg 7.) Platelets (x 103/mm3) Leukocytes (x 103/mm3) Neutrophils I (7) Neutrophils M (7.) Lymphocytes (%) Eosinophils (7) Monophils (7) Basophils (7) Atypical cells (7) Nucleated RBC (7) Fasting Glucose (mg 7.) SCOT (IU/t) SGPT (IU/i) Alkaline Phosphatase (IU/i) BUN (mg 7) Bone Marrow Myeloid/erythroid ratio 194 202 194 194 194 194 194 164 202 202 202 202 202 202 202 202 202 194 194 194 194 202 24 47 4-7 . 4 -7 4-7 4-7 4-7 4-7 4-7 4 -7 4-7 4-7 4 -7 4-7 4-7 4 -7 4-7 4 -7 4-7 4-7 4 -7 4 -7 4 -7 5 -9 6.7 1.3 6.8 1.3 6.7 1.3 6.7 1.3 6.7 1.3 6.7 1.3 6.7 1.3 6.9 1.3 6.8 1.3 6.8 1.3 6.8 1.3 6.8 1.3 6.8 1.3 6.8 1.3 6.8 1.3 6.8 1.3 6.8 1.3 6.7 1.3 6.7 1.3 6.7 1.3 6.7 1.3 6.8 1.3 7.7 1.4 a/ Data collected between September 1971 and December 1974. Observed Results Mean s.p. Range 5.49 0.77 . 0.75 0.58 42.3 3.4 13.6 1.3 78.0 9.8 25.1 3.7 32.0 1.6 3.28 1.16 10.7 3.5 0.68 1.28 57.17 10.29 37.07 10.15 2.23 2.14 2.19 2.14 0.01 0.10 0.14 0.49 0.04 0.20 98.9 15.1 23.6 7.6 25.2 7.1 72.8 19.6 12.7 3.5 3.27 - 7.75 0.04 - 5.05 32 - 51 11.0 - 18.6 55.8 - 128.4 17.1 - 41.6 28.7 - 40.4 1.08 - 7.95 3.8 - 26.9 0-7 31 - 84 10 - 61 0 - 13 0 -9 0 -1 0 -4 0-1 55 - 130 6 - 49 8 - 49 30 - 146 4 - 25 1.5 0.4 1.1 - 2.4 662.31-1 S'Sd %* TABLE J ABSOLUTE AND RELATIVE ORGAN WEIGHTS OF MALE BEAGLE DOGsV Organ Weight Mean S.D. Absolute Range Liver (gm) Spleen (gm) Kidneys (gm) Adrenals (gm) Thyroids (gm) Testes (gm) 264 51 60 29 52 10 1.12 0.26 1.06 0.34 6.57 4.88 166 - 384 22 - 167 32 - 71 0.74 1.75 0.55 - 2.41 1.32 - 18.00 Relative (per kg bodv weight) Mean S.D. Range Liver (gm) Spleen (gm) Kidneys (gm) Adrenals (mg) Thyroids (mg) Testes (gm) 27.8 4.8 6.2 2.4 5.5 0.9 118 28 111 34 0.66 0.42 19.6 - 42.3 2.8 - 12.5 4.0 - 7.7 70 - 165 56 - 211 0.13 - 1.67 a/ Data collected between September 1971 and December 1974 from 34 dogs, weighing 9.6 1.9 kg, used as control animals. Ft&S 112800 18 TABLE K ABSOLUTE AND RELATIVE ORGAN WEIGHTS OF FEMALE BEAGLE DOGSi/ Organ Weizht Mean S.D. Absolute Range Liver (gra) Spleen (gm) Kidneys (gm) Adrenals (gm) Thyroids (gm) Ovaries (gm) 224 t 46 51 21 44 9 1.11 0.28 0.91 0.27 -0.80 0.25 106 - 322 16 - 103 24 - 71 0.49 - 1.65 0-.55 - 1.91 0.38 - 1.27 Relative ( ter kg body weight) Mean S.D. Range Liver (gm) Spleen (gm) Kidneys (gm) Adrenals (mg) Thyroids (mg) Ovaries (mg) 28.8 4.6 6.3 2.5 5.7 1.1 144 38 117 32 103 36 20.7 - 38.8 3.1 - 10.9 3.7 - 7.9 67 - 215 75 - 219 54 - 222 a/ Data collected between September 1971 and December 1974 from 33 dogs, % weighing 7.8 1.5 kg, used as control animals. R&S 112801 19 3083 U SSU TABLE l HEMATOLOGY. CLINICAL BLOOD CHEMISTRY VALUES. AND BONE MARROW (MYELOID/ERYTHRQID) RATIOS OF MALE ALBINO RATS--^ Humber S tudied Erythrocytes (x lO^/nam^) Reticulocytes (%) Hematocrit (vol 7.) Hemoglobin (gm 7.) MCV (H3) MCHb (ppg) MCllbC (mg 7.) Platelets (x 103/mm3) Leukocytes (x 103/mm3) Heutrophils I (%) too Neutrophils M (7.) Lymphocytes (X) Eosinophils (%) Monophlls (%) Basophils (7.) Atypical cells Nucleated RBC (%) Fasting Glucose (mg 7c) SGOT (IU//) SGVT (IU/i) Alkaline Phosphatase (IU/i) BUN (mg 7.) Bone Marrow Myeloid/erythroid ratio 343 343 343 343 343 343 343 288 264 264 264 264 264 264 264 264 264 71 71 71 71 71 49 Male Rata Age Body Weight (gm) (weeks) Mean StD,____ 5 _7 5-7 5 -7 5 -7 5 -7 5-7 5 -7 5 -7 5 -7 5-7 5-7 5 -7 5 -7 57 5 -7 5 -7 5-7 10 - 12 10 - 12 10 - 12 10 - 12 10 - 12 155 24 155 24 155 24 155 24 155 24 155 24 155 24 154 25 151 25 151 25 151 25 151 25 151 25 151 25 151 25 151 25 151 25 358 78 358 78 358 78 358 78 358 78 10 12 341 48 aj Data collected between September 1971 and December 1974. Observed Results Mean S.D. Range 5.93 0.52 2.82 1.72 45.7 3.4 13.6 0.9 76.8 7.7 23.2 2.5 30.0 1.8 5.06 1.03 14.4 2.8 0.12 0.40 13.8 4.6 83.60 5.30 0.63 0.93 1.49 2.00 0.01 0.14 0.01 0.12 0.11 0.49 133.1 16.4 122.8 39.5 34.8 20.1 87.2 30.4 17.5 5.5 3.24 7.60 0.30 - 6.83 40 - 58 11.8 - 16.9 62.3 - 104.6 19.2 - 41.0 21.1 - 36.9 2.30 - 7.95 6.3 - 20.8 0-3 4 - 29 52 - 96 0 -6 0 - 13 0 -2 0-1 0 -4 94 - 165 63 - 223 17 - 120 . 32 - 153 8 - 41 1.8 0.4 l.l 2.6 V TABLE M ABSOLUTE AND RELATIVE ORGAN WEIGHTS OF MALE ALBINO RATS^ Organ Weight Mean S.D. Absolute Range Liver (gm) Spleen (gm) Kidneys (gm) Adrenals (mg) Thyroids (mg) Testes (gm) 10.07 2.01 0.64 0.11 2.54 -0.37 52.8 8.9 23.9 4.9 2.89 0.40 7.18 - 15.09 0.34 - 0.89 1.84 - 3.58 21.9 - 73.5 14.3 - 37.7 1.76 -- 3.81 Relative (per 100 gm bodv weight) Mean S.D. Range Liver (gm) Spleen (gm) Kidneys (gm) Adrenals (mg) Thyroids (mg) Testes (gm) 2.81 0.34 0.18 0.04 0.71 0.10 15.L 3.5 6.8 1.6 0.82 0.16 2.09 - 4.01 0.10 - 0.30 0.22 - 0.88 5.8 - 22.4 4.2 - 12.7 0.23 - 1.09 a/ Data collected between September 1971 and December 1974 from 71 rats, weighing 360 74 gmf used as control animals. R&S 112803 21 T TABLE N PRESENCE OF VARIOUS SUBSTANCES IN THE URINE OF MALE AND FEMALE DOGS AND MALE RATS Species: No. of Animals: No. of Collections: 365S/ 365 Dogs 81 424^ Glucose:; < 250 mgZ > 250 mg 0.3 (D^ 0.5 (2) 0.5 (2) 0.2 (1) Protein: < 100 mgZ > 100 mg7. RBC:--^ Moderate Recessive WBC:--^ Moderate Excessive 12.6 (46) 0.3 (1) 21.1 (77) 3.8 (14) 20.5 (75) 4.1 (15) 8.7 (37) 0.7 (3) 15.1 (64) 4.0 (17) 18.9 (80) 3.3 (14) EpitheliumiS/ Moderate Recessive 17.0 (62) 6.6 (24) 16.3 (69) 2.6 (ID Crystal Moderate Excessive 0.3 CD 0.3 (1) 0.9 (4) 0.9 (4) Casts: Positive 0 0.9 (4) Rats/ 66^7 13 66 38^ 00 00 13.6 (9) 18.4 (7) 00 4.5 (3) 7.9 (3) 00 0 2.6 (1) 00 0 5.3 (2) 00 0 2.6 (1) 0 2.6 (1) 00 a/ Pooled sample of 4-6 rats. b/ Baseline data collected from all animals employed between September 1971 and December 1974. c/ Data collected at weekly intervals for 4-7 collections from control dogs employed between September 1971 and December 1974. d/ Data collected at 2-week intervals for 2-4 collections from control rats employed between September 1971 and December 1974. e/ Percent of total (number of samples). f/ Normal, 10 or less cells; moderate, 10-100 cells; excessive, > 100 cells/ field (x 440). jj/ Normal, 5 or less cells; moderate, 5-25 cells; excessive, > 25 cells/ field (x 100). h/ Normal, none; moderate, 1-5 crystals; excessive, > 5 crystals/field (x 100). R&S 112804 22