Document G5aEw86bOVd1E83LRKV2k7Or7

Chemical Biology Research DOW CHEMICAL U.S.A. Midland, Michigan RAT AND HAMSTER INTRATRACHEAL AND INTRAPERITONEAL INJECTION STUDIES ON TITANIUM PHOSPHATE AND ASBESTOS by J. M. Norris and G. L. Sparschu April 1972 ST0 0 00 I 02 TABLE OF CONTENTS Introduction ..................................................................................................... 1 Summary and Discussion ............................................................................. 1 Materials .......................................................................................................... 2 Methods - Intratracheal Injection Study ............................... 3 Results - Intratracheal Injection Study ................................ 4 Methods - Intraperitoneal Injection Study ........................... 8 Results - Intraperitoneal Injection Study ........................... 8 References..............................................................................................................11 Bibliography ...................................................................................................... 12 ST0000I 03 1- - INTRODUCTION Titanium phosphate (Ti3(PO.).) fibers are being considered as a replacement for asbestos'in commercial applications such as brake linings and construction materials. A study employing the Sherman rat and the Golden hamster, herein reported, was conducte on a sample of titanium phosphate, prepared at the Western Division, to determine whether the fibers would produce a fibrogenic response or compound-induced neoplasms when introduced into the lung. To assure deposition of known quantities of the titanium phosphate, the material was administered to the animals by intratracheal injection. Subsequently, an intraperitoneal injection study, using rats and hamsters, was conducted to assess and compare the effects from the two routes of administration. Crude chrysotile asbestos was used as a positive control in all phases of the investigation. SUMMARY OF RESULTS AND DISCUSSION OF THE INTRATRACHEAL AND INTRAPERITONEAL INJECTION STUDIES Titanium phosphate, under the experimental conditions of the study herein reported, produced a granulomatous foreign body response of a non-fibrogenic nature in intratracheally and if) intraperitoneally treated rats and hamsters. No compound-induced neoplasms were observed in the animals so treated. 0 CD The pulmonary response to the titanium phosphate in the intratracheally treated animals consisted of: _ CD CD (1) A moderate macrophage response j- (2) Multifocal inflammatory reaction in the rat and a diffuse inflammatory reaction in the hamster (3) sequestration of the fibers by atelectasis (4) focal obliteration of alveolar structure in the animals receivir. the top dose and preservation of the alveolar structure in the animals receiving the lower dose. (5) slow clearance of the fibers from the lung. The granulomatous response in the peritoneal cavity of the rats and hamsters was accompanied by minimal collagen deposition. In contrast to these responses, asbestos produced mesotheliomas in the intraperitoneally treated rats and a fibrogenic response in intratracheally treated rats and intraperitoneally treatedhamsters. Observation of mesotheliomas in the peritoneal cavity of rats has been reported by Wagner.1 The fibrogenic response to asbestos was characterized by (1) a reticulin stromal reaction followed by collagen deposition, (2) sequestration by scar tissue, (3) abundant macrophage response, and, in the lung, (4) destruction of alveolar structure, and (5) slow clearance. 2- - In contrast to the responses in the rat lung, asbestos in the hamster lung produced a mononuclear inflammatory reaction without collagen deposition. Gross and deTreville noted these pulmonary observations with asbestos and ascribed them to a less effective pulmonary clearance mechanism and greater reactivity of the pulmonary tissue in the hamster than in the rat.2 Furthermore, the results obtained on the intratracheally treated rats and hamsters in the study herein reported are the same as reported by Gross et al.2 In discussing the possible mechanisms of carcinogenic action of asbestos, Harington and Roe have associated the activity with the presence of (1) natural oils found on asbestos fibers and (2) metal complexes in the asbestos. For this reason, the asbestos used in the present study was crude and non-acid washed. The iron and iron complexes in mined asbestos are suspect carcinogenic agents. Hematite, which contains 70% of ferric iron, has been suspected of causing lung cancer in man. Harington and Roe also suggest that the ratio of f|ic+o ferrous iron may be of significance, in that a low Fe :F may be associated with carcinogenicity. The sample used in the present study was, therefore, analyzed for various trace elements including iron compound arid the ratio of iron in the ferric and ferrous states. The ratio in the case of each of the three iron compounds found in the asbestos was high ferric to ferrous. The asbestos, however, did produce a fibrogenic response in the peritoneal cavity of the rats and hamsters and in the lungs of the rats, and d^use+|bdomin mesotheliomas in the rats. The association of low Fe :Fe+ ratio with carcinogenicity, therefore, appears questionable. MATERIALS The titanium phosphate sample used in these investigations was identified by reference #GP12-66-67 and batch #30. The fibers which measured 10-20y by 0.2-0.3u are acidic, insoluble in water at room temperature and have a density of 2.76 + 0.02 (Midland Analytical Laboratories report AL579-476). Trace elements in the sample were identified by emission spectroscopy (Spectrographic report 195058) : SO I00001S Aluminum Copper Iron Magnesium Manganese Nickel Silicon Titanium Vanadium 0.07% <0.005 <0.001 <0.1 Present <0.005 0.03 >6.0 <0.01 but <0.01% The sample of chrysotile asbestos used in these investigations was identified as "Hooker #1" non-acid washed asbestos that was mined in Morrisville, Vermont by the G.A.F. Corporation. An aqueous slurry of the asbestos was prepared and, with the use -3- of a Waring blender, the.asbestos was ground to a fiber length of less than lOOy. The water was then separated from the asbestos by suction aspiration through #40 Whatman filter. The asbestos was washed three times with 100% ethanol, dried by washing with ether, and ground through a #40 Tyler mesh screen. The ground asbestos fibers were morphologically characterized by electron microscopy (Midland Analytical Laboratories report, A1S86-352). The fibers measured 150-400A by 500A-70y and were commonly found in bundles. The sample contained electron dense particles, measuring from O.lu to 10y, which could not be identified from morphological characteristics. Analysis of the sample for iron compound(s) content revealed three such compounds, one of which was magnetite (Fe^O.), one an unidentified ferric compound and the remaining one, an unidentified ferrous compound (Letter Report File #68115-01, Radiochemistry Research Laboratory). The relative amounts of the iron compounds were A/B/C = (1.0+0.1)/(2.01+0.2)/(7+1); where A = unidentified ferrous, l|+= unidentified ferric and~C = magnetite. The ratio of the Fe to Fe+ was 2.1+0.3 in the magnetite and 1.8+0.2 in the two unidentified compounds. INTRATRACHEAL INJECTION STUDY Preparations Administered to Animals STOQOO I 06 Suspensions of the titanium phosphate were prepared with 0.85% saline solution so that 0.5 ml of the suspensions would delive 50, 10, and 2.5 mg of the titanium phosphate/kg body weight of the animal. Suspensions of the ground asbestos were prepared in 0.85% sali solution so that 0.5 ml of the suspension would deliver 10 mg of the asbestos/kg body weight of the animal. Experimental Design The study was conducted in two phases. Phase I was a short term study, using the hamster, that was designed to follow the course of the inflammatory response over an eight week period subsequent to a single intratracheal injection of the titanium phosphate suspensions, the asbestos suspension or the saline solution. Phase II was a long term study to determine whether the titanium phosphate and asbestos fibers would produce a fibrogenic response in the hamster as well as in the rat. The animals used in these studies were randomly selected. The design of both these studies has been summarized in Chart I. Experimental Procedure Th rats and hamsters used in the Intratracheal Injection Study were housed three and five per cage, respectively, and a standard laboratory chow and water was made accessible to them at all times. -4- The animals, prior to treatment, were lightly anesthetized with methoxyflurane and then were treated intratracheally with titanium phosphate suspenion or asbestos suspension, or saline solution. The technique used involved insertion of a sterile trocar into the trachea to within approximately 0.5 cm of the bifurcation of the trachea. A dose of 0.5 ml of the test suspension or saline solution were slowly deposited, using a tuberculin syringe attached to a piece of 11 mm plastic tubing, in the designated region of the bifurcation. The rats were placed on an inclined plane after dosing and whenever necessary, given artificial respiration until breathing appeared normal or the rats had recovered from the anesthesia. To facilitate uniform dispersion of the various suspensions or the saline solution throughout the three lobes of the lungs of the hamsters, these animals were rotated laterally immediately after deposition. The hamsters were then handled in a manner similar to the rat. Spontaneous deaths were recorded. Body weights were taken weekly and the terminal body weights were analyzed by the Student's "t" test. In the short term study, at the designated necropsy times, the hamsters were lightly anesthetized with methoxyflurane, the trachea clamped off using a bull-dog clamp and the animals killed by decapitation. The lungs and mediastinal lymph nodes were removed and fixed in 10% formalin, sectioned at 6p and stained with hematoxylin and eosin for histopathologic examination. In the long term study, at the designated necropsy times, the hamsters and rats were killed by decapitation and examined for gross pathological changes. The lungs and mediastinal lymph nodes and portions of the following organs and tissues were removed and fixed in 10% formalin, sectioned at 6u and stained with hematoxylin and eosin for histopathologic examination: pituitary, aorta, brain, adrenal, accessory sex glands, mesenteric lymph node, stomach, small intestine, pancreas, thyroid, colon, kidney, heart, testes, spleen, liver, spinal cord, peripheral nerve, urinary bladder, thymus, skeletal muscle, and all lesions that were observed. Results CO PHASE I - short Term Study Using Hamsters Titanium Phosphate study O O o o The hamsters in the various treatment regimens, except for two O hamsters that had been treated with 50 mg titanium phosphate/kg -J and one that had been treated with 10 mg asbestos/kg, survived till the designated necropsy times. Histopathological examination of sections of lungs removed one hour after treatment showed the titanium phosphate fibers clumping -5- in the terminal respiratory bronchioles and adjacent alveoli. Within 24 hours polymorphonuclear leukocytes (PMNs) and macrophages were present causing obliteration of alveolar architecture, pulmonary edema, and focal areas of atelectasis containing titanium phosphate fibers and macrophages. The edema was less severe at the 10 mg/kg levels than at the 50 mg/kg level. At 72 hours the giant cells were forming and much of the titanium phosphate was within alveolar macrophages. The alveoli in the terminal respiratory bronchiole areas were obliterated by minimal edema, macrophages and PMNs and proteinaceous debries. At the end of one week there was no pulmonary edema, but there was an increase in the number of giant cells. The inflammatory reaction to 10 mg titanium phosphate/kg was less severe than to 50 mg titanium phosphate/kg. By 8 weeks there was a granulomatous response consisting of mononuclear cells, giant cells containing the titanium phosphate fibers, and a minimal number of PMNs. The granulomatous inflammatory response focally obliterated the alveolar architecture. Asbestos Study Histopathological examination of sections of lungs from hamsters treated with asbestos revealed severe pulmonary edema and numerous PMNs within 24 hours. Throughout the first week the inflammatory reaction was diffuse and consisted predominantly of PMNs. After 2 weeks, foci of mononuclear cells, and PMNs, were present and by 8 weeks these inflammatory foci consisted of mononuclear cells with scattered lymphocytes, plasma cells, and golden structures interpreted as asbestos bodies. PHASE II - Long Term Study Using Rats and Hamsters Mortality - The mortality data on the rats on the long-term study are presented in Table I. The study was concluded after 17 months due to the increase in spontaneous deaths primarily caused by endemic chronic murine pneumonia. The number of rats surviving until the designated necropsy times are given in Table II. Several animals were sacrificed prior to their designated necropsy times due to injury and/or apparent ill health. The times at which these animals were necropsied are also included in Table II. The spontaneous mortality data on the hamsters on the long"term study is presented in Table III. The mortality data of the titanium phosphate and asbestos treated hamsters is similar to the saline controls. The random mortality distribution suggests that there is no direct correlation with treatment. The study was concluded at 18 months to be consistent with the long term rat study. The number of hamsters that survived until the designated necropsy times are given in Table IV. ST0000I 08 Body Weight - The terminal body weight data for the 6, 12, and 18 month necropsies are presented in Tables V (rats) and VI (hamsters). The statistically significant body weight differenc in tdie rat weights are interpreted as not of practical significance with respect to treatment. The male hamsters treated with 50 and 2 mg/kg titanium phosphate, and those treated with 10 mg/kg of asbestos and necropsied at 12 months had significantly decreased body weights. The females treated with 10 mg/kg asbestos had significantly increased body weights at the 6 month necropsy. The significant body weight differences in the hamster weights are interpreted as not being of practical significance as there was no dose response in the titanium phosphate treated males at 12 months and no significant body weight differences in either sex dosed with the titanium phosphate or asbestos at the 1718 month necropsies. Gross Pathology - The principal lesion seen grossly in the rats and hamsters treated with titanium phosphate, asbestos or saline solution and necropsied at the designated times consisted of focal consolidation of the lung (Table VII). Pulmonary consolidati was a consistent finding in control rats and reflected the high incidence of chronic murine respiratory disease in these animals. Consolidated pulmonary foci were not observed in hamsters treated with 10 or 2 mg/kg of titanium phosphate. Histopatholoqy Titanium Treated Animals - Histopathologically, the pulmonary response m rats to intratracheal injection of 50 mg titanium phosphate/kg at 6 months consisted of multirfocal granulomas, macrophages and giant cells containing titanium phosphate fibers with scattered PMNs. The areas of inflammation were discrete foci that obliterate the alveolar architecture. The granulomas did not contain collagen. Scattered focal areas of atelectasis containing titanium phosphate fibers were observed. The inflammatory response at the 10 and 2 mg/kg levels consisted of scattered macrophages and giant cells, containing the titaniusjo phosphate fibers, within the alveoli and alveolar wall in the --! area of the terminal respiratory bronchiole. The alveolar architecture was not destroyed. O O CD The reaction to the titanium phosphate was essentially the same C3 12 and 17 months as at 6 months. At 17 months there was minimal amount of collagen within the focal granulomas at-the -" CD 50 mg/kg level. The titanium phosphate fibers did not appear to be readily cleared from the lung as evidenced by their presence at the 10 and 2 mg/kg levels after 17 months. At 6 months and throughout the remainder of the study, titanium phosphate fibers were present within macrophages of the mediastinal lymph nodes of the rat. There was no proliferative or inflammatory response to these engulfed fibers. -7- The spontaneous rat neoplasms are tabulated in Table VIII. No neoplasms were associated with the intratracheal titanium phosphate treatment in the rat. Histopathologically, the pulmonary response to titanium phosphate in the hamster was similar for the 6, 12, and 18 month test period. The response was mononuclear in nature and not unlike that observed at 8 weeks. The titanium phosphate was present within macrophages and giant cells and focally the granulomatous reaction at the 50 mg/kg dose obliterated alveolar architecture. The reaction at the 50 mg/kg dose was more diffusely scattered throughout the lung, whereas, the reaction at the 10 and 2 mg/kg doses was located mainly in the areas of the terminal respiratory bronchioles. The titanium phosphate fibers did not appear to be readily removed from the hamster lung, but were, rather, engulfed by macrophages and sequestered in the lung. There was no evidence of a fibrogenic response or collagen deposition in relation to the titanium phosphate fibers. After 8 weeks, scattered macrophages containing the titanium phosphate fibers were found in the mediastinal lymph nodes. No proliferative reaction was associated with this finding throughout the 18 month test period. The spontaneously occurring neoplasms in the hamster are given in Table IX. No treatment related neoplasms were observed. Asbestos Treated Animals - Histopathological examination of the lungs of the rats treated with asbestos and necropsied after 6 months showed scattered circumscribed collagenous-nodules and a collagenous thickening of alveolar walls throughout the lungs but more commonly near terminal respiratory bronchioles. Associated with the areas of collagen deposition were asbestos bodies and scattered mononuclear cells. Morphologically, the lesions in the lungs were the same at 12 and 17 months. Asbestos produced no lesions in the mediastinal lymph nodes. Spontaneously occurring rat neoplasms are tabulated in Table VIII. There were no neoplasms induced by the intratracheal injection of asbestos in the rat. Histopathologically, the hamster treated with asbestos and G__Oj necropsied at 6 and 12 months showed multifocal areas, mainly r--> near terminal respiratory bronchioles, of mononuclear cells wit^o a few PMNs and asbestos bodies. A scant amount of collagen i--> deposition was associated with the mononuclear reaction.- At o 18 months there was a slight increase in the amount of collagen -- present within the inflammatory foci, but the reaction was -- predominantly mononuclear. Asbestos bodies or inflammatory fociO were not observed within the mediastinal lymph nodes. The spontaneously occurring neoplasms in the hamster are given in Table IX. No neoplasms were associated with treatment. Saline Solution Treated Animals - Histopathologically, the rats treated with saline solution showed the existence of chronic murine respiratory disease. The spontaneous lesions, -8- which were found in all groups of rats from 6 months on, consisted of peribronchiole and perivascular lymphoid collections throughout the lung. There were focal areas of hyperplasia of the bronchiole mucosa with mucus and PMNs on the mucosa and in the lumen. Occasionally there were focal areas of PMNs and mucus in the alveoli. Much of the interstitial tissue of the alveolar walls was thickened with mononuclear cells. Spontaneously occurring neoplasms in the rats are given in Table IX. Through the 18 month test period, a minimal number of control hamsters treated with saline solution had small foci of PMNs C/3 scattered within and on the bronchial mucosa. Occasionally a 6animal would have peribronchial lymphoid infiltration. No neoplasms were observed among this group of hamsters. o o INTRAPERITONEAL INJECTION STUDY o Preparations Administered to Animals Suspensions of titanium phosphate and asbestos were prepared with 0.85% saline solution so that 0.5 ml of each would deliver 10 and 2 mg/kg body weight of the animals, respectively. Experimental Design The Intraperitoneal Injection Study was designed to follow the course of the response to titanium phosphate and asbestos when suspensions of these materials were injected into rats and hamsters. A group of rats and hamsters that were left untreated served as control animals. The design of the Intraperitoneal Injection Study has been summarized in Chart II. Experimental Procedure The rats and hamsters on the intraperitoneal injection study received one injection of either a sterile suspension of titanium phosphate or asbestos in saline solution. A group of untreated rats and hamsters were used as controls. The rats were housed three/cage and the hamsters were housed five/cage. Standard laboratory chow and water were made accessible to the animals at all times. Spontaneous deaths were recorded. At the designated necropsy times, the animals were killed by decapitation and examined for gross pathological changes. The organs and tissue saved from these animals and the histological techniques used were the same as described for the intratracheal injection study. Results Mortality - The spontaneous mortality of the rats on the intraperitoneal injection study are given in Table X. The study -9- was concluded in the 17th month because of the increased incidence of spontaneous deaths. The major cause of spontaneous death was endemic chronic murine pneumonia. Due to the increased mortality among the control rats during the 13-17 months of the study, no definitive statement relating mortality to treatment can be made. The number of rats surviving until the designated necropsy times are given in Table XI. The spontaneous mortality data on the hamsters on the intraperitoneal injection study is given in Table XII. There ^ was no excess mortality associated with the titanium phosphate__^ or asbestos treatments. The number of rats surviving until th^ designated necropsy times are given in Table XIII. q Gross Pathology O O Titanium Phosphate Treated Animals - Gross pathological -- examination of the titanium phosphatetreated rats revealed ro scattered abdominal adhesions and white raised foci on the abdominal viscera. The gross observations were similar at each necropsy from 6 weeks to 17 months. Gross pathological examination of the titanium phosphate treated hamsters revealed minimal scattered abdominal adhesions and grey-white, slightly elevated foci on abdominal viscera. The gross observations were similar at all the various necropsy times. Asbestos Treated Animals - Gross examination of the asbestos treated rats revealed scattered abdominal adhesions and smooth, slightly elevated white foci. A number of rats that-were necropsied at 12 and 17 months and those dying spontaneously during that period of time had white nodular masses throughout the serosal surface of the abdominal cavity. Gross examination of the asbestos intraperitoneally treated hamsters revealed minimal abdominal adhesions and slightly raised scattered white foci on visceral surfaces. The gross observations were similar at each necropsy from 6 weeks to 18 months. Histopathology Titanium Phosphate Treated Animals - Histopathologically, the titanium fibers invoked a granulomatous foreign body inflammator response in the omentum and on the serosal surface of the abdominal viscera in the rat. The inflammatory reaction consisted of giant cells and macrophages containing the titanium phosphate fibers, along with scattered plasma cells, lymphocytes and occasionally focal axeas of PMNs. The response was similar for each necropsy interval and there was very little collagen deposition present. There were no titanium phosphate treatment-related neoplasms in the rat. A male rat necropsied at 17 months had a pulmonary -10- reticulum cell sarcoma and another male rat had a subcutaneous fibroma of the axilla (Table XIV). Histopathologically, the inflammatory reaction in the hamster induced by the titanium phosphate fibers at 18 months was similar to that seen at six weeks. Titanium phosphate invoked a granulomatous foreign body reaction characterized by the fibers being present within giant cells surrounded by macrophages, and scattered plasma cells, lymphocytes and occasionally focal areas of PMNs. The granulomatous reactions were not encapsulated and there was very minimal collagen deposition. One male hamster at 10 mg/kg, had a leiomyosarcoma of the small intestine after 12 months on test. Malignancy was based on the extreme anaplastic nature of the cells in conjunction with several mitotic figures. The neoplasm appeared to have originated in the smooth muscle of the intestinal wall. The leiomyosarcoma was interpreted as not being related to treatment. A female that received 10 mg/kg titanium phosphate died after 14 months on test with generalized reticulum cell sarcoma, a spontaneous neoplasm (Table XV). Asbestos Treated Animals - Histopathological examination of the rats necropsied throughout the test period revealed asbestos produced collagenous nodules and plaques on the abdominal serosal surfaces. Asbestos bodies and scattered mononuclear cells were associated with the areas of collagen deposition. l/> In addition to the inflammatory reaction, 8 male and 6 female O rats had diffuse abdominal mesotheliomas that were interpreted ^ as being related to treatment. The first mesothelioma was observed after 8 months on test (Table XIV). ^ All of the mesotheliomas were predominantly of a mesenchymal "T type with oval to spindle shaped cells. Several mesotheliomas had tubulopapillary areas with epithelial type cells. Mitoses were common and areas of collagen were present within the neoplasms. The mesotheliomas tended to spread along serosal surfaces with infiltration of contiguous structures. Occasionally liver metastasis were found. Histopathologically, the lesions in the hamster produced by asbestos consisted of mature collagen surrounded by a minimal number of mononuclear cells, plasma cells and lymphocytes. Asbestos bodies were observed embedded in the collagenous foci. The fibrogenic response produced by asbestos in the peritoneal cavity of the hamster was not severe or progressive in nature. No neoplasms were observed in the asbestos intraperitoneally treated hamsters(Table XV). No neoplasms were observed in the control animals (Tables XIV and XV). -11- REFERENCES 1. Gross, P. Westrick, M. L., McNerney J. M. Progression of experimental silicosis in the rat. Industrial Hygiene J., 19 :201-204-158. 2. Gross, P. and deTreville, R. T. Experimental asbestosis. Arch. Environ. Health, 15, 638-649, 1967. ST0000 I I h -12- BIBLIOGRAPHY Churg, J., Rosen, S. H., and Moolten, S. Histological characteristic of mesothelioma associated with asbestos. Annals New York Academy of Sciences, 132, 614-622, 1965. Faulds, J. s. and Steward, M. J. Carcinoma of the lung in hematite workers. J. Path. Bact., 72, 353, 1956. Gross, p. Chronic pathologic responses of the lung to inhaled materials. Paper presented before Gordon Research Conference, Toxicology and Safety Evaluations Session, July 30, 1969, Meriden, New Hampshire. Harington, J. s. Chemical studies of asbestos. Annals New York Academy of Sciences, 132, 31-47, 1965. Harington, J. s., Roe, F. J. C. Studies of carcinogenesis of asbestos fibers and their natural oils. Annals New York Academy of Sciences, 132, 439-449, 1965. McCaughey, W. T. E. Criteria for diagnosis of diffuse mesothelial tumors. Annals New York Academy of Sciences, 132, 603-613, 1965. ------ Wagner, J. C. Experimental production of mesothelial tumors of the pleura by implantation of dusts in laboratory animals. Nature, 196: 180, 1962. S T 0000I15 exper im en tal desig n of the t it a n iu m phosphate intratracheal in je c t io n study u >1 <N to 0U >. 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V -U n 10 rl i. 0) 0 4-> (0 V A cn JSL 0) c tH pH 10 w 0E o n n (N irt in w r> vo pH cn r* TABLE I I ne in in th S T 0000I19 c* C 0 H 0 C TJ E n v in in n in ^ + cn in bob >3a* *0H) cn CpMH a VO XL hU XL r- *> *T EW> u o0 Q) pH 01 2 C 01 E 2 H M Eh ou rcHn -h <T5 H oc> Hu cO' 3 -H 3 2 H H co -a u 3 -a tj sJ A* H H 1H <*H C O -H O 1C lH E (0 u<8 0) &H 2 in vo in + in uo VO in vo m in 0u) W a oi n o ? SO C U h O 2OJ 0o1 tj mCL, 0181 U0 0in1 C UO oca E ce -a 2 C EWh g aO Eh z r1H0 *"c(THHnJ *<h3m CN ^CN (N ^CN ^CN 'CTN WhpH npH Wvf--i ipnH WmpH vrHo **r CN CN wpCHN OpHV CN CN cw\ rmH N 01 e O E c rH fa c >1 VO O w < 2o wO 22 CO a. in 0 a ai lH 0 o U M 01 O Eh e JChO X u 0 >H TJ a, 01 SIuSL-Sb 2 fa 2 fa OH 0 01 oBO w OH o TJ *H O 0) *0 uh u 2 O' Xy* ra 0) JO H -H n O 01 JO tfl "v OL Oin O pH CN CN & <8 <8 E to CO aE pH iH H (0 (8 (8 a H(H8-t T0J1 jHoji Vaf-i' * Hoa-- n <8 C 2w H E- CO 0 CO a (0 < 0cH1 pH C1/83 EE H H *H CCC (8 18 O 01 01 ^ OC C rf inn h ib n h in HT <N >a3H Eh V5 n Z o > u au> UEMh c HI <N H H H ^ N N rt n cn o PJ Z QJ s H 4 Eh <W XU< IX E- 2 EZ >JV0J tOfli C 10 O' - mH -H CN o a Hi -l O O Ph O (N O Hf u N O H rl H O O hH Eh ST0000I 20 WW J X Eh Eh Z o Xuw Eh \ >H f--t I--I Hf Hf ^ rT <N<N<NCN<NIN IB O' O' ID <f if HT Hf wCN CN O <*1 m CN <N \r- \r~ Cu O >< Eh J <Eh Cu Cu Cu Cu Cu o 3w ouz IS O s TABLE IV STOOOOI 2 I a Z CM uEU* o 2M <w ou w zU0) 4cJ n i/UTMr n r N m 'T mm uE <H0 Ct-N4 C z uu <sr a tn Ec<:- 36 C VO VO ID IP ^ t vo vo m 20 2e- E VO 2 u H\ u 'O 2 OJ 0H) 0 ato,4aj u z E-i z c ocn: U0 0<0) a wi OH riHn ^W <3 E TT 5T Tf (WM CN (\N \M \N (\M co un rH in pvHo o OJ o f-H rs CM \-^T \.H i-H iH <*Nr CmN \NpvHO Ewt2fl < 6H -hC c< < o K ZO 2 OCu W w 2w X QJ 2 b 2 b 2 b 2 b 2 b E-* t/J > o 10 c 2M . ob n H E* CO 4oJ a> 10 oj cH CO H ifl <w TERMINAL MEAN BODY WEIGHTS OF RATS ON THE LONG TERM INTRATRACHEAL INJECTION STUDY u 0) -- > 2 O' (fl a o 'O Mo Om 0) T a* 4J 4-1 co o U U) a 2 to a 0 T3 uO o CQ ai O (N <J\ CM +1 m m n* V0 4J 4-4 c0 0 Vj 01 O 4J CM i 3 Z m ^ r* in i--i (Nr*-cm *tr o + l+ha o o in o GO CM 10 in m ^m m in rM CO CM in V0 +i m CN ov HJ O' H 01 >, 2 W a. O' 0 o uO oa 01 2 10 o CN (N 4- I O O o m in 4J 4-1 co U (fl 0) 4J 'O IS 3 Z in VO *9* GO H 4T VO o cd in niom +1+1+1 oo o O CO ^O O .-4 V0 tt in in m m in o c* ID cn +i o m m in in X 0) C/1 0) O' O' O' 01 O' 10 m H 0 01 Ji c a O Ji 1 \w 4J \ Ua ai 4J o c H pH O O' O' O' oi O' Cu E E E ai E 10 c <0 H O O CM W CM <0 CO &- in h < * rn or^H 00 CM in cv co CM CM 01 10 10 in mm 4-1 o + 1+1+1 OO O +1 o 01 o o 3 Hin r* ^ cn CM 01 VO COON CN CM cm n n m a > m CM CM oij jj c m oo oe <r> c\ VO CM +1 o CM o CM in n h co omn o^^ nui^ +1+1+1 oo o n co tt * in ao rHO^f mmm in in 10 in co m +i r^* (N in m 10 4-1 001) 4-> C4-> T031 3 4J C/1 o o CM +i o tT ID CM J* CO in oo co -h n- m *-M CM -M +1+1+1 ooo in 'c id nh C** CM pH cm n n ^ il fN | r* I<n, +1 ; m m 4J cf0-- 4Ht4l H c O' u 0) Cl CM T3 (0 J4 o> H 14 in vo in in V0 0) 4J 01 (0 Cb tb Cb Cb Cb oci OX' MO' JOO ' o O' o> o' a- e e e -(0u0 O' J\i O' a> 6 10 m H O O fSI 01 CM V) E- lf> ~t < 4WJ 3 4J HO' 0O) j<: oi 3 u10 o E0 oi ST0000I 22 TABLE V I Xa D M-> OZt-t t* uu -3 z < w u<a (-1 $ h z K U O Zo _3 U P z o e: u E- W E < b. c 3 (H u 2 X o o03 z < < z M E a w U x: O' H X a> in s o a 0 >i u 'O 00 a; ca z 4J U-l c 0 in ou E u a) 0 4J 30 tn fH E E 3 re ZX VO r> +i 0 H in i-4 r-4 VO HJ O' > 0) ~ to S tJ a o >1 u TD 00 0) c z JJ V_) C0 ou E U CJ QJ 4J cv J3 03 H1 E 3 flj zx Ol CO r* +i o VO oi 'ey pH in Cl ^ Cl Ol 00 Ol r- r* cn H <N H + 1+1+1 r* o tn ny 04 co n m oj pH pH pH MTIO pH O 00 VO O 03 pH tn vo co pH pH pH + 1+1+1 omo CD OJ m ci in o^h pH pH pH in Tr *<r co Cl in pH +1 o ^y m 04 pH m * VO TT pH Ol +1 VO 04 Ol pH m jj O' h X 0) -- to 2 O' a oX u o o0 0) n z 4J 4H c 0 (0 0x Euo 0) Jj C i 3 ZX o VO o <N +i O CO --H m pH VO X <D E W - oo ro ^ on * VO vo fO pH 04 04 +1+1+1 o oo in ro ro co cm in 04 co rpH pH pH VO VO VO EEE pH r*^ +i o ro in p> i t , ro i f ; rH a O' O' O' tn O' ro tn XXX O JX H 0 0 *\\\ 4J \ ua c O O' O' O' tn O' <U '#4 eu E E E V E 4J 0 pH PI re c pH O O 04 tn o E re w Hinn *G --* i pH pH o pH +l O' vo VO Ol pH O <N ro co in ro ro fl'HH + 1+1+1 in in in 04 O' 04 * oj r* O' 04 04 04 pH pH pH ro 04 pH If) o o v a tn o 3 rrHe > VO 04 pH 04 04 +i O Ol o 04 pH m vo ro Ol pH +1 o vo in pH in ^ CO ^ *r pH ro o co oO CI * pH pH O' + 1+1+1 0030 o vo m ro m m OHO pH pH pH in ro --' w om CO 03 Ol * Ci vo ro Ol pH ro + 1+1+1 ooo in 'T in > <rtf O CO ny Cl vc in ^ pH o 'T ro pH +1 in O' * o pH pH TT -- ro VC O' +1 ro ro in H o u XJ c o o E oJ-l Hx tn 0) jj XJ (0 c <D >a 3 XJ M >i XJ C C(3J u-i C O' tn a) T3 ittn tn H X VC o XJ trne x jj c Ui Ui Cu fu O' O' O' tn O' J* J* 0 JX o \s\ XJ \ c O O' O' O' tn O' H a- E E E a) E pH -) CO H O O Ol r* m pH tn < poH T3 i<tU X re E tn <y 3 re > (/> O CD O O ro co TABLE VII INCIDENCE OF PULMONARY CONSOLIDATION IN RATS AND HAMSTERS ON THE LONG TERM INTRATRACHEAL INJECTION STUDY Material and Dose Saline i Ti3PO. 50 mg/kg 10 mg/kg 2 mg/kg Asbestos 2 mg/kg Animal1 Rat 6 Month T---- IT Month- Necropsy Necropsy 0/10* 5/8 17 Month Necropsy 4/4 5/11 3/10 1/10 5/10 ! i i j 4/7 2/10 4/6 5/10 2/2 3/4 6/8 7/8 Saline Hamster 0/11* 0/10 -- TijPO. 50 mg/kg 10 mg/kg 2 mg/kg ! Asbestos 10 mg/kg 1 j 1 1/12 0/11 i 0/10 i i 1/9 i 2/10 0/7 0/6 1/9 -- -- -- 1Sexes combined. *Number of observations/number of animals examined. 18 Monthj Necropsy ! : -- ___ 1/12 6/11 j 0/13 ; 0/14 ; j 1/6 CO o o o o PO -er TABLE VIII NEOPLASMS IN RATS ON LONG TERM INTRATRACHEAL INJECTION STUDY Material Injected Ti3(PO,,) ,, I j ! !1 Asbestos Saline Dose mg/kg 50 10 2 10 Total # Rats Sex Examined* M 11 F 13 M F M F M F M F 1 14 15 12 16 12 19 9 13 Neoplasm Classification (2)1 - Reticulum cell sarcoma (9 months and 10 months)2 (1) - Cortical adenocarcinoma of adrenal (12 months) (1) - Carcinoma of thyroid (17 months) (1) - Uterine myoma (16 months) (1) - Diffuse lymphosarcoma (17 months) (1) - Pheochromocytoma (17 months) -- (2) - Fibroadenoma of mammary gland (17 months) (1) - Abdominal carcinoma with lung metastases (11 months) (1) - Adenocarcinoma of mammary gland (16 months) ST0000I 25 *Necropsied or dying spontaneously from 6th-17th month of- test. Number in parenthesis indicates the number of rats with the indicated neoplasm. lumbers in parenthesis indicate month after treatment in which observation was made. TABLE IX NEOPLASMS IN HAMSTERS ON LONG TERM INTRATRACHEAL INJECTION STUDY o ro cn Total Number Material Dose Hamsters Iniected mg/kq Sex Examined* Neoplasm Classification Ti3(P04)4 50 M 20 (1)'-Islet cell adenoma o pancreas (18 month)3 (1)-Pheochromocytoma (18 month) F 17 10 M 17 (1)-Hemangioendotheloma o spleen (6 month) (1)-Pheochromocytoma (18 month) F 19 (1)-Islet cell adenoma of pancreas (18 month) (1)-Undifferentiated sar- coma of abdomen (18 month) 2M 21 F 14 (1)-Adenocarcinoma of uterus (12 month) Asbestos 10 M F 15 (1)-Pheochromocytoma (18 month) (1)-Adrenal cortical adenocarcinoma (18 montl 15 Saline M 18 F 16 *Necropsied or dying spontaneously from 6th-18th month of test. 'Number in parenthesis indicates the number of hamsters with the indicated neoplasm. aNumbers in parenthesis indicate month after treatment in which observation vras made. zoMfuu* zM j c a; fca, 5 X zM oz CEOi $ fa O H f<Ha O DOwzCO H< zo a. to STUDY r- ro O' CO o> fca fa S pH (0 (0 ,fca >u QI1Q1 9Uwc(oQ ifQCal> fEfealJ* Nfra1C MO ^ 'f MM *C0caOj f0a fufOoiCuaOaa' e vX0fi1l O O OO O fa faa> D rzS3Q .Xtr 3 \0 m iH nh ro <n pH f0a \JfcaSn 'O f<caQj w 9o fma IQ fa cfao co fa \o \ vO m CfaO \\ CO pH <N <N \\ in H pH pH W4 Z fa fa fa o <3 II TABLE ST0000 I 27 pH o fam V * iH o 0 faV Q) cr- n <Q H M CO o 4J CO o X W __________ pH o fvai c 0 u ST0000I 28 0 a o o r~ H rl U CO O t-4 & 0) Ha 0a h n vo in in n to rN XQ H U rH1s0 (0 z oM Eu< 0 13 a 0) vo H in in wW ("3 a z U o 0 u< zn a z o W 4J -n VO VO VO VO E-i (0 K es W VW 0 X E-> o s u z z in to ht to in in > u E-i H Zs o 13 0) to P TJ E-> W (U 3 a jj u0 V H3 0u) oDu at H z w VO VO \n \n VO VO \mm\ 'C \cm \cm CO w H (N vo o\ m gM Eh rH 10 cm m IT3 a aH H c m cm h X c< < C&O o 0z z Uu z X a< AA m d) > w 4J 0 e (N <N (N pH CN CN wDH Q (0 Hc <r-*1* OMz EUU-i l z 3 0) 0 0c 4(0J HB H a 0 g c 0 tao 4VJ 1C3 vC U1 o CN om o O'.* <a zo UH H ? o 0) o r* a H 0) Q ,g S-l %3 0) 3 o -U ron om Z OQ) r- rH c ra -<J3 iUo E 0 CH fr< o a) cCn u^A z3 X a/ b w e v^ nSo 6 oH QE in <n n \\ in vP pH \ b 00) x M CN T3 * ^ ** 1H3 U 4VJ U * bo 0J 1) *--' 4J 1- n <3 C iH Hh cn 0 4J cn 01 A cn _______ pH u0 A 3 0 O ro pH in TABLE X III ST0000I 30 O in Hr e jfjlj fN <--i in + PO ++ po in ha VH -- a>* ian o OE z H XOI Oi cn 0) z z o W) -I o H E UE wn cpt CO z tf Xi 0) O V C JX O 4J Ec -rt u *r >, pH a) iaon sx u x> o D cc H U T03) 10M -vCwIJ TcJ; H ^ U >m| U ft !d cn CO 10-1) <t a 5 -> CO j= 4J IT3 c .c ro o a pH to x: jj g W E-1 to s < aion j<a->o> oO mm rr in in m <r CHO wII) r <D CP c JC C o 4J >H E A AO \ VD \ro \C7NN \ o ra CM CM rH IN pH E > C -H -C *H > 4J Xl >1 3 pH CCOO C>O A O A U W Z Ec cH < < zo zo Cl. Cl. T3 u a KoH C c *H lH o TC130 *0 v u 03 4 -<xi OJ *p| J H 0) Xl E > a> us a C3 <o in jC ooc C50 pH -O <0 V * CO 0 O -> E - fw M JJ o 4J 0 Xi u a, (0 lH 0) HI <U 4J Jj n .3 c <0 c H (0 0 M Eh <u TABLE XIV NEOPLASMS IN RATS ON INTRAPERITONEAL INJECTION STUDY Material Iniected Ti3 PO*)^ Dose Number Rats mq/kq Sex Examined* Neoplasm Classification 10 M 21 (1)1-Pulmonary reticulum cell sarcoma (17 month) (1)-Subcutaneous fibroma F 23 of axilla (17 month) -- Asbestos 2 M F 20 21 Control M9 F 13 (8)-Diffuse abdominal mesothelioma (l-10mo.; 3-11 mo.; 1-14 mo.; 3-16 mo.) (6)-Diffuse abdominal mesothelioma (1-8 mo.; 2-12 mo.; 3-16 mo.) (1)-Generalized reticulum cell sarcoma (8 month) -- } Necropsied or dying spontaneously from 6-18 months on test. 1Number in parenthesis indicates the number of rats with the indicated neoplasms. 2Numbers in parenthesis indicate month after treatment in which observation was made. oo o CD CD CD CO TABLE XV NEOPLASMS IN HAMSTERS ON INTRAPERITONEAL INJECTION STUDY Material Iniected Ti3(P04)4 Dose mqAa 10 Sex M No. Hamsters Examined* 18 F 19 Neoplasm Classification (1)`-Leiomyosarcoma of small intestine (12 month)2 (1)-Reticulum cell sarcoma (14 month) Asbestos Control 2M F Mix 13 20 11 ---- -- *Necropsied or dying spontaneously from 6th-18th month of test. `Numbers in parenthesis indicate the number of hamsters with the indicated neoplasms. 2Numbers in parenthesis indicate month after treatment in which observation was made. GO O O o o CO ro Toxicology Contact - H. C. Spenc I? THE DOW CHEMICAL COMPANY BIOCHEMICAL RESEARCH LAS. MIDLAND. MICHIGAN <U QUEST FOR TOXICOLOGICAL STUDIES _________mi bloc. "T5IT53; ' DD-54 I file n !t34.19-DD-54-( C OF MATERIAL to ac testeo Titanium Phosphate - TlTfPO.^A JUSMfTTED RT [iW| Present contact - R. G. Heitz ' _________ -P.--B.--AXC- __________ SuFCIvisO* T|0b* ** 12&L Western Division 'PROJECT J*Nlr cOOROimaTCR V. K. Rowe C&TitE&TEO C01T iho.ooo _________ I L. L. Pitchforth i; account LEDCERi^AXll^iJIWaBI Wl. i! Western PO 7448^ FROILCM ttO. ..l TOTOBXEICUOSLEODGYBT 99 810.00 5 ----------2 09 DO not write below this line complete all INFORMATION cn Rf'ERSE ROUTE OF aOmiaiJT#*TlOM Single dose TP and Intratracheal LENGTH OF TfaTu|nT Tnuantir to ~2 vea*rs---------------------------- ("rTTN F rabbit ,a6B,T Df guinea p,c Of NOTES, CHANGES. ADVERSE EFFECTS NOTED 7-31-6Q: To date, eroups of rata and hamsters. injected IT or IP, have been necropsied 6 weeks ^ 6. Q or 12 months following treatment. 18 k 24-raonth necropsies are scheduled. DOG D" f MONKEY D" Df (maMSTeS) Om Bf OTHER DOSli 0, 50, 10, 2 mg/kg CHECK POINT AMPLE RECIVED TOKlCOLOCr 1 j DATE 1967 9-15-69: l(V13/69: 11/4/69: Animals continue on expt. as schedule Animals continue on expt. as scneauiet "Epidemic" of pneumonia has developed in the past week, with considerable mortality. Tetracycline is being; admi lstered in drinking water. Susan McCollister """" TWE ESTIMATED REPORTED DATE REVISED ESTIMATE REPORTED ! S/68_______ 1 1/19,20/69 - Due to the high mortality, it was decided 1970 J that there would not be enough animals to continue the I i experiment as originally scheduled. Therefore, the REPORT CRAFTED REPORT ISSUED ______________FATE OF SAMPLE RETURNED OESTROYEO REFERENCE SAMPLE STORED I REFERENCE SAMPLE TO K STORAGE NOTES DATE animals are being necropsied these dates (approximately 18 months following treatment). Susan McCollister 12/8/69 - Tissues are being processed for slide preparation. G. Sparschu and Susan McCollister 1/6/70 - Tissue preparation still in progress. Susan McCollister 2/13/70 - No Change . Susan McCollister - --r ^15/70 No Change. Susan McCollister tn O -oo 4/12/70 - No Change. Susan McCollister __ ----------------------------------------------~----- ------------------------------------------ p in 0TI PROJECT COuPlCTCO FORM C-JJ'TO MimTEO N u.L*. Ml SAMPLE INFORMATION Titanium Phosphate MOLECULAR FORMULA -Tl,gQj< STRUCTURAL 'OlAUUCl COMPOSITION MOLECULAR Cl&HT "^amPlE SkE'EilftdE. SOURCE an OR iCEnTi^CaI'O* * : ESTIMATED plRiTt of sample SOILING PC'NT ax mm *i < A. . ^ g I CORtOJ'vENESS '* ce**** **ij o iN 0. o | CHEMICAL REACTIVITY o- w2 I* jTaHli Tl Ini. j SAMPLE AMALFI | RESULTS [yIyes . EXPLOSIVE LtMl TS -V-OL-iUTM- E IN AJR) I Imp -F-LASH POINT See Pile tCHlTrOM |* mP. H PHYSICAL state c (5!. 'MMiilL TING POINT 1 i *c "cSToS" .late ._ , ,!.- yfcobl'7.^W I .n hG 25C p to>coio?'C:< itvdies Aee4*4* IacIh** stoqe o* Vii^sn ipewr*' pioot*^ w*. iWd el Nm(W- De raw * e< oy ****# eW*c* *** This study was requested to evaluate the flbrogenlc potential of Tl, fPO*^. Included as controls are aabeatos and saline, oo o o o o CO ___; PROTOCOL COMPLETED ID ANIMALS ordered ID ANIMALS STARTED on test ! 1 NECROPSY DONE FQNN CM?fO PPlNTtO : VIA HI ! statistics oone ' CLINICAL TESTS REPORTED HEMATOLOGY TESTS REPORTED I I SLIDES STARTED D SLICES TO BE REACT I I SLIDES READ | MISTOPaTh reported ORA ALL OONE. REAOY TO WRITE (BACK) marc. !! && SIXTH DATE: ACC IN OAYSc STARVED, (0 0 , QO-$<f- u) ^LOOO COUNTS ( ) SOME MARCO* COUNTS ( > BIOCHEMICAL RESEARCH DEPARTMENT ANIMAL M.l MATERIAL: Y * - . - GROSS EXAMINATION f/O' DATE:' ' C0NHT1QN: OOY WTj . 6Rams KILLED iY DECAPITATION ( 1 OR EXAMINATION OF ORGANS: APPEARANCE OF VISCERA: TISSUE EXAMINATION $/b FILE NO. T EXPT. Ml SIMM. OATS ~^ MICROSCOPIC EXAMINATION FORMALIN! IOC DATE REMOVED FROM FIXATIVE____ EMREDOEDs PARAFFIN! IOE SECTIOMEDIY STAINS: HDUTOXTLM AMD EOSIN ( oc EXAMINATION of section* ) WED I KILLED ( ) WT. LUNG () Sjqqr\ OQA) TISSUE SAVED DEGREE OF DEG. SLIDE PREP. DEGREE OF DEG. VENTRAL DORSAL ume* HEART: i- - LIVER: EIONEY* SFLEE* ADRENALS: PANCREAS: TESTES. . DONE MACRO*: STOMACH: ' 3 '- LUNG: HEART: LIVER: HONEYS: SPLEEN: ADRENALS: PANCREAS: TESTES: (ONE MACRO*: STOMACH: T ^ -H Q CD O CD ----CO CTI DATED in*-* Pawns II a.s.1. SIGNED DATED SIGNED GROSS NECROPSY Animal #DD54 210 brain /' There was a subdgrl hemorrhage approximately 1/2 centimeter in diameter on the left mid-cerebrum. The left ear canal was filled with a yellowish-green somewhat caseous material which extended up into the brain following the route of the 8th nerve. Diagnosis - Otitis Interna. c/J o o o CD Philip B. Conran, D.V.M. October 9, 1968 MICROSCOPIC EXAMINATION Animal #68-427 brain This section of brain is taken through the hippocampus and foeally there is an area of acute necrosis characterized by coagulation necrosis and focal hemorrhage. Iasnedlately adjacent to this is a moderate amount of sub-dural hemorrhage. brain This section of brain is characterized by focal areas of hemorrhage and necrosis with occasional polymorphonuclear Inflammatory cells being noted. Pocally there appears to be a . thrombotic vessel. Immediately adjacent to this vessel are focal areas of hemorrhage. brain This section Of brain is characterized by having the presence of the third in the two lateral ventricles and suspected of being ante: to the hippocampus section. Focally there is a wedge shaped area of necrosis with pin point areas of hemorrhage. Associated with this area of necrosis, there is massive subdural hemorrhage. The distribution of the lesion is characteristic of possible vascular occlusion. Within the necrotic area, there are occasional polymorphonculear inflammatory cells noted, however for the most part the lesion is very acute and the tissue appears to have died in <ltu without inflammatory exudate. REPORT CONTINUED P P.B. Conran November 13, 1968 MICROSCOPIC EXAMINATION Page 2 Animal #68-427 (con't) brain brain This section of brain is taken through the hippocampus and again there is a focal area of necrosis with pinpoint hemorrhage. Within the central area of the necrotic area there is a thrombosed vessel. This section of brain is taken through the cerebellum and there appears to be severe congestion of the cerellar vessels. Pinal Diagnosis: Infarction, focal, hemorrhagic, acute, cerebrum, brain, due tc thrombosed veins. ~.iilip''B. Conran, D.V.M., November 13, i960 STOOOOI 37