Document EmDmL2G9ereY5dKXQbzOYMk90

FILE NAME: ALCOA (ALC) DATE: 1968 Jan DOC#: ALC007 DOCUMENT DESCRIPTION: Memo with Comments and Published Newspaper Article and Journal Articles PITTSBURGH OFFICE ROCKDALE WORKS COTIDENTIAL January 16, 1968 In connection with your letter at January 12, 1968, concerning employees who use an asbestos-Lunnlte ceaeat in crucible and trough linar rapair a, wa hava nada contact with Johns-Manville concarning thair No. 352 canant which wa uaa. Tha asbastoa conponent at this canant is cryaotila. It contains no crocidolita. Tha abova asbastoa, if inhalad ovar a large nunbar of years in axceasiva anonnts, will produce asbestosis. Tha currant Threshold Llnit Value is 5 Million parts par cubic foot for a weighted 8-hour exposure. There is at present controversy ovar tha allowable exposure to crocidolita, but since this is not involved in tha canant now being used, it does not enter into tha picture. Internit tent exposures to asbestos dust would not be expected to produce asbestosis unless the dally weighted average exceeds the TLV*s. Even so, asbestosis fron crysotile is not produced with in a short nunber of years nor is it possible to be exposed to enough dust during the handling of dirty clothes to give rise to a hazardous situation. In the repair of crucibles and troughs where asbestos dust is Involved, it is desirable to correct the condition, when practical, by exhaust ventilation. However, sone intermittent situations do not lend thenselves to this approach and respirators oust be worn. The respirators used for this purpose should be those approved by the Bureau of Mines for the protection against pneunoconiout producing dusts, that is those that would be adequate for pro tection against silica dust would also be editable for asbestos dust. The Martindale respirator is not recognised as being suit able in this application. While it is effective for nuisance dusts, it is not reconnended for either toxic or pneunoconiosisproducing dusts. Suitable respirators can be reconnended by your Safety Director. However, should you wish us to consent on a particular respirator, please let us know. Dr. Dawson will be in touch with Dr. Richards concerning this subject. One of the things that disturbs us is the abundance of incorrect infornation now being circulated in relation to . asbestosis. Probably the nost outstanding feature printed now in newspapers is cancer known as nesotheliona. Sone researchers believe that crocidollte is responsible for the nesothelionas However, there is not enough information presently available to establish such a relationship and we must await further investi gation. On the other hand, many of the so-called asbestos bodies which are found on autopsy are probably in no way related to asbestos. We now know that many fibers, organic or inorganic, can produce these bodies and they are more and wore being referred I PLAINTIFF' exhibit arch l8742 FROM H . A . SEMKEN ROCKDALE WORKS DR. L X y . CRALLEY MEDICAL DEPARTMENT -- P ITT SHJRGH\OFFICE Jan u ary 22, 1968 CONFIDENTIAL RE: POTROOM CRUCIBIE REPAIRS_____________________________________________ A ttached is a copy of th e newspaper a r t ic le re fe rre d to in our l e t t e r of Jan u ary 12 concerning employees who u sa^ a sb e sto s-lu m n ite cement) in c ru c ib le and trough lin e r re p a irs. Dr. R ichards is prepared to t a l k to th i s employee as soon as th e necessary arrangem ents can be made. H . A . SEMKEN HAS:S A ttach 0A LC O A AR CH 18744 ,w ancer Lause Puzzle to Doctors Dy THOMAS 0 TOOLE Washington Post WASHINGTON - A fatal cancer of the chest and abdomir:;'; cavities is cropping up among the nation's asbestos workers. So rare that 20 years ago it wasn't even in the medical textbooks. ' The cancer is called Mesothelioma, and seem s almost peculiar to people exposed to asbestos. Ironically, those stricken with the deadly disease include a nurse whose father worked in. an asbestos plant and a man who handled asbestos for no more than a month while he insulated the plumbing in his own house. Mesothelioma was first spotlighted in 1960, when South African doctor found 32 cases among the people living and working in the Cape of Good Hope asbestos fields. Alerted to the problem ,. this sam e doctor turned up 57 more cases in the next two years. Nobody knows how many Americans have had Mesothelioma. Even today doctors have trouble spotting it -- whether they're trying to diagnose it in a person still alive or pin it down at an autopsy. In three separate studies in the last three years, doctors in three states have turned up 71 cases. Oddly, 11 of them appeared to have had no real exposure to asbestos. The other 60 could ail be traced to asbestos contact, either because they worked with it, lived with people who did or lived in neighborhoods where the air was heavily laden with asbestos dust. The first American study w a s one run in 1964 by Dr. Irving J. Selkkoff of New York's Mount Sinai Hospital, who began the study to see if cancer in any way affected the lives of the asbestos insulation workers in the New York Metropolitan area. Tracing the lives of C32 m embers of Lie asbestos workers union who were on the union rolls before Jan. 1, 1943. Dr. Sclikoff found that 255 of them had died by Jan. 1, 1903. Some of what he found did not surprise him -- that 12 of the 235 died of asbestosis, a long-known but decreasingly fatal (due to antibiotics) disease brought on by years of breathing asbestos dust. Dr. Selikoff also found that 45 cf the mean had died of lung cancer, a rate 6.6 times higher than that in the normal adult male population. Even more surprising, he found five, deaths due to M esothelioma/ "This incidence of more than 1 pet cen t," said Dr. Selikoff, "is strikingly high for a tumor which is generally considered to be extremely rare." Dr. Selikoff then launched a follow-on s t u d y of 307. consecutive deaths among asbestos insulation workers throughout t h e w h o l e ' Northeast. Again, to his surprise, he turned up 10 deaths due to Mesothelioma. About the same time, the Pennsylvania State Department of Health began a. blind study to find how Mesothelioma m i g h t be affecting the more than 5000 asbestos workers scattered about the state. To 162 hospitals in the, Southeastern part of the stale they sent a questionnaire, asking the hospitals to report all Mesotheliomas diagnosed between 1958 and 1903. Back cam e 42 documented cases of the disease, only 11 of which could not be traced to asbestos exposure. Ten victims of the disease turned out to be men who worked in asbestos plants. Eight were people who lived near asbestos plants most of their lives. Three were members of families who had one cr more asbestos worker members -- a three-year-old girt whose- father was an asbestos engineer, a nurse whose father had worked for 35 years in an asbestos plant and a woman with two sons who worked as asbestos insulators in a shipyard. Of 10 deaths which seemed "questionable" a s b e s t o s exposures, two turned out to be almost bizarre. One was a Swiss cheesemaker, w h o worked almost his entire life around a large boiler covered with Asbestos insulation that flaked off at the touch. Another was a worker in a brewery, where it turned out that beer is filtered through pulp filters made essentially of cotton fiber and asbestos. The most convincing study is the most recent one -- conducted by a team of Middlesex General Hospital m , New Brunswick, N.J., a few miles from the nation's . .T upsdav, largest asbestos plant, one No state lists Uie disease m that employs 5000 workers. its workmen compensation In the past three years, no j fewer than 19 cases of Mesolheiioma passed through Middlesex General. Only two laws, so nobody afflicted with it can be paid damages for his affliction. Even now, the first test cases are in the courts, of the 19 were not workers at where they may conceivably , the nearby asbestos plant, one a woman who apparently inhaled asbestos dust in laundering her husband's work clothes, the second a spinster who lived one block from the plant. stay for the next five to 10years. The fact that nobody knows how much exposure to asbestos can bring on the disease h a s c r e a t e d controversy, too. A relatively "new" disease. Most laws stipulate that Mesothelioma is also one of the most difficult diseases to confront modem medicine. It is almost impossible to* treat. Middlesex General's Dr. asbestos workers be in an environment where there is no more than five million ., , Part,des oi asbestos-dust per Maxwell Borow says that even one cubic foot of air -- a the most potent anti-cancer regulation that many in the drugs have no effect on it. asbestos industry fought years About all that radiation. t0 set approved treatments do, he says, is Bat with Mesothelioma, the remove some of the fluid that disease might be brought on builds up in the lungs from, by exposure to as little as one- the disease. tenth or one-hundredth that "The disease U 100 percent "We're ref y over a fatal," Dr. Borow states, " and J?3115 on,,f!?IS one> slates one usually takes less than a vea: doctor' Because we dou t after diagnosis to run its know hw Jmuch exPsure ] causes the disease, we don't .1 know what to recommend." Nobody knows why tt Despite obstacles like these, attacks s o m e asbestos research has b in a workers and not others, or handfuI * places t0 (ind out why some victims of the more about the ailment. disease ,, get . . .it after Mount Sinai's Dr. ScU'-tvif is apparent^ mild exposure to now monUcrin,, ;lie asbestos. Tne University of 130Q members of the Aso, slc , Michigan School of Medicine w 0 r k e r s U n i o n in cites one case of a man who metropolitan Ne,v York. The was exposed to asbestos for Phiiade,phja Iocat of t!u- same about a month while h e ----- -- . . ............... insulated his house. Six years <union has started a similar later, he- was dead Mesothelioma. of program with Hahneman Hospital to uncover more Even the nature of the information, disease is little understood."1 _ While it's generally known that j _ M l d d I e 5 e x < * * * } s . f hr' itlhiae adiisceaJsel attacks the lliinniinnrg, Borow W * " t he is in the of the chest and abdom en,. process of setting up a nobody knows how it g e ts, traveling exhibit to bring there. j attention to the disease. He's When asbestos dust is doing it with, a 3500 dollar inhaled, the fibers get trapped grant from the Papermakers in the lungs, where they form Union, which represents the gummy d e p o s i t s . T h i s ! asbestos workers in the New explains how asbestosis gets Jersey plant Dr. Borow started, and perhaps e v e n . studied. It wasn't easy getting the lung cancer. One theory is _ __ . , t.1h-aft the gummy ,, , money. Dr.Borow went to 40 substance it that builds up around the organizations before he got it asbestos fiber lodged in the ' -- including the American lung can cause : Cancer Society and the Public cancer Health Service. elsewhere in the bod)'.* But Any attention the disease how docs it do it? Nobody : gets might come none too knows. ; j soon. More than 1 million tons Because it is such a new and little understood disease. of asbestos is consumed those Mesothelioma is a l c r e a i* , ^ 30fl(J ^ h?i,, , . !: ^rtedfor,hef3St`3ro'vin5 ARCH D e c . 21!- .la w 18745 / {(H ft'l y v .g Identification and Control of A sbestos Exposures LEWIS J. CRALLEY, Ph.D. Bureau of Occupational Safety and Healthy Public Health Service, Department of Health, Education and Welfare, 1014 Broadway, Cincinnati, Ohio 45202 tg Asbestos can be used safely in modern industrial technology if adequate pre cautions are taken to prevent excessive and unsuspected exposures. To distinguish between asbestos and other fibers, new techniques must be applied to electron micros copy and diffraction, emission and atomic absorption spectrophotometry, electron microprobe, and neutron activation analytical procedures. Standards and evaluation techniques should be based on air-borne fibers and the use of the membrane filter and phase contrast microscopy for sampling and counting. Controls should include procedures for the safe transport of asbestos; exhaust ventilation and personal pro tection a t work sites; the safe disposal of waste dusts; and the prevention of com munity contamination. 'T 'HE UNIQUE PHYSICAL and chemical _L characteristics of asbestos along with its abundance as a natural resource has con tributed much to technological advances since the beginning of the century. This is es pecially true of industrial fiber technology which has had a tremendous growth since the 1940's. From data developed through epidemiologic and laboratory research it is now apparent that special health problems may be associated with exposure to designated fibers and that the biologic response to some fibers may be quite distinct from that of particulates of the same chemical composition but of random spherical shape. The biological consequences of fibers in the lungs must be determined and understood for establishing rational control standards. It is equally important that knowledge of the control of fibers in our environment be de veloped and applied. Otherwise, adverse ex periences from health problems associated with tlie use of asbestos may inadvertently de prive us of the benefit of this natural resource and alscji cast a shadow that could impede future advancements in the application of fiber technology. Identification and Measurement o f Exposun Numerous analytical techniques are av able for identifying and distinguishing physical and chemical characteristics of lib Because of the minute size of individual l pirable fibers, often under 1 micron in ameter, and the similar characteristics many of the fibers, as in the amphib group, one technique by itself is seldom finitive in identifying individual fibers. The ubiquitous nature of respirable materials has been recently reported.1'3 eral fibers may be present and dominant ` number of ores of commercial value, various forms of asbestos, or may be pr in lesser but appreciable amounts in mercial minerals such as talcs and clays. No problem is generally involved in identification and measurement of air! levels of fibers associated with an indu process where they are a dominant part the mineral involved, such as asbestos, is not true in instances where the fib present to a lesser extent in the mineral 1 processed, where more than one type mineralogy may be present in the fora of the ore, or where mixtures of minerals -- be processed. Without such knowledge an Industrial Hygiene Association Journal 83 by definitive identification and mea- particle size ranges have been used to mea ^ ients, unsuspected hazards may be pres- sure asbestos dust levels. When such an in M from air-borne exposure to fibrous min- strument is used it is essential that the ratio ^Is. Conversely, the mere presence of fibers of the asbestos fibers to the over-all partic fcpot indicative that a hazard exists. ulates remain relatively constant. These in Analytical techniques useful in identifying struments should be frequently correlated and individual fibers include electron microscopy calibrated against data using the membrane jnd diffraction, emission and atomic ab filter and phase contrast microscopy method ruption spectrophotometry, electron micro- of fiber measurement. The main advantages niobe and laser microprobc in conjunction of the automated dust counters over the wjlli a time of flight mass spectrometer, and manual method are in their saving of neutron activation analytical procedures. The manpower and their capability of giving me of such techniques requires specialists in immediate data for assessing the proper func the instrumentation involved and is time con- tioning of dust control procedures. mning and costly. This data however, pro vides an indispensable bridge of knowledge on Co n tro l of Exposures tbe physical and chemical nature and rela Special precaution should be taken to pre tionship of fibers in the air at industrial opera vent exposure to asbestos fibers when they are tions. Less complicated methods of measure handled in a loose (unbound) form. Such ment can then be applied as an index of ex operations include mining, milling, shipping posure upon which rational standards can be and warehousing, processing into products, based. and mixing and blending asbestos fibers with This aspect of knowledge is especially im other materials. No special problem is nor portant since past research* has shown that mally encountered when the asbestos is firmly tos fibers differ greatly from one type Ether in their chemical composition and neous metals associated with the ores, bound with other materials, ie, vinyl asbestos tile, plastics, cement products, except when these materials are reshaped or are diminuted due to their hardness, the fibers may and the fibers may become uncoated, free, de and become contaminated with the tpd released through friction, destruction, de '1 alloys in which they are processed. The analytical techniques are useful in deitg the changing millieu of the fiber processing. terioration, and the like. The control of air-borne asbestos fiber is not greatly different from that of other par ticulates having a similar aerodynamic size, ch over the past several years in though special problems and technologies may Jnited Kingdom and the United States be involved in processing and handling the shown that air-borne fiber levels are a 1 fibers. The basic principles of preventing ex better index of exposure to asbestos posures to asbestos include exhaust ventilation, **an over-all particulate exposure since enclosure and isolation of exposures, wet atio of fibers to over-all particulates in methods, good housekeeping and mainte may vary greatly depending on the nance worker education, good work and sani of die process and the materials in- tation habits, and supplimentary personal res Revised standards for asbestos ex pirator protection where indicated. Asbestos in these countries4'5 also stipulate should be handled and processed with ade .ods for the collection and measurement quate controls to prevent community con r-borne samples. The procedures"'1 state tamination. .the samples should be collected on mem- Exhaust ventilation is the control of choice filters and, after subsequent treatment, since the asbestos fiber is captured before it ers should be counted using phase con- can disseminate and contaminate the im microscopy at 400X magnification. mediate and adjoining work areas. Require measuring dust counters that are ments have been established for the proper le of discriminating between different ventilation design, fiber capture and carrying An Experimental Approach to Study the Toxicity of Nonparticulate Air Pollutants I. R ationale and M ethods Ahmed N. M. Nasr, MD, PhD; Bertram D. Dinman, MD, ScD; and I. A. Bernstein, PhD, Ann Arbor, Mich This report describes an experimental m odel lor ctuciying the toxicity of nonparticulate air pollu tants, in terms of a 'biochem ical lesion.' Changes In chemical composition of lung tissue homoge nate, following inhalation of a noxious gas, could neither be traced to a particular tissue component, nor b e regarded as solitary unrelated incidents. Biochemical changes In a single kind o f cell, separated from the respiratory tract after the animal's exposure to poisonous gas, may be more directly related to the toxic mechanism of the investigated gas. A technique for isolating epi thelium lining the rat trachea has been adapted from a method used to separate tracheal and bronchial epithelfa from human autopsy material. Ozone was chosen a s test gas In this experimental approach because of its importance in photo chemical sm og and Its presence In certain in dustrial environments. T h i s IS th e first of two reports dealing with the developm ent of an experim ental model for the study of the toxicity of nonparticulate air pollutants. H ie second report deals with the effect of inhalation o f ozone Submitted for publication May 14, 1970; accepted June h From the Institute of Environmental and Indus trial Health, the University of Michigan, Ann Arbor. D m Nasr and Dinman are also affiliated with the Department of Internal Medicine, University of Michigan, Ann Arbor. Reprint requests to W5640 University Hospital, Ann Arbor, Mich 48104 (Dr. Nasr). on the levels of the coem ym e, nadide phos phate (N A D P ), in the tracheal mucosa of the rat. Experim ental studies on the toxicity of air pollutants have been largely lim ited to changes in structure and functions of the lungs and bronchial tree, mucous secretion and ciliary m otion, deposition and clearance mechanisms, and chem ical changes in lung tissue homogenates. Research tended to focus on those effects which were earnest to measure, often a t the expense of the m ore delicate and less detectable changes. The investigation of respiratory function, for example; has probably been overemphasized in comparison to other physiological and biochem ical param eters.1 Specific therapy or prophylaxis for diseases caused by toxic inhalants (and affecting the respiratory system ) is lacking, in spite of numerous investigations that have been con ducted in tins field. T he study of biochem ical changes in vari ous tissues has been one m eans o f elucidat ing disease mechanisms. In reference to air pollutants, som e investigators, eg, Scheel et al2 and B uell e t al,3 have described changes in the chem ical com position o f lung tissue hom ogenates follow ing inhalation of ozone. T he interpretation of their findings, in terms of the mechanism of toxicity o f ozone, pre sents several difficulties. T he lung, unlike Arch Enuiron Health--Vol 22, May 1971 fon BOO) (Jiff ena ves; 1 the et; ack tioi hie bio chi ica the al hui tio. pic fro * go; an toi dii ti> ba we pn cu tai oil an P* in of of fo ni w) al fe ar ai late grille, nadide phosxacheal mucosa of n the toxicity of air argely lim ited to i functions of the , mucous secretion ition and clearance al changes in lung a those effects which cn a t the expense of detectable changes. ratory function, for a overemphasized in logical and biochem- lylaxis for diseases (and affecting the taking, in spite of h at have been con- ia l changes in varimeans o f elucidatIn reference to air atom, eg, Scheel et ' described changes tion of lung tissue ihalation of ozone, r findings, in terms id ty of ozone, preT he lung, unlike NONPARTICULATE AIR POLLUTANTS--NASR E T AL 539 the liver, for example, is composed of a more heterogeneous assortment of cells and tis- f sues that are dissim ilar in structure as w ell ?as function. Thus, a change in the chem ical ? composition of lung tissue homogenates can ; neither be traced to a particular tissue com ponent, nor could it be regarded as a soli tary incident. I t may represent a profile of a multitude of changes, a "vector" of several forces operating sim ultaneously. Further more, unless the chem ical changes in the different components of a lung tissue homog enate are additive* they m ay elude the in vestigator by nullifying each other. The approach adopted by Spencer48 in the study of the role of smoking in the etiology of bronchial carcinoma has been adapted to the purpose of this investiga tion. H e attempted to detect precancerous `biochemical lesions' by the correlation of biochemical changes in tracheal and bron chial epithelia with sm oking habits and clin ical records. Spencer's approach* required the development of a method for the remov al of tracheal and bronchial epithelia from human autopsy specim ens. In this investiga tion his technique has been modified for the preparation of epithelial cell suspensions from the trachea of the rat. T his investigation was planned with two goals in mind. The first goal was to develop an experimental m odel for the study of the toxicity of air pollutants, in terms of `bio chemical pathology.' A second goal was to try to elucidate some of the aspects of the basic mechanism of ozone toxicity. Ozone was chosen as the test gas because of its prevalence under various environm ental cir cumstances, notably its prominence in cer tain kinds of `smog.' I t also shares w ith other air pollutants, eg, oxides of nitrogen and organic peroxide compounds, the two properties of being both an oxidant and an irritant. Ozone is an im portant component of the Los Angeles smog.7-8 During periods of high photochem ical activity, it accounts for 90% or more of the total oxidant, w hile nitrogen dioxide is the main component when photochem ical activity is low or absent.8 T he presence of ozone and its ef fects on human health in inert-gas-shielded arc welding have been described by several authors.9-10 M ethods Isolation of Tracheal Epithelium,--Thirtysix young, adult male rats of the CFN strain were used in this investigation. T he animals ranged in age from two to four months, and in weight from 200 to 395 gm. T he rats were killed by stunning, and the tracheae were im mediately excised through a midline longitudi nal incision. A stream of ice-odd isotonic sucrose-O.QlM edetic arid (EDTA) solution was squeezed into the trachael lumen from a plastic bottle through a fine-tipped nozzle, in order to remove the mucus and blood aspirated by some animals during death. The esophagus was then excised and the trachea slit open along its dorsal surface. The trachea was then trimmed and further cleaned of mucus in a P etri dish, using a soft camel-hair brush and ice-cold su crose-edetic acid solution. T he technique used for separation of the epithelial cells lining the trachea was adapted from the one reported by Spencer.* A large rubber stopper 4 inches in diameter, covered w ith filter paper, was used (Fig 1). The open trachea was fixed on the rubber stopper with insect pins, the trough formed facing upwards. Small volumes of ice-cold sucrose-edetic acid solution, 0.01 to 0.02 ml, were instilled on the mucosal surface through disposable capillaryglass micropipets. The epithelium was brought into suspension using a No. 2 camel-hair brush, w ith bristles trimmed to a length of approxi m ately 4 mm. The sequential steps of instilling sucrose-edetic acid solution, brushing off the epithelium, and pipetting the cell suspension out of the trachea were repeated u ntil a total volume of 0 2 to 0.5 ml had accumulated. The sides, as well as the bottom, of the tracheal trough were brushed in order to harvest the greatest number of cells. The following procedure was used for cell counting: After repeated pasting of the cell suspension in a microsyringe, 0.03 ml of 0.1% methylene blue in the isotonic sucrose-edetic acid solution, pH 7.4, was added to an equal volume of epithelial suspension in the same medium. In order to achieve thorough mixing and further cell dispersion, the contents of the tube were again passed several times through a microsyringe. Counts of nuclei were made in a hemacytometer chamber in the same fashion as in counting white blood cells. T he standard error of counting was estimated by the method of Berkson et ah11 Exposure iff Animals to Ozone.--The ozone exposure apparatus is illustrated in Fig 2. Ozone was generated from pure oxygen (U ltra High P urity Oxygen) obtained from a commercial Arch Environ Health--Vol 22, May 1971 540 NONPARTICULATE A IR POLLUTANTS--NASR E T AL insect pin trachea 7" filter paper -- rubber stopper fig 1.-- Apparatus used for iso. lation of epithelial cells from the trachea of the rat. fig 2.-- Diagram of the ozone exposure apparatus, showing ozone generator (A), bell jar (IS), mixing vessel with glass beads (C), and animal Inhalation chamber (D). manufacturer. Pure oxygen was preferred to air for the purpose of generating ozone in order to avoid simultaneous generation of nitrogen oxides (from the nitrogen in the air). Svirbely and Saltzman1* reported th at substances associated with ozone production from air (including ox ides of nitrogen) are unlikely to influence the toxicity of ozone in laboratory animals. Since this study was planned to investigate some as pects of the basic mechanism of injury by ozone, and as an experimental model for tox icity studies of other inhalants, the above-men tioned precaution was taken. An ozone generator (Welzone R-2), labeled A in Fig 2, was obtained from a commercial manufacturer. I t was housed in a glass bell ja r (B in Fig 2) tightly fitting over a circular glass plate, on which the ozonator was placed. The inflow of pure oxygen to the bell ja r was controlled by a two-stage valve regulator con nected to the oxygen cylinder and measured with a gas flowmeter. Ozonized oxygen flowed through flexible tub ing from the bell ja r to a spherical mixing vessel (C in Fig 2). This vessel is 2,000 m l in capacity and was filled with glass beads 6 nrm in diameter. Filtered partially-dried a ir was introduced into the mixing vessel through an other in let This arrangem ent enhanced effi cient mixing of air and ozonized oxygen. A stopcocked outlet, through which the air-oxy gen-ozone m ixture flowed to the animal inhala tion chamber (B in Fig 2), was built in the mixing vessel a t a level higher than the upper most surface of the glass beads. The air used for diluting ozonized oxygen was supplied from a compressed-air line. I t was passed through activated charcoal to remove any m ineral oil that might have contaminated it from the com pressor, and through silica gel to dry it to a relative humidity of 40% to 50%. H ie a ir was then filtered through a membrane filter, 0.8a in pore size. A glass desiccator, 8,000 ml in capacity, was used as an animal inhalation chamber. A thick sheet of aluminum, with several hides drilled Arch Environ Health--Vol 22, M ay 1971 num sh nniT**al and hel ^Tbe op ' fitted w ; entered ,, gtopcocl which placed, infialati and Ga Vinyl m ateria tire ap) chfunbe. cury thsured v industri humidit and we' were o compan (Coffin' Sixto the ra (N A D I (NADI animals control while 1 337 1 at rand hour to ture, su rate of minute, centrati timi chi fered-p< S altzm t Duri became soon tt aminat ozone hemorr effect c concen ination after e: vealed ed with AL NONPARTICVLATE AIR POLLUTANTS--NASR E T AL 541 --Apparatus used for iso. epithelial cells from the if the rat. ito it, was placed on the protrusions in the ide of the glass desiccator approximately 4 aches above its bottom. This perforated alumi num sheet served as a platform on which the animal was placed during exposure to ozone, and helped to separate the ra t from its excreta. The opening in the lid of the desiccator was r fitted with a glass-to-glass cover through which V entered inlet and outlet tubes equipped with : stopcocks. A ir flowed from the outlet of file T he extent of removal of the epithelium by this method was studied by examining histological sections of th e trachea before and after separation of the cells, fig u re 5 illustrates the appearance of the trachea of a normal rat. Figures 6 and 7 show the ap pearance of the trachea after the epithelium had bean removed by the brush method. H ie submucosal layer remained intact. T he ab .' inhalation chamber into a laboratory hood in sence of contam ination of the epithelial cell I which the whole exposure apparatus was placed. The use of a glass desiccator as an inhalation chamber was adapted from Diggle and Gage.13 Vinyl tubing (Tygon) was used as th e tubing material for flexible connections in the en tire apparatus. Tem perature in the inhalation chamber was measured w ith an ordinary mer cury thermometer. Relative humidity was mea suspension with connective tissue was veri fied by phase-contrast m icroscopic examina tion of unstained cell smears and examina tion of stained preparations. T he appearance of the cells soon after they were separated is illustrated in F ig 8. The brush technique resulted in removal of sheets and clumps of epithelium. Repeated passing of the cell sus sured with an indicator m anufactured by an pension in a micropipet effected partial sepa ii-a industrial instrum ent company. T he relative ration of the cell dumps. Repeated passing humidity indicator was calibrated w ith a dry through the orifice of a microsyringe resulted and wet bulb thermometer. A ll gas flowmeters were obtained from a commercial scientific company and calibrated with a spirometer (Collin's). Sixteen animals were used for comparison of toe ratio of reduced nadide phosphate (NADPH) to oxidized nadide phosphate (NADP*) in normal animals to th a t ratio in animals poisoned with ozone. The weight of the control animals (mean SE) was 331 20 gm, in better separation of the cells. Figure 9 demonstrates the effect of repeated passing of the cell suspension through the orifice of a micropipet, while Fig 10 shows the results of using a microsyringe for the same purpose. Considerable breakage of the cells took place when the microsyringe was used to disperse the dumps. The cells were ground in tissue homogenizer in preparation for the assay of while th at of the experimental group was N A D P+ and N A D PH . Figure 11 illustrates 337 14 gm. Exposure to ozone was assigned their appearance after hom ogenization. > a spherical mixing vessel is 2,000 ml in ith glass beads 6 mm rtially-dried air was ;g vessel through au gment enhanced effi- ozonized oxygen. A a which the air-oxy- at random. The animals were exposed for 1 hour to 33 ppm ozone in an air-oxygen mix ture, supplied to the inhalation chamber a t a rate of 1,000 m l air and 50 m l okygen per minute. Samples for analysis of the ozone con centration were taken a t the inlet of the inhala tion chamber and analyzed by the neutral buffered-potassium iodide method described by Saltzman.14 T he technique developed for the assay of coenzyme made a total cell count neither feasible nor essential. However, it was con sidered desirable to obtain an estim ate of the total number of cells isolated from one trachea. Such an estim ate was only approxi m ate, since com plete separation of the cell clumps into individual cells was not at to the anim al inhala2), was built in the -gher than the upperbeads. The air used en was supplied from was passed through tove any m ineral oil ated it from the coma gel to dry it to a to 50%. The a ir was imbrane filter, 0.8/ in Results , During the exposure period the anim als became lethargic after a few m inutes, and soon their breathing was labored. Gross ex amination of the lungs after inhalation of ozone revealed congestion and subpleural hemorrhages. Figures 3 and 4 illustrate the effect on the lungs of ozone inhalation a t a concentration of 33 ppm. H istological exam tained. T he number of cells that could be isolated from a trachea was of the order of 1.56 X 10 2% (mean C V ). In this study the coefficient of variation is the standard error, rather than the standard deviation, calculated as percentage of the m ean. For the purpose of th is investigation, we considered th is way of expressing the coefficient of variation more appropriate for assessm ent of reproducibility. ination of the trachea, excised im m ediately ) ml in capacity, was ion chamber. A thick several holes drilled after exposure of the anim al to ozone, re vealed no abnormality that could be detect ed with the light microscope. Comment The relevance of studying the epithelium Arch Environ Health--Vol 22, M ay 1971 NONPARTICULATE AIR POLLUTANTS--NASR E T AL Fig 3.-- Lung of a normal rat (reduced from X 160). Fig 6.-- Transverse section in trachea of normal rat, after removal of the epithelium by brush method. A part of the mucosa, from which ciliated epithelium was not removed, is shown (reduced from X 400). Fig 7.-- Transverse section in trachea of normal rat after removal of the epithelium by brush method. The submucosal layer is intact (slightly reduced from x 400). I Fig 9.-- Epittu [of the rat. Cell [times through a Fig 4.-- Lung of a rat which inhaled ozone for 1 hour a t a concentration of 33 ppm (reduced from x 160). Fig 5.-- Transverse section in the trachea of a nor mal rat (reduced from X 400). of the trachea could be questioned, since serious injury to the respiratory tract results from the pulmonary rather than the tracheal lesions. Tracheal epithelium is one of the earlier targets of contact with a highly reac tive inhalant It is biochem ically active; it secretes mucus and transports it through ciliary motion. Thus, although tracheal epi-j Arch Environ Health--Vol 22, May 1971 ilium is no' ten total ini Ived in imp ainst toxic i the trachea an contamc NONPARTICULATE A IR POLLUTANTS--NASR ET AL in trachea of normal lium by brush method. iich ciliated epithelium educed from X 400). in trachea of normal lium by brush method, (slightly reduced from Fig 10.-- Epithelial cells isolated from the trachea of the rat. Cell suspension had been passed several times through a microsyringe (reduced from x 400). Fig 8.-- Epithelial cells isolated from the trachea the rat (slightly reduced from x 640). Fig 9.-- Epithelial cells isolated from the trachea the rat. Cell suspension had been passed several nes through a micropipet (reduced from X 640). Fig 11.-- Epithelial cells isolated from the trachea of the rat. Cell suspension had been ground in a tissue homogenizer (reduced from x 400). ; questioned, since <ratory tract results :r than the trached ium is one of the with a highly reaclem ically active; 8 asports it through lough tracheal cp*; ments. D etection of a `biochemical lesion' in such a preparation may thus be more mean ingful with respect to the basic mechanism of in ju ry caused by th e inhalant in question. ilium is not the site of maximal damage ten fatal intoxication takes place, it is in bred in important mechanisms of defense ainst toxic inhalants. Also, the epithelium the trachea can be isolated, relatively free an contamination by other cellular ele- This investigation was supported in part by re search grant UI-00450 bom the National Institutes at Health, by general research grant 6-SO1-FR-05447, bran NXH to the School of Public Health, the Uni versity of Michigan, and by American Cancer So ciety institutional grant IN-40H. The experiments reported in this investigation are part of a thesis submitted by Dr. N asr to tbe Uni versity of Michigan in partial fulfillment of the re quirements fra the degree of doctor of philosophy in industrial health. Arch Environ Health--Vol 22, May 1971