Document DdDJrE6RvMK9RB8MnQEmXVJgN

1 11 *0 C CO I B II ce v. ss 5C5-fOcC s *o.2 **->** *g :1 cKo 0.0 !i 4| |1l1 o Cu sM? 6.s_s -i E JSJS H 2 Buccal j1 cavity i n1 74 5 $ 2 3 8 e 3 L ;2 8 :2 " i c x > d d o i d d o * - ~ ~ I S S 3S Sa!53SJ8g 5!'32S235USS r oo i - t- t - oo d cti oo no !>.' 'iSSSSSSgfeS-S_i I l;85SfiP .SS S8ffi 2 = = d -S i2 = 2 2 2 :-:?!? = h S g g S S S -J=2 sd " =2ES!= II! i1'11ji1lu ! 38SU S5283S2S sissisU SsisssiJisi S ?.?S 8o*oS 2S S 1-: X 1-. - ^ / / / d d ?1 n i 5i sig a s s s s f c s . t F . K si ?? ? ? j ?i ?{ S S S S S JS S S E S U RS3R3U388SS S'?gS =^3??S! ansssssssss ssssststesils i^srssusssss I N1 II ip*> ssssa ssssss 22222228823 i i iis 2 ? S S ? g $ 3 ':5 3 5 5 S .O *0 .C ^ d .C C.o d d i 1 fjt ! i--i--1-' SV sSSSK K aK Sil: 3.o* c 1 r ??,=2ris5!g'-g2 22322^ ^o2'5'3--3--=2223'2-i -SSSSSSSSSRS !3 83S S3?$S2i2;S S 2 ssiri3 a3 n ?ia?i 5 !:S 3 f!!;2 JS 8 5 2222^232222 s a a a a ssissR s M rtC O M nK M K K 3333S33S335 - * 3 -:m- -- I N1 l-iiipi |ii it S feS S saaaS ftS S i^2 2 2 3 2 2 2 2 g E irs s g '-s g k s ed dddi-w dcrxoe srssa sssssK 83S3S3SSS38S3S3S3S3 lf^ S '2 ? S S g S 2 n c-i c-i c-i d d d d d d d d 3- 3'32- 8- J35- J2:J: 88'-S<3 gi: =Sa?3!eSS8 55 -S S S 8 3 8 8 S 3 iili223SS2S SU3S383SSSS83 as iiiii-iiii MiMMiii I ii L FEDERAL SECURITY AGENCY U. S. PUBLIC HEALTH SERVICE Public Health Bulletin No. 253 l/' THE RELATIVE TOXICITY OF LEAD AND SOME OF ITS COMMON COMPOUNDS By L A W R E N C E T . F A IR H A L L , P rin cip al In d u stria l T oxicologist and R. R. SA Y E R S, S en io r S u rg eo n W ith a sectio n on P ath o lo g y by J . W . M iller, P ath o lo g ist F rom th e D ivision o f In d u stria l H ygiene National Institute o f Health P R E P A R E D HY D IR E C T IO N OK T H K S U R G E O N G E N E R A L UNITED STATES GOVERNMENT PRINTING OFFICE W A S H I N G T O N : 1910 9m aale by th e S u p e rin te n d e n t o f D o c u m e n ts, W a sh in g to n , I). C. Price 25 cenOt i !/ iI ORGANIZATION OF THE NATIONAL INSTITUTE OF HEALTH T h o m a s P a it r a n , Surgeon General, United States Public Health Service L. R . T h o m p s o n , Director, National Institute of Health Division of Hiologics Control C h i e f , S e n i o r S i n g . W . 'I '. H a r r i s o n . Division of Chemistry-- C h i e f , P r o f . C . S . H u d s o n . Division of Infectious Diseases-- C h i e f , S e n i o r S u r g . R . E . 1 ) y e k . Division of In d u stria l Hygiene- C h ie f , S e n i o r S u r g . R . R . S a y e r s . Division of Pathology- C h i e f , S u r g . R . 1 ). L i l l i e . Division of Pharmacology-- A c t i n g C h i e f , C h i e f P h a r m a c o l o g i s t M . I. S m i t h . Division of Public Health M ethods-- C h ie f , (5. S t . J . P e r r o t t . Division o f Zoology C h i e f , P r o f . W i l l a r d H . W r i o h t . National Cancer In stitu te C h i e f , P h a r m a c o l o g i s t D i r e c t o r C a r l Vo e o t l i n . HU) CONTENTS I n t r o d u c t i o n ________ ______________ _____ _____ ______ _______ ____________ - . . Industrial e x p o su re to le a d . ____ -- ......................... .. F acto rs i n f l u e n c i n g t h e a b s o r p t i o n o f l e a d . . ..................... ...... ........... E x p e r im e n ta l p r o c e d u r e . ............. ..... ........................................ ........... ............ - C h em ical m e t h o d s o f a n a l y s i s ......... .. ...................................... - -- E xperim ental re s u lts ___________ . -- .. - ................. S u m m a r y ...... ......................... . . .................. - ................................. ......... B ib lio g r a p h y ___________________ ______ _ - . -- ------ ----------------- -- -- -------- A c k n o w le d g m e n ts . . ........ .. Pathologic c h a n g e s in d u c e d by le a d a n d b y so m e of its c o m p o u n d s by J . W. M i l l e r ______________ __________ __________ __________________________ (Vj ] `auc I 3 "> 7 11 12 2K 30 32 33 THE RELATIVE TOXICITY OF LEAD AND SOME OF ITS COMMON COMPOUNDS INTRODUCTION The economic importance of lead and many of its compounds is such that the degree of industrial exposure is of hygienic significance. The extent to which exposure may occur may be inferred from the figures for the annual production of lead and from the diversity of its industrial application. The amount of refined primary pig lead available for consumption in the United States during 1937 amounted to 452,129 short tons (1), while the total consumption exceeds this figure owing to the return of secondary lead from lead-consuming industries. The amount of secondary lead recovered in the United States in 1937 amounted to 275,100 short tons (2). The amounts of lend used by various industries in the United States during 1937 were as follows (3): Purpose: White lead__________ Red lead and litharge, Storage batteries. _ Cable covering__ __ Building________ ____ A u to m o b ile s_________ Railway equipm ent. Shipbuilding. __ Lend C07Isumrd ishorl tons) 80, 000 . 57, 000 192, 000 . 93, 000 . 45, 000 12, 000 3, 800 300 Ammunition____ __ T erneplate.. _ .__ ___ Foil_______ . 39, 500 6 , 400 21, 700 Bearing metal, , Solder___ Type metal C alk in g .. C a s ti n g s ___ __ Other lines___ _______ . 15, 000 22, 000 17, 000 15,000 6 , 000 . 50,000 081, 700 Mere examination of the major industries in which lend is used, however, does not properly convey an idea of the varieties of lead compounds used, nor of the most usual type of exposure. M any lead compounds have application beyond their usual specific use. Lend peroxide is used in the match industry as well as in the manufacture (1) I of accumulators; lend chromate is used in pyrotechnics as well as in the pigment and textile industries; lead sulphate is used in the manu facture of leaded zinc oxide as well as in the manufacture of paints. In general, however, workers are exposed to lead fume and dust in the melting of lead, to lead carbonate, lead sulphate, red lead, and lead chromate in the pigment industry, to lead arsenate dust in the manufacture and use of insecticides, to lead silicate in the pottery industry, to litharge, red lead, and lend peroxide in the storage battery industry, and to the sulphide, carbonate, and oxide of lead in the mining and milling of lead ore. During the past 2 or 3 years the field and laboratory studies under taken by the Division of Industrial Hygiene of the National Institute of Health have revealed a lack of definite information concerning the relative toxicity of various lead compounds used widely in industry. Therefore in the fall of 1930 we began a study of the toxicity of lead and of the following lead compounds: Lead arsenate, lead carbonate, lend chromate, lead monoxide, red lead, lead peroxide, lead phosphate, lead silicate, lead sulphate, and lead sulphide. Exposure to lead and its compounds takes various forms. In industry in general we are accustomed to consider the dusty trades as the most hazardous with respect to lead poisoning and doubtless a greater number of cases of lead poisoning arise from this source than from any other. However, a certain amount of lead inevitably enters the system by mouth and comparison of poisoning by the two different routes serves the useful purpose of placing the emphasis where it is necessary in prevention and control. It was felt further more. that further light would be thrown on the absorption and path ological behavior of lead and certain of its compounds by a study of its effects when injected intraperitoneally, since in our past studies of fibrosis-producing dusts (4, 5) much information was secured from the biological response of these dusts. It was anticipated that a study of this nature would give basic information necessary for the improvement of methods of control in indust ry. INDUSTRIAL EXPOSURE TO LEAD The mode of entry of lend into the system in industrial exposure which is chiefly associated with lead poisoning is now generally con ceded to occur through the respiratory tract rather than through the alimentary tract and controversy over this point has largely died out. Formerly, however, it was held th at the greatest danger of exposure to lead in industry was th at arising from lead carried into the digestive system by eating with dirty hands, smoking at work, or chewing tobacco. One of the chief proponents of the latter theory, Sir Thomas Oliver, while agreeing th at some of the finer particulate material en tered the lungs, was of the opinion th at most of the dust breathed in is held by the mouth and throat (6), swallowed, and thus enters the digestive tract. This opinion has also been held by several German authorities. The experiments of Lehmann, Saito and Gfriirer (7) showed th at during breathing only 12 percent of the lead reaches the lungs and that about 70 percent is found in the alimentary canal. This degree of retention compares favorably with th at found by Sayers, Fieldner, Ynnt, and Thomas (8). The latter found th at the average retention was slightly less than 15 percent of the lead dust in the inhaled air. Experiments of Legge and Goadbv (9), however, showed th at the rate, of poisoning by inhalation of dust is greatly in excess of the rate of poisoning by feeding, even where poisoning by feeding actually occurred. Later work by Aub and his collaborators (10) has also shown that the inhalation of lead dust constitutes the most frequent type of exposure. The preponderance of opinion at the present time may be said to consider that lead intoxication by inhalation is the most serious in industry. The prevailing ideas concerning the relative degree <>j Uuirity of various lead compounds are less clear than that of mode of entry. Examination of the literature for many years back shows th at a con siderable degree of confusion has prevailed. Some have maintained that lead compounds are all equally toxic, others that while some are admittedly toxic, other compounds are much less, if at all, toxic. Rambousek (11), an outstanding authority on problems of industrial hygiene, considered that the degree of toxicity depended upon the solubility of the salt in water and in hydrochloric acid. On this basis, "lead acetate, lead carbonate, lead oxide, lead dross, and minium arc relatively the most poisonous. Lead sulphate and lead iodide are relatively less poisonous, although bv no means innocuous. The least Is OUT'1 in i Si 4 poisonous, if not altogether innocuous, is sulphide of lead, because it is an insoluble salt." Again (ib., p. 293) Rambousek states, "as lead sulphide is the only nonpoisonous lead compound it is a duty to take advantage of this fact for purposes of lead prophylaxis." In Occupa tion and Health, of the International Labour Office (12), the following statem ent concerning lead sulphide is made: "Statistics relating to several thousands of workers in the mines of Pribam, and recorded over a period of 10 years, tend to show complete absence of cases of colic and only a few of gastritis and constipation." According to M urray (13), carbonate and oxide ores are much more apt to cause poisoning than sulphide ores. Hanzlik and Presho (14) found th at equivalent amounts of lead chloride, iodide, sulphide, car bonate, or acetate were less toxic for pigeons than metallic lead. Hanzlik (15) further concluded th at the beneficial therapeutic effect of magnesium sulphate and of calcium sulphide was due to the di minished solubility of the lead in their presence and hence diminished absorption. Blum (16) states th at solubility in water alone is not the only criterion of lead poisoning since lead oxide and lead iodide are in soluble and produce lead poisoning. On the other hand, lead sulphide is insoluble both in water and (according to the information available to Blum at that time) in the digestive juices. The innocuous nature of lead sulphide has been vigorously con tested by Alice Hamilton (17), who states: "This (lead sulphide) was long considered quite harmless, but we know now that it can be ab sorbed by the human stomach and set up poisoning." Or. Hamilton considers (ibid., p. 13) that in addition to solubility the physical character of the lead compound (i. e., particle size) must receive consideration since one compound may be more soluble than another and dose for dose more poisonous, yet if its physical charac ter is such that it does not remain suspended in air, it may be less harmful. It has been affirmed that, because of its insolubility, lead sulphate should he substituted for white lead and that the lead in this form is nontoxic. This somewhat prevalent idea has been contested by several investigations. Oliver (18) stated: "In my own experiments 1 found lead earbonate to be more toxic, than sulphate. Goadby found cats more readily poisoned by lead sulphate than by carbonate. Blum, on the other band, found sulphate of lead to be less poisonous than the carbonate. Carlson administered lead sulphate paint dust and basic lead carbonate paint dust in meat to dogs, and be found that the dogs which received lead carbonate paint dust developed severe symptoms of lead poisoning within 24 to 48 hours after the first feeding; whereas dogs fed upon sulphate paint dust did not show symptoms of poisoning until from 72 to 96 hours afterwards. 5 Cats were exposed to a similar ordeal, with the result th at lead carbonate and lead carbonate paint dust were found by Carlson to be more distinctly poisonous than basic lead sulphate and lead sulphate paint dust. 1 have given the details of these experiments owing to the controversial opinion expressed by various writers in regard to the comparative toxic values of lend carbonate and lead sulphate." Comparatively recent work in this field has shown th at there is but little toxic difference between these two compounds. Lehmann (19) states that lend sulphate is similar to white lead in the dosage and length of time necessary to cause poisoning and death. Koelsch, Lederer, and Koelsch (20) carried out both feeding and inhalation animal experiments with the two commercial lend com pounds. No significant difference in relative toxicity of the two substances could be found. They state that the commercial de scription of white lead sulphate as "safe and practically non-poisonous," or as "relatively harmless" is unjustifiable. FA CTO RS IN FLU EN CIN G T H E A B SO RPTIO N OF LEAD The purpose of the following investigation was to determine whether lead or some of its compounds showed variations in toxicity. Three phases of the problem were explored- -toxicity by ingestion, by inhalation, and by intrnperitoneal injection. As stated above, opinions heretofore have been various with respect, to the relative toxicity of lead and its compounds. Legge and Goadby (21), further in this regard, state that, "All compounds of lead are not poisonous in the same degree; the more easily soluble compounds are more poisonous than the less soluble * * *. The fineness of division in which the compound of lead exists is another factor affecting its poisonous nature; the more finely divided particles find their wav into the lung more easily than the coarser particles." T hat differences in toxicity are associated with variation in particle size and in solubility is the opinion of a number of investigators in this field so (hat if, may be said that the bulk of considered opinion at the present time is that toxicity is related to both solubility and particle size. In considering solubility, however, it is not sufficient to consider solubility of the compound in water, for its solubility in the tissue fluid of the lung may be far different from its solubility in water alone. For instance, if we consider the solubility of lead oxide in th at form known as litharge, it has been found that, if dissolves to the extent of 1.1620 grams per liter at 25 in blood serum (22), while its solubility in water alone at the same temperature is only .0171 grams per liter, or practically only 1 percent of its solubility in blood serum. This it i (i however, is on1v a special case and does not apparently apply to such compounds as lead sulphide or lend chromate. Furthermore, solubility of the various lead compounds so far as intoxication hv inhalation is concerned is of more importance than the size of the particles. Relatively large amounts of lead compounds of a material of very small particle size (such as lead phosphate') may he retained in the lungs with small or only slow signs of intoxi cation (see table 5 and fig. 8). Because of this low degree of solubility the substance does not show the degree of toxicity which we find associated with some of the other lead compounds. A further effect, which is not necessarily related to the degree of solubility, although in many instances this appears to he the case, is that of rule <>{ eolation. The solution rate with respect to many lead compounds would seem to he of considerable importance from the point of view of toxicity. When the solution rate is relatively high, the system is flooded with lead beyond the point of physiological tolerance and pathological changes follow. With a low solution rate, on the other hand, the system has an opportunity to immobilize the load to some extent by storage or to remove it by excretion so that it is not surprising that some slowly soluble lend compounds show an apparent low degree of toxicity. The rate of solution of particulate material in contact with the tissue fluid of the lung is not only a function of the particle size, i. e., surface, but depends also upon the amount of solid already present in solution. As ordinarily considered the solution of a solid involves two processes: (1) the reaction between the solvent and solid, which takes into account hydration, or the degree of solvation, and (2) the rate of diffusion of the solute away from the solid. Dust or fume in such a physiological site as the lungs presents further differences with respect to solubility to that of a solute in pure water. The tissue fluid not only contains a variety of dissolved substances, but in a few cases contains an ion in common with that of the lead compound, as in such cases as lead carbonate and lead phosphate. In addition to this there is a high degree of phagocytic activity to be taken into consideration. To what extent peptization occurs, or to what extent solution of the lend compound occurs due to the formation of easily soluble, undissociated or molecular lead compounds, owing to the presence of organic acids in the cell secretion, is not known. Certainly all these factors must be taken into account and it is to be hoped that future investigation will throw light on the mechanism of this important phase of haul absorption. I! ( EX PERIM EN TA L PR O C ED U RE (iuinea pips wore iisot1 f-liroiiplioiif as experimental animals. Some experiments with respect to inhalation were made with white rats in the later stapes of the inhalation experiments, hot these were dis continued in favor of guinea pigs as experimental animals. The guinea pig is sensitive to the toxic action of many substances, and perhaps often too sensitive to epizootic diseases, but for the purposes of this investigation it seemed more suitable than many other of the usual laboratory animals. Data were secured with respect to the weight changes of these animals as well as changes in blood morphology and the common signs of lead intoxication. At death the pathological changes, the blood calcium content and distribution of lead in the tissues were ascertained. huji'xtuin. For the ingestion experiments the difficulty arose of feeding a known (but very small) quantity of lead compound to each animal each day. This was obviated by diluting a known amount of the lead compound with flour, so that a scoopful represented the requisite am ount of lead compound, and after moistening with a drop or so of water rolling this up in a small piece of lettuce leaf. Each group of animals was provided with a box having a series of compart ments, so th at each animal could he fed in his individual compart ment. In this way it could be ascertained whether or not each an imal had consumed his daily dose of lead compound. Inhalation. For the inhalation of lead the dosage had to be arrived nt. in a more indirect manner than in the ingestion experiments. For this purpose the length of time of exposure was noted and the dust or fume content of the air breathed by the animals was determined hv analysis. The inhalation animals were exposed in groups to lead fume or dust in exposure chambers of the type shown in figure I. The front nf the exposure chamber was made of heavy glass fitted against a soft rubber gasket and was easily removable for purposes of cleaning or of transferring the animals. 'Phe dusting apparatus was th at designed by P. A. Sanitary Engineer .1. M. DallaValle (23) and the arrangements for exposure and sampling were carried out in consultation with him. This apparatus yielded finely particulate dust of uniform size at a steady rate (fig. 2). (Aimpressed air, in a number of cases dried by means of a calcium chloride tower, nnd measured with a flow meter, was led into the glass con tainer holding the lead compound through a cone-shaped apparatus from the bottom of which four very small tubes carried the air up I 8 through the lend compound. A cyclonic effect was thus imparted and the dust was carried up into an elutriating device whence it proceeded to the exposure chamber arranged beneath. In the case of lead arsenate, and to some extent lead monoxide, trouble was experienced in obtaining a dusty atmosphere, or, having once obtained it, the dust settled very rapidly. In spite of prolonged heating, drying, and fine grinding in a ball mill the lead arsenate proved especially refractory. Dried air blown through the powdered material seemed to push through without carrying dust into the air. Grounding the lead arsenate apparatus in order to carry off the elec trical charge afforded no improvement. Hence the lead arsenate in halation experiment failed to yield the results which might have been anticipated from heavier exposure. None the less, sufficient exposure was secured for some comparison with the other lead compounds. The lead compounds used were of reagent quality and with the exception of the lead silicate were chemically pure compounds. The lead silicate used in the injection experiments was chemically pure, while for the inhalation experiments a commercial lead frit was used. With respect to the sampling of the dusty air, the arrangement shown in figure 3 gave very satisfactory resultsi. Samples of one ii I' t'^Y< !) cubic foot in volume ns measured by n meter in each case were drawn through filter paper discs and the latter analyzed for lead. With this type of apparatus therefore a degree of control could be exercised conveniently with respect to the aerial lead concentration, ns well as control with respect to particle size. For the formation and control of lead fume the following apparatus was devised: An arc enclosed in a glass balloon (fig. 4) was struck between two carbon electrodes, the lower one of which had been drilled out and filled with metallic lead. Direct current (110 volts) was found to give a steadier arc than alternating current. Nitrogen gas was lead into the apparatus at (A) and air was added at (13). While it is commonly thought th at oxidation of lead fume is quite rapid, under the condition of this experiment the lead was carried along as particulate metallic lead, and the particles were very small TO PUMP F k ju re 3.-- D ia g ra m .showing a rra n g e m e n t for sam p lin g d u sty air. in size. That the particulate material was metallic lead was shown by treatment with diluted silver nitrate solution. Observation under the microscope showed a minute silver tree forming from each par ticle, clearly indicating that the particles consisted of metallic lead and not oxide. The weight changes of these animals were recorded and they were observed, as in the case of the other experimental animals, for sym p toms of lead poisoning. Particle size measurements were made of samples of the various compounds taken at intervals in which the method of examination of Bloomfield and DallaValle (24) was used. Owing to the very fine state of subdivision of metallic lead it was difficult to measure the size of these particles. The Owens' jet ap paratus is out of the question for lead fume owing to the very rapid transformation of the lead to lead hydroxide which occurs when the I 10 lone] fmnt' is drawn over the surfure of the moistencr. Satisfactory slide preparations of lead fume were finally made by simple exposure of cover slips anil rapid mounting in piperine. Iutrnperitoneal injection.-- The third set of animals was injected intrnperitonenlly in the first experiment according to the technique of Miller and Savers (2.r) with 0.2 gram of metallic, lead or its equiva lent in the various compounds of lead. VVe are indebted to Acting Asst. Surg. J. W. Miller for kindly injecting these animals for us. In the case of the first injection there were nine animals on experi ment for each compound. A short time later another group of ten ti 11 animals was injected with one-tenth the former quantity of lead arse nate and this was followed hy another complete set of animals of ten in each group. Each animal of these groups was injected with half the original amount of lead or of lead compound th at was given in the first case. T h at is, each animal received 0.1 gram of lead or of its equivalent ns lead compound. The weight changes of these animals were followed and they were also observed for toxic symp toms. No attem pt was made a t death to determine the distribution of the lead in the tissues of these animals as the risk of contamina tion was too great to attach any importance to the chemical analysis of the individual animal tissues. However, it was possible to free the greater part of the carcass of the animal from possible contami nated lead. The animals were skinned and carefully eviscerated, well washed, both with water and with ammonium acetate solution, and finally the carcasses were examined chemically for lead. The tissues were removed for pathological examination and the anterior abdominal wall, to which the lead compound was usually attached, was removed and set aside for microscopic study. In all the preceding experiments also, portions of the tissues were removed for pathological study as well as for chemical analysis. The tissues which were analyzed for lead were principally the liver, kidney and bone tissue and, additionally, in the case of the inhalation experi ments, the lungs. ( H K M K 'A I, M E T H O D S O F ANALYSIS Owing to the wide range of lead concent ration dealt with, no single method of analysis was applicable throughout. Instead the method of analysis used was adapted to the quantity of lend being determined, since in our experience it is useless to attem pt to use one method of analysis suitable for the determination of micrograms for the, evalua tion of milligrams of lend or vice versa. In sampling the dusty air to which the animals were exposed, one cubic foot samples were taken at intervals daily from each exposure chamber. These samples represented in many instances a lead content of several milligrams and wore analyzed by separation and titration of the lead as chromate (2(i). In the analysis of those tissue samples where the lend content was highest, as proved to be the case with the lungs of animals that had been exposed to the cllist, or fume, the above method was also used. With other tissues where the lead content was represented by micrograms the chromate method was obviously not suitable and the tissue lead content values were obtained either by the photometric method of Webster (27) or a modification of Fischer's dipbenyltbiocarbazone method (28). IM ill 7 III 12 EXPERIMENTAL RESULTS SIGN S OK I.KAO INTOXICATION T h e anim als in all these experim ental groups were observed for marked outward signs of lead poisoning, such as muscular incoordina tion and peripheral motor paralysis of the extensor muscles, colic, loss of w eight and loss of appetite. The physical signs of lead intoxi cation usually associated with severe lead poisoning, such as lead palsy or m uscular incoordination, were not apparent in any of these groups of animals, since the attem pt was made to seek information regarding a lower grade of lead intoxication rather than merely to produce severely poisoned animals. Hence, more weight was attached to th e com parison of the degree of lead distribution in the various animal tissues. The fact that, in all these cases of exposure to lead, the anim als were absorbing lead in varying quantities, i. e., the am ounts of lead taken up by the blood stream and deposited in the tissues, in itself constitutes an index of toxicity. The tissue content constitutes a clue to the concentration of the "lead stream " to which the animal has been exposed. In those tissues in which the organism as a w hole seeks to dem obilize the unexcreted lead (m ainly in bone) in order to reduce the concentration of the lead stream , one would therefore expect to find lead stored in proportion to th a t degree that lead has succeeded in invading the organism . H ence more im portance was attached to the am ount of lead deposited and to the lead distribu tion in the tissues than to any other single factor. In view of the pronounced degree of lead poisoning found by various experimenters with the subcutaneous injection of lead compounds, it is of interest th at anim als 12 to 12 m on th s after the injection of 200 m illigram s of lead or its eq u ivalen t were surviving in each group w ith ou t outward m anifestations of lead poisoning after having sustained an apparently normal increase in w eight. As more than five hundred anim als were used in this investigation, it is im practical to give individual values. T he results therefore are reported as average figures for the groups of anim als so exposed. lilood changts. The blood changes ordinarily associated with lead poisoning were followed with particidar reference to punctate stippling, polychromasia, and morphological changes. The degree to which these changes were apparent in the different types of exposure and to different lead com pounds is indicated in table 1. A t the present tim e it is wrell recognized th a t while basophilic degeneration of the red cells is of great assistance in the diagnosis of lend poisoning, it is n ot pathognom onic of the disease itself but must be considered in relation to other ob jective findings. On the other hand, the absence of basophilic grounds does not indicate absence of the effect of lead on the organism. Basophilia, as well as the other changes noted, was m ost pronounced following exposure to lead and its com pounds by inhalation. T a b l e 1. R e d -b lo o d -c e ll c h a n g e s in g u in e a p ig s follow ing th e in g e s tio n , injection, or inhalation o f lead and its com pounds Stippled ('ells 1`olychroinasia Morphologic changes 1mil's- Injec In h ala i imi's- Injec Inhala Inges Injec ln hala t.ion tion tion tion tion tion tion tion tion M etallic lead + + + + 4- 4- I/cad arsenate . - . . . No d ata N o d a ta -- 4- -- I<ead carbonate + -- + + + + 4 4- f -- -- + + + Lead chromate -- + 4-4- + -- -- Litharge .. -- --+ 1 4- 4-4- -- -- + Red le a d ___ f -- ++ f -- 4-4- -- -- ++ Lead peroxide -- + + + 4-4- + -- -- -- Lead silicate C) -- -- + 4-4- ---- Lead phosphate -- + + + 4-4- -- -- +-+ Lead sulphide + -- + + -- + 4-4- -- -- + + Lead sulphate .. C o n tro l.............. .. ---- ---- -- --+ -- ---- + 4- 4- 10C1MpNoeordrope5th--0eo0xSlopRtgoip.iscpRealde.l dCbcy.hCaiennlglgse'e,ss:t(i--o(n)--. n) onneonfeo,un(d+,) (+) 1-2 per 5(H) U. R. O., pule, slight anisocytosis, (4-4-) 3-6 per 500 R. R. slight poikiloeytosis, ( + + (+4-4-) ) slight anisocytosis, slight, poikiloeytosis plus 1-3 nucleated red (tells, (H--h-f-) above changes accompanied by many nucleated red cells. In the present in stan ce, th e blood ch an ges as show n in the table indicate th at lead carbonate, red lead, and lead sulphide when taken through the lungs have a definite irritant effect upon the hem atopoi etic system because of the frequent occurrence of basophilia, polyehromasia, and morphological changes. M etallic lead, litharge, lead peroxide, and lead phosphate do not have this degree of response, whereas lead arsenate, lead chrom ate, and lead silicate revealed no c.ytological changes different from the controls. T he appearance of these blood changes in anim als exposed to these com pounds raises the question as to w hether m inute am ounts of lead entering the blood stream constantly are not more concerned with the formation of basophilic grounds than m assive doses of lead. It is well known, for instance, that blood changes arc frequently associated with early exposure and may be absent with increasing exposure. W hile changes in th e red blood cells were marked in the inhalation groups, only a mild degree of hem atopoietic irritation w as shown in the group of animals exposed by ingestion, while animals exposed by injection revealed the least change. Blood calcium.-- Blood-calcium determ inations were also made on the ingestion and in halation groups at post m ortem in order to deter mine w hether any possible changes occurred which could he related to lend absorption. T h e blood-calcium values nre given in table 2. T he blood-calcium value fluctuated around the norm al values for guinea pigs in all cases where the anim als were exposed to lead com 14 pounds either by ingestion or by inhnlation. No significant, departure from th e norm al is a p p a ren t in any case. T abi.e 2 . - Blond ca lciu m values o f guinea pigs following th e ingestion and inhalation o f lead and its com pounds Metallic lead. Lead arsenate Lead carbonate Lead chromate. Ivead monoxide Red lead............ I>ead dioxide__ lead phosphate. Lead silicate .. lend sulphate. Lead sulphide Normal __ . Substance Blood cal cium (mgs. percent) ingestion Blood cal cium (mgs. percent) inhalation 9.1 11.1 10. H 10. 4 10.3 10.4 10.4 11.1 10. 4 9.4 10. (i 9. 0.7 10.5 10. 1 10.0 10.7 10. 1 T H E IN T R AP E R ITO N EA L IN JE C T IO N OF LEAD AND ITS C O M PO U N D S The am ount of lead or of its compounds th at was injected in each case is shown in the following table, together w ith the degree of survival. The quantities of m aterial injected into each animal varied from 200 milligrams of lead, or its equivalent, to one-tenth this am ount in the case of one group of lead-arsenate animals. The m aterial was injected as a sterile suspension in normal saline solution. A num ber of deaths occurred am ong the animals early in the experiments, but sim ilar deaths occurred among the control animals. In most cases (particularly in the 200-milligram-exposure group) these deaths were due to pneumonia th at was prevalent a t th at time among the control as well ns injected anim als. A few cases of abdom inal abscesses developed w ith the latter anim als also. The surviving animals seemed to rem ain in good health and showed no gross sym ptom of lead poisoning. Those findings are somewhat different from the results obtained by E rlenm eyer (20) and later by S trau b (30) w ith respect to the subcutaneous injection of lead salts. The former, who injected cats w ith lead carb o n ate, and the latter, wdio injected ra b b its w ith both lead carbonate and lead sulphate, both reported these subcutaneous depots as inducing lead poisoning readily and considered them as the sources of a " lead stream " and th a t the degree of lead poisoning varied w ith th e concentration of this lead stream . Aub and his co-w orkers (31), however, found th a t th e subcutaneous injection of lead carbonate failed to produce any toxic effects, except for the appearance of a lead line in a few cases. T he la tte r results are more in conform ity w ith the intrap erito n eal injection results obtained in the present instance. j j / j j j J : j j i j 1 I j j j j j i j 15 T able 3.-- M ortality of guinea pigs at various intervals following injection w ith lead and equivalent a m o u n ts o f various lead com pounds Substance injected Total number of aniraals in jected injected (milli grams) Total mortality Number of animals surviving at end of exiterlment. 1 After 18 hours After 4 weeks After 4 months 0 months 7months Metallic load. . /( 10 200 100 0 0 1 0 2 3 ft Dilead ortho arsenate.. ........... 11 1S 380 100 9 10 0 6 ft 18 38 4 7 7 2 I/oad carbonate________ /9 l 10 250 124 0 0 f7t 7 2 8 1 U-ad chromate ___ { 1? 1/cad monoxide..... ............ lied lead.......................-- . { r\ so to 310 150 210 108 220 118 0 0 1 0 0 0 0 390fi 0 0 0 9 4 5 4 4 0 0 2 3 1 l/ead dioxide.................. { 230 115 0 0 5 ft 0 3 ft 3 U ad orthophosphate . !1 190 200 131 0 0 0 0 8 3 7 1 bead sulphate . { 290 0 0 7 140 0 l 9 2 bead sulphide -- { I.ead silicate . 10 220 113 130 0 0 0 4 f2t 5 2 0 8 9 2 Control { 041 3 0 3 5 4- 1Two groups of anim als (except in the case of the lead arsenate experim ent In which case there were 8 groups) were Injected, the experim ent lasting 6 m onths in one case, 7 in the other. It is of interest that such a large amount of a lead compound could he injected into the abdominal cavity and remain in the animal for periods of time as great as one year without gross symptoms of lead poisoning. In many instances this material was walled off and in all cases, with the single exception of lead arsenate, it behaved as an inert substance. In several cases also it was distributed in small clumps at various places in the abdominal cavity manifesting no more irritant reaction than response usual with other inert materials. This resistance of guinea pigs to intraperitoneally injected lead compounds w ith reference to lead poisoning is of the greatest interest in relation to th e co m p arative ease of producing lend poisoning by other means. The animals th at were thus injected with the com pound suspended in norm al saline solution were killed a t the end of the experim ent and the degree to which diffusion of lead into part of the tissues had occurred was determ ined by chem ical exam ination of the carcasses. E v ery effort was m ade to m inim ize contam ination by com pletely eviscerating the skinned anim al w ith the enclosing pellicle of tissue and w ashing well w ith am m onium acetate solution as well as w ith distilled w ater. T h e am o u n t of lead th u s found dis tributed through the m uscular tissues and skeleton was sm all in all cases and usually less than one milligram. (See table 4.) In general the am o u n t th u s found in the group killed afte r 6 m onths was som e 1(1 w hat less than in the group killed one m onth later. This probably indicates th at w hatever dissolved lead entered the circulation was adequately excreted and that no particularly great storage occurred. T able 4. ('areass c o n te n t o f lead follow ing th e in tra p erito n ea l injection of lead and its com pounds Substance injected Amount injected (milli T otal lead found in grams) carcass (milligrams) ' Iroup I Group ll Group 1 (after 7 months) G roup 11 (after 0 months) M etallic lead I*ead arsenate Lead carbonate Lead chromate I/cad monoxide Red lead Lead dioxide Lead phosphate I/ead silicate. Lead sulphate Lend suphide . . it HI 100 0.31 0. 00 380 100 ' (0. 005) 250 125 1.01 0. 73 .112 150 . SO .82 210 .05 230 IIS . 21 .03" 230 115 . 30 .01 202 131 . 25 .71 137 . 42 202 140 .53 .07 220 113 .05 .33 Injected with Ho quantity piven Group I animals. The results obtained with lead arsenate were different from all the o th er cases. In the first group of 9 anim als each injected w ith 380 m illigrams of this com pound, all the anim als died overnight. A second group of 10 anim als was then injected with half the am ount of lead arsen ate, or 190 m illigram s, and all 10 anim als died overnight. A third group of 9 anim als was then injected with one-tenth the original am ount of lead arsenate, or 38 m illigram s each, and while there was no im m ediate effect, only two survived for the 0-month period. The toxic effect would thus appear to be clearly due to the arsenic and the im m ediate action is probably due to the effect of soluble arsenate split off from the lead arsenate in co n tact w ith tissue fluid, as solubility experim ents now in progress indicate. The m ortality rates of the intraperitoneally injected anim als are shown in table 3 and are grouped in 18-hour, 4-week, anti 4-m onth periods. W ith the single exception of an anim al in tin* lead monoxide period which died of infection, no other animals were im mediately affected besides the lead arsenate group. While a num ber of these anim als died before the completion of the experim ent, the immediate cause of death was with few exceptions acute respiratory disease and no signs of lead poisoning were evident. Indeed, representative anim als of each of the heavily injected groups, with the single excep tion of lead arsenate, were alive 12 m onths after injection with no sign of lead intoxication. T he lead compound was found to he walled off on the an terio r abdom inal wall to some ex ten t (fig. 5), h u t was also found to he d istrib u ted in the peritoneal cavity a t various points (fig. ti). T here was no appearance of irritation in the surrounding r>--- N a i m , - i l c o l o r p h o t o m i c r o g r a p h o f g u i n e a p i g t i s s u e 7 m o n t h s a f t e r m je c tio n with 0.:il g ra m lead c h ro m a te . T h e yellow m asses a re solid lead chrom ate. 17 tissues. The inertness of infra peritoneally introduced lead com pounds is, therefore, of considerable in te re st when one, recalls the behavior of these com pounds in either the respiratory or gastrointesti nal tra c ts and the co m paratively g reater ease; of producing lead poison ing at these respective sites. INGESTED LEAD AND ITS COM POUNDS The distribution of lead in the tissues of those guinea pigs th a t had ingested lead or its com pounds is shown in tab le 5. T h e to ta l am ounts of lead ingested in m illigrams and the ratios of tissue lead co n ten t to ingested load are given in table 6. --T able 5. The d i s t r i b u t i o n o f lead in the tissues of gui nea pigs folloiring the ingestion and inlialation of lead and its compounds Compound Lung Ingestion (mgs/10 grams tissue): Control Metallic lead . IiCatl arsenate Lead carbonate Lead phosphate Lead chromate L ith a r g e ..................... Red lead . . .. .. . Idiad dioxide Lead sulphate Lead sulphide Inhalation (mgs/10 grams tissue): Control Metallic lead Lead arsenate . . . . . . Lead carbonate . ___ Lead phosphate Lend chromate Litharge Red lead _______ Lead dioxide...................... .. Lead silicate................ . I,ead su lp h id e ..................... 0. 001 . 070 . 229 . 781 5. 490 1. 491 . 248 1.09 . 921 . 340 . 072 0. 004 017 .014 .017 . 045 .022 .031 .019 . 039 .041 .010 . 000 . 022 . 029 . 085 . 000 . 040 . 032 . 007 . 002 . 004 . 093 0.004 .046 .045 . 099 .077 .078 . 117 .051 .111 . 107 .070 . 005 . 055 .070 . 164 . 127 . 101 . 108 . 180 . 121 . 150 . 134 0. 000 . 080 . 054 . 390 .. 097 222 ! 413 . 124 . 157 .484 . 135 .000 . 134 . 158 .578 . 218 .238 . 538 .277 .298 . 152 . 204 T 6.able The total amount of lead ingested and the ratio of the lead con- Xt e nt of 10-grarn por t i on s to t h e t o t a l ingest ed lead 1,000 Pb......... .. P b ll AsO 4 PbCO, PPbbOCrOi 1*1)304 l*bOj___ Pb3(PO)f PbS04 .... l*bS............ ompotind At death of ani mal total Pb ingested aver age mgms. Ratio tissue, content to total ingested Pb X 1,000 Li ver Kidney Bone 89 0. 08 0. 74 10 .90 2. 90 123 . 14 .79 73 . 30 1.07 78 . 39 1. 60 80 22 . 59 109 . 35 1.02 72 . 03 1.07 109 . 37 .98 92 . 17 .70 0. 90 3. 48 . 73 3.01 5. 27 4. 18 I. 43 1.35 4.44 1.47 As will he noted in these tallies, the concentration of lead in the tissues, th a t is, th e am o u n t of lead per 10-gram portion of tissue, is least in the liver, greater in the kidney, and greatest in the bone 18 substance. T his relationship occurs with some regularity in prac tically all the cases (see table 7) and shows an average liv e r: k id n e y : bone ratio of 1 :3 :7 . T able 7.-- The com parative distribution of lead in the tissues following ingestion or inhalation o f lead or various lead com p o u n d s (the licer:kidney:bone ratio) Substance Metallic lead Ivead arsenate Lead carbonate Lead chromate lead monoxide Red lead . Lead dioxide 1/ead phosphate Iiead silicate. . Lead sulphate I/ad sulphide Ingestion Liver Kidney 1 2.7 1 3. 2 1 5.8 1 3. 5 1 3.8 1 2. 6 1 2.8 1 1. 7 T 2. 6 i 43 Bone 4.7 3.8 5. 4 10.0 13. 3 6.5 4. 0 2.6 11.8 8. 4 Inhalation Liver Kidney 1 2. 5 1 2. 4 1 1.9 1 2.2 1 3. 4 1 2.8 1 2.0 1 2.0 1 2.3 1 3. 1 Bone 6. 1 5.5 6.8 5.2 16. 8 4. 1 4.8 3.6 2.4 2. 8 U nfortunately it was difficult to get the animals to ingest exactly equivalent am ounts of lead compounds, so th a t no final conclusion can be reached regarding the relative storage of lead. However, certain of the lead compounds, notably lead carbonate, lead monoxide, and lead sulphate, cause the deposition of more lead in the bone and to a less m arked extent in the kidney, even though the total am ount ingested was not invariably large, than was found to be the case with the rem aining lead com pounds (fig. 7). B ased on th eir solubilities alone, it is n o t surprising th a t both lead carbonate and lead sulphate would be more readily absorbed than the other lead com pounds. Carlson and Woelfel (32) found both these salts to he appreciably soluble in hum an gastric juice. From its chemical behavior it would be expected th a t lead oxide would be appreciably dissolved and abosrbed in its passage through the gastro intestinal tract. On this basis then, lead carbonate, lead oxide, and lead sulphate would be indicated as somewhat more immediately toxic than the remaining lead compounds th at were studied. In the case of lead arsenate, although the total am ount ingested was much smaller than was the case with the other compounds the proportion of kidney lead to ingested lead was higher than with the other lead com pounds (table 6). I t would appear th a t with a similar degree of exposure to th a t of carbonate, oxide, or sulphate, its toxicity rating would be greater. T H E INHALATION OF METALLIC LEAD AND ITS COMPOUNDS One of the m ost interesting phases of the toxic effect of lead and its com pounds, in view of its industrial im portance, is th a t of inhala tion of the substance, as dust or fume. As a practical means of F ig u r e 6.--X-ray photograph of a guinea pig 2 weeks after the injection of 0.24 gm. of red lead. 19 ____ BO NE L,/Vfi_______ KIDNEY cavrnaL pt, Pbsu,^ ft>co3 f t C r O , ft>o P tjO , r ta , fts o , /1>S FiauttK 7 .- T issu e d is trib u tio n of lead fo llo w in g th e in g e stio n of lead o r its com pounds. 20 producing lend poisoning, the inhalation of lead d o st or fume has been regarded by some as of small importance.. MeilhVe (33) noted that some absorption from the lung may occur, hut considered it of no (Treat eonscipicncc. Carlson and Woelfcl (34) also considered the gastrointestinal tract the chief source of lead absorption even though the lead originally entered the respiratory tract, due to the fact that material was coughed up and swallowed, or due to the retention of the substance in entering the nasal passages or m outh. M inot (3.r>) m ade a direct attack on this problem by ligating the esophagus and introducing a suspension of lead carbonate directly into the trachea. The degree and rapidity with which the absorption of lead occurred under these conditions leaves no doubt as to the importance of direct absorption of lead from the lungs. T he distribution of lead in the tissues of the guinea pigs exposed to lead fume or to the d u st of various lead com pounds is shown in table 5 and the relative' concentrations are plotted in figure 8. O wing to d illieulties in ob taining a sulliciently high concentration of fum e or dust in the case of m etallic lead and of lead arsenate, respectively, these particular experim ents were som ew hat less conclusive than had been anticipated. The anim als were exposed for a period of one hour per day, live d ays per week, over a period of several weeks. The range of exposure to the various com pounds is indicated in table 8: T aiii.i: 8. Effect o f d u r a t io n o f e x p o su r e u p o n th e tis s u e d is t r i b u t i o n of h ad in 2 g r o u p s o f g u in e a p ig s folloicing t h e i n h a l a ti o n o f lead f u m e or of lead-compound dust I.(>>fI distribution Njiiiu*of >->m11xiiin>i j I! U n l i t ........... | exposure [ Liver Kidney Lung | Hone hours I Short range of expo ure Nrsenate ............... C h ro m ate ......................................... Monoxide ...... ... Sulphide .............................. . . ( 'arbonate Silicate ............... Lead Peroxide, Kcd lead Phosphate Longer ranee, of exposure: Arsenate .............................. C h ro m ate M onoxide Sulphide Carbonate ............................. Silicate . . . .......................... l/cad ............................................ Peroxide ................................. Red lead Phosphate ........... ----- ------- --------------------- 2 It 1 lfi M2 4 14 2 14 2 14 1.5-14. 5 .V14 2-14 1.5-10.5 24. 5-38. 6 10-39 18-118 24. 5-38. 5 22 39 14. 5-38. 5 28. 5-118. 5 24. 5-38. 5 14. 5- 38. 5 17 43 Oiiilligrams/10 grams tissue) -- ----------- - ---------- n (Kill . 0115 022 .078 . 052 . 024 .044 . 048 . 007 . 035 . 022 .043 Oil . 101 . 210 . 102 .015 .075 .089 .079 t). 104 .070 .045 080 .077 . 127 .084 . 120 . 132 . 152 0.163 .474 .097 799 .297 .403 .067 .281 1.273 7. 545 . 181 . 103 . 145 . 101 352 . 108 .00 . 121 . 219 .082 -- .273 .470 . 393 .522 .760 .293 .082 .381 1.063 5.055 0.209 .050 .OftH .244 .092 .247 .489 .538 .362 .531 . 152 .226 .347 .251 .249 21 To some extent we were able to secure com parative d ata respecting tlie d istrib u tio n of lend in the tissues in term s of degree of exposure. These d ata are also given in table S. In general an increase in con centration was noted with increasing exposure. I'ARTICI.K Si/.K Since elu triato rs were used in all tin1 experim ental work, the partid es of the various lead com pounds do not vary greatly in size. In fact th ey show a surprising uniform ity with the. single, exception of m etallic lend which was not elutriated hut was generated directly ns fume and which consequently is much sm aller in particle size. M eas urem ents (200 to each slide of the com pound exam ined) were m ade following the procedure of llloomficld arid DallaValle (30). These results are shown in (able it. T a b i ,K 9 . / `a r t i c l e s i z e m e a s u r e m e n t s Dust nr fume Avertigo pari icle size (microns) 1 1Jcviation 1Just or fume Avertigo particle size (microns) I Jovial ion Metallic lead Load arsenate. ..... Lead carbonate. Load chromate................ Lead monoxide 0. .. H4 1) fcO. II Med lead . 34 Lead dioxide. . .33 Load phosphate 1. 0 3 0 Lead silicate 1.0 . 32 Load sulphide 1. 1 rfcO. 33 1. 1 dr. 37 1.09 . 11 .36 ..3345 .Judging from m easurem ents made at different times, the particle size values did not depart significantly from the values indicated above. This and the degree of uniform ity apparent between the different compounds clearly indicates the very satisfactory degree of efficiency attained with this type of dusting apparatus. In two cases, nam ely in the case of litharge and of lead carbonate, the am o u n t of lead deposited in hone tissue was more than half a milligram per It) grains of bone tissue in each case in spite of the fact that these two groups of animals were not subjected to the same degree of exposure that, some of tin* o th er groups received. As in the case of the ingestion experim ents, it was impossible to subject the guinea pigs to precisely the sam e am ount of exposure. V ariations, n o t in particle size, b u t in other physical properties, rendered it difficult to m aintain a uniform concentration of aerially dispersed lead with the various lead compounds. An attem p t was m ade to estim ate the theoretical am ount of lead breathed in by the guinea pigs, knowing the tim e of exposure and concentration of air-borne lead. It seems reasonable to assume a 22 tidal volum e of 2 liters per hour for an average (300 gram) guinea pig, hut w hatever volum e of air is moved in the ordinary respiration process it would he som ewhat uniform throughout as the pigs were initially uniform in size. On this basis the am ounts of lead that could have been theoretically inhaled are tabulated in table 10. The am ount found in the lungs by analysis at the end of the experi ment represents a factual value. It represents retention in a m echani cal sense and does not discriminate between loss of material coughed up and loss of material due to absorption. It is of interest, however, th at the percentage retention is som ew hat higher in those cases where the m aterial is least soluble (table 10), nam ely, in the cases of the arsenate (11.5 percent), chromate (14.4 percent), phosphate (47.4 percent), and silicate (10.0 percent), while in the case of the dioxide the retention was low er (5.1 percent). T a b i.k 10. -- T h e a m o u n t s o f lead re ta in e d in th e lu n g s o f g u in ea pigs exposed to lead fu m e or dust t'oinixiurid lx*ftl arsenntc l/oud carbonate Lead chromate Lend monoxide Load, red Load dioxide Lead phosphate I/'-ud silicate Lend sulphide U ad Lead content of air mgs/cu.m. Theo retical amount of lend inhaled, mgs. Actual amount of lead found in lungs, Total average exposure, hours Reten tion (percent) mgs. Solubility product constant 102. 7 324. 1 2.42 2.52 0..2208 11.8 3.9 11.5 8.4X10-" (25 C.) 7.9 3.3X10-" (16 ( '.) (37) (38) 87. 6 2. 64 .38 15. 1 14. 4 2.0X10-" (18 ('.) (39) 82 6 538. 0 28..4007 1..0129 1115..01 7.7 1.32X10-'* (17 r . ) 12.7 5.3X10-*' (17 C.) (40) (41) 204. 1 4.86 .25 11.9 5. 1 3.2X10 (17 C.) (42) 182. 2 7. 70 3. 70 21.3 47.4 3.2X10-*' (43) 99.3 318. 1 13.8 82.. 58 66 .62 . 43 . 54 .05 9.2 14.0 22.5 166..26 8.1 2.2X10-8 (25 r .) Solubility 1.4X1(7 4 (44) (45) inols. per liter. M oreover, the concentration of lead in the hone tissue, which seems to be a fair index of absorption, is higher in the case of lead carb o n ate and lead monoxide th an in any of the o ther cases (fig. 8). It would seem quite probable, in these two cases where the system would tend to be flooded with lead, th a t the organism would adjust itself to m eet the situation by dem obilization, or storage, as well as excretion, while the slower solution of more physiologically inert m aterial could be somewhat more adequately met by the usual pro cesses of elim ination, whether by simple solution alone or by phagocytic activity. Microscopic examination of the lung tissue of the inhalation animals showed areas of deposited m aterial. It proved somewhat difficult to prove the plumbiferous nature of this particulate material by the usual chemical procedures w ithout destroying the structure of the surrounding tissue. Finally, it was found possible to do so by im m ers ing the section for some days in a solution of potassium chrom ate 2.'! TISSUE DISTRIBUTION OF LE A D FOLLOWING THE INH ALATIO N OF LEAD OR IT S COMPOUNDS AS FUME OR DUST. M ILLIG RAM S LEAD PER TEN G R AM S TISSU E. \ 0 ! 0 M ILLIGRAM S OF LEAD. LUNGS BONE ||p 1 LIVER KIDNEY CONTROL PMiAsQ, A&TO, /VC rt* / * 0 /*>jO, F * 0 , /* O s S .O , FU S F i g u r e 8 .-- Tissue distribution of lead following the inhalation of lead or its compounds as fume or dust. 24 acidified with acetic acid. Under these conditions crystalline lead chrom ate is slowly formed. In figure 9 crystals of lead chrom ate can be seen d istrib u ted in a lung section of a pig which had inhaled red lead. In a few cases the crystal of lead chrom ate can be seen growing from the m other particle. As in the ingestion experim ents, the distribution of lead in the tissues was found to be such th a t the lead was least in the liver, greater in the kidney, and greatest in the bone tissue. T he ratio of liv e r: kid- T/ME IN W EE K S F igukk 10.---C umulative d e a th s of guinea pigs exposed to lend by ingestion, peritoneal ingestion, and inhalation. j ney : bone lead was 1 :2.5:5.8 (table 7). I t should be added th a t these experim ents were of longer duration than has occurred in some animal experim ents where the system was flooded w ith ingested lead in order to produce poisoning. F o r instance, in a recent experim ent where a dog was fed large quantities of lead carbonate, in other words, when the system was flooded w ith lead, the ratio of lead in sim ilar weights of liver, kidney, and bone tissue was 1:'/2:10: T h is is in agreem ent with opinion regarding the im p o rtan t tra n sito ry role of the liver and the trend towards demobilization of lead by storage in bone (issue. KiUiiK 9.-- Particulate l'li;,04 converted to lung tissue. crystalline lead chromate in F u rth er evidence, regarding the severity of poisoning; by inhalation as com pared w ith either poisoning; by ingestion or by intrapcritoneal injection is shown on exam ination of the d eath frequencies of all anim als of the three groups and also in the rates of grow th of all anim als subjected to the. three types of exposure. 'The d eath frequency (fig. 10), while no t conclusive for the ingestion or the injection groups, because of sim ilar rates of death among the controls, is consistently higher for the inhalation group of anim als as Fuji itio I I . Peronutiige increase in weight of guinea-pig groups in th e various types of lead exposure. a whole. The deaths were not due to lead poisoning per se, but chiefly due, as was the case with the other groups of animals, to respiratory infection. How ever, it seems reasonable to consider th a t, since the death frequencies were higher for the inhalation animals, their resist ance to respiratory disease was lowered as result of this exposure. T he effect of breathing lead dust, or fume, as contrasted with other types of lead exposure, is m ore m arkedly shown in the guinea pig growth curves (fig. II) of all the anim als. T he inhalation anim als 26 ns n group rem ained practically stationary in weight during the entire experimental period and showed a maximum increase of only about 4.5 percent. This contrasts sharply with the other types of exposure and would seem to indicate a nutritional im pairm ent of m arked degree. PHYSIOLOGICAL TOLERANCE FOR LEAD In the production of experim ental lead poisoning in animals one is frequently im pressed by the relative degree of tolerance which the anim al displays tow ards lead. W hile this is in m any cases due to the fact th a t the anim al is able to block the entrance of the poisoning by walling it off ns in the case of subcutaneously or intraperitoneally introduced lend or (in the case of very insoluble com pounds) is able to keep the am ount of circulating lead a t a minimum by im mobiliza tion as stored lead or by excretion, there also is apparently built up a degree of tolerance towards the toxic m aterial. W hether the ability of the tissues to convert it to more insoluble and therefore less im m e diately harm ful forms, or w hether it is excreted more readily, or w hether n greater tolerance is built up for it, it is impossible to say. Any one of these processes would assist in lowering its general toxic effect.. Instances of such physiological tolerance are no t unusual, nor is th e idea novel, for it has been cu rre n t from an early tim e, b u t perhaps because of its ancient origin it has usually received scant attention. Nicotine, alcohol, morphine, atropine, and arsenic arc cited by C'usluiy (4ti) as instances of poisons tow ards which a tolerance of varying degree is acquired. The idea of tolerance with respect to lead has from time to time been impressed upon those who have made much study of lead poison ing. Meill&re (47) states: " C ertains sujets possedent done une v eri table idiosyncrasie; une im munity naturelle ou acquisc, qui en fait des M ithridates du satum ism e." Leggc and Goadby (48) in particular have given em phatic expression to this point: " We show in a la te r ch a p te r th a t the excretion of lead in persons tolerant of the m etal takes place through the medium of the bowel, and th a t probably those individuals who are engaged in w hat are recognized as dangerous processes in lead industries, and yet show no signs of illness, have established a kind of balance between the intake of the poison and its excretion by the bowel. It is rarely possible in such persons to find any lead excreted through the kidney. " Persons who gradually acquire tolerance go through the stage of anem ia without exhibiting any sym ptom s of colic or paresis, and w ith o u t any tre a tm e n t the hem oglobin and the num ber of red cells gradually pass back to a more or less norm al condition. "Such persons are either immune from the commencement, or they have established a certain degree of tolerance towards the m etal; the la tte r supposition is the more probable, as there is reason to think several of them suffered from n mild form of poisoning during the earlier years of their em ploym ent." L ater work by Weller (49) has dem onstrated an experim entally induced tolerance to lead poisoning in respect to m eningocerebral m anifestations as they occur in the guinea pig. A ttention has also recently been drawn to the question of tolerance for lead by M inot (50), who, in review ing previous work in this con nection, concludes that: "Observations of this type suggest th at continued exposure may decrease the danger of small am ounts of lead." In the present investigation, the variation in slope of the growth curves in the ingestion, the injection, and the inhalation groups points to a period of read ju stm en t which m ay or m ay n o t be of significance. Furtherm ore, the general physical appearance and behavior of the animals gradually improved rather than the contrary with the contin ued adm inistration of their daily dosage beyond the initial period. It is qu ite likely, therefore, th a t one should consider physiological toler ance as a factor where the attem p t is m ade to produce experim ental lead poisoning in the guinea pig. RELATIVE TOXICITIES Insofar as we can consider the storage of lead in bone tissue as an index of th e ex ten t to which the system has been flooded with lead in a given space of time, taken in relation to other factors indicating possible dam age, it is possible to arrange the toxicides of various lead com pounds into two groups. As indicated above, allowance m ust be m ade for the fact that, it is very difficult, if not impossible, in anim al experim ents to duplicate all the conditions exactly. H ow ever, careful consideration of the d a ta in the above experim ental work indicates th a t while no exact numerical order can be assigned to the toxicity, certain lead com pounds may be set a p a rt as more toxic than others. T able ll .-- The relative toxicity o f lead and its com pounds Order of toxicil y Injection Mot hod of introduction Ingestion j Inhalation Most toxic I,etui arsenate Of .similar but a lower decree of toxicity. Metallic lead bead carl>onate Lead chromate I/ead monoxide Red lend I/ead dioxide Lend phosphate Lead sulphate Lend sulphide. Lead arsenate Lend carbonate Lead monoxide I/oad sulphate Metallic lead Lead chromate Red lead Lead dioxide Lead phosphate I/ead sulphide Lead carbonate. Lead monoxide. Metallic lead.1 Lead arsenate.1 Lead chromate. Red lead. Lead dioxide. Lead phosphate. Lead silicate. I/ead sulphide. Inconclusi' 28 The toxicity of lead arsenate following intraperitonea 1 injection is outstanding. The effects are rapid anti definite. However, this acute toxicity can only he ascribed to the arsenic component ol the molecule, for similar effects were not produced by any of the other lead compounds. It was thought that lead chromate would exhibit similar, if less pronounced, results but there was no indication that lead chromate on intraperitoneal injection behaved otherwise than as an inert substance. On autopsy in some instances portions of lead chromate were found distributed through the abdominal cavity, some enfolded in the liver with no sign of irritation in the surrounding tissue. Lead arsenate, lead carbonate, lead monoxide, and lead sulphate appear to he somewhat more toxic than lead or other lead compounds when fed to animals. These compounds are perhaps more soluble in the digestive juices. It would be expected that lead chromate would dissolve more or less in the hydrochloric acid of the gastric juice and therefore be more completely absorbed. It doos, in fact, exhibit a greater degree of toxicity as measured by absorption than the remaining lead compounds and therefore occupies a position intermediate between the two groups. It should be indicated that while lead arsenate showed no great difference from certain of the other lead compounds so far as invasion of the tissues by lead was concerned, consideration of the toxicity of lead arsenate in these experiments was wholly limited to lead poisoning. Consideration of the kidney storage of lead with respect to the total amount ingested (table ti) indicates a possible toxicity rating comparable to the more toxic compounds. With respect to inhalation, lead carbonate and lead monoxide again appeared to he more toxic than the remaining lead compounds. SUM M ARY The foregoing investigation comprises an inquiry regarding the relative toxicity of lead and various commonly encountered lead compounds. More than 500 guinea pigs were studied during and following the administration of these substances by mouth, by intra peritoneal injection and by inhalation. Symptoms of lead poisoning such as muscular incoordination and anemia were sought, blood changes with respect to the red cells par ticularly basophilic stippling, polychromasia and morphologic changes were noted and blood calcium determinations were made. Body weight changes and mortality figures were collected. The lead com pound was administered in measured amounts in both the ingestion experiments and in the intraperitoneal injection of the animals, anti the degree of exposure in the inhalation experiments wois determined by sampling and analyzing the dusty air for lead at intervals during 2!) the exposure period. Finally the lead was traced out in the various tissues and analytical determinations were made of the amounts present in the liver, kidney, and hones in order to find the degree of absorption that lmd occurred. In the case of animals breathing leaddust laden air, determinations of the lead content of the lungs were also made. Particle size determinations were made of the suspended lead fume or dust and identification of dust particles in situ in the lung tissue was established in the case of animals exposed to lead by inhalation. The toxic effect of lead compounds was more evident on inhalation than when administered either by mouth or by intraperitoneal injec tion and emphasizes the importance of this type of exposure in dusty trades employing lead. Lead arsenate proved to be extremely toxic on intraperitoneal injection us compared with the other lead compounds investigated. The order of toxicity as shown by feeding experiments did not vary much for a number of the compounds studied. However, lead car bonate, lead monoxide and lead sulphate were shown to be more toxic by mouth than lead or the remaining lead compounds. Lead carbonate and lend monoxide were more toxic following inhala tion than the other compounds studied. BIBLIOGRAPHY I M inerals Y earbook, 1938, U. S. B ureau of Mines, IT. S. G overnm ent P rinting Ofliee, W ashington, p. 119. 1938. 2 , M inerals Y earbook, 1938, U. S. B ureau of Mines, 1T. S. G o vernm ent P rin tin g Ofliee, W ashington, p. 110. 1938. 3. M inerals Y earbook, 1938, U. S. B ureau of Mines, IJ. 8 . G o vernm ent P rin tin g Office, W ashington, p. 119. 1938. I Miller, .1. W., ami Sayers, H. R .: M ieroseopie app earan ce of experim entally produced d u st nodules in the peritoneum . Public H ealth R eports, 50: 1019. 1930. 5. Miller, .1. W., an d Sayers, R K.: T he physiological response of peritoneal tissue to certain industrial and pure mineral dusts. Public Health R eports, 51 : 1077. 1930. 0. O liver, Sir T hom as: Lead poisoning. H. I<. Lewis, London, p. 110. 1914. 7 L ehm ann, K. lb , Saito, Y., and G frorer, W .: U eber die q u a n tita tiv e A bsorp tion von S ta u b nus der L u ft durch den M enschcn. Arch. f. H yg., LX X V. p. 152. 1912. S. Sayers, R It., F id d lier, A. C ., Y ant, W. P., and T hom as, B. G. II.: Experim ental stu d ies on th e effect of eth y l gasoline and its com bust ion products. R eport of th e LT. S. B ureau of Mines, p. 115. 1927. 9 I.egge, T M., and G oadby, K. W.: Lead poisoning and lead absorption. K dw anl A rnold, London, p. 102. 1912. 10. Aub, ,1. C ., F airhall, L. T ., M inot, A. S., an d R e/.nikolf, P.: Lead poisoning. W illiam s an d W ilkins, B altim ore, p. 48. 1920. 11 R am bousek, ,1.: In d u stria l poisoning. E d w ard A rnold, London, p. 181. 1913. 12 Occ u p atio n and H ealth . In te rn a tio n a l L abour Office, G eneva, p. 122. 1934. 13 M urray, A rth u r L.: R elation of lead poisoning in U tah to mining. Bur. M ines R epts. Inves., Serial No. 2274. 1921. 14. Ilanzlik, P. ,L, and Presho, K .: C o m p arativ e toxicity of inorganic lead com pounds and m etallic lead for pigeons. Jour. Pharin. and Exptl. Thorap. 21 : 129. 1923. 15 lla n /lik , P. .1., an d Presho, K.: T h erap eu tic efficiency of various a g en ts for chronic poisoning by m etallic lead in pigeons. Jour. P harin. and E xptl. T lierap., 21 : 131. 1923. Ill Blum , K.: fib e r das Schicksa! des Blei iin O rganism us. Wien. Med. W elm sehr. 5.', : 537. 1904. 17. H am ilton, Alice: W om en in th e lead industries. B ulletin Bur. Labor S tats. No. 253, U. S. D ept, of Labor, p. 14. 1919. 15. O liver, Sir T hom as, loc. cit. p. 109. 19 Lehm ann, K. Ib: Vergleichendc U ntersuehnngen tiber die G iftigkeit des B leisulfats und des Bleiweiss. Arch. f. H yg. 1 0 1 : 197, No. 4. 1929. 20 Koelseh, F., Ledcrer, E., and Koelsch, R.: Vergleichendc U ntersuehnngen iiber die G iftig k eit von Sulfobleiweiss und K arbonatbleiw eiss. Arch. f. H vg. 1 0 1 : 234, N o. 4. 1929. 21. Ix'ggo, T. M., an d G oadby, K. W. loc. cit. p. 34. (30) 22. Fairlm ll, F. T .: T h e solubility of various lead com pounds in blood serum . Jour, Biol. Chem . //) : 482, No. 3. 1924 23. D allaV allc, J. M. (In publication.) 24. Bloomfield, J. J ., and D allaV allc, J. M.: Tiie d eterm in atio n and control of industrial dust. Public H ealth b u lle tin No. 217. 1935. 25. Miller, J. W ., an d Sayers, It. It.: T h e physiological response of th e p eritoneal tissues to dusts introduced as foreign bodies. Public H ealth R eports 49 : 80 89. 1934. 20. F airhall, L. T .: T he estim atio n of m in u te am o u n ts of lead in biological m aterial. J. Ind. Hyg. 4 : 9. 1922. 27. W ebster, S. II.: (U npublished) In th e case of these values w hich were kindly estim ated bv Dr. W ebster, use was m ade of his visual m ethod which is now superseded by m ore refined a p p a ra tu s. 28. Fischer, H elm ut: Die M ctalv erb iu d u n g cn d cr D ip h en y lth io carb azo n s und ihre V erw endbarkeit fiir die eheniische Analyse. Wiss. VcrdfTenti. a. d. Siem ens-K onzern. 4 158. 1925. 29. F rlenm eyer, F .: K xperim entelle S tudien fiber den M ech an ism u sd crch ro n isch en Bleivergift.ung. M iinch. toed. W ochenschr. HO : 1114. 1913. 30. S tra u b , W.: G ift und K ra n k h e it nach H eobachtungen an experim cntellor chroniseher H leivergiftung. M iinch. m ed. W ochenschr. 61 : 5. 1913. 31. Aub, J. C., F airhall, I .. T ., M inot, A. S., an d ReznikofT, P .: loc. cit. p. 03. 32. C arlson, A. J., and Woelfel, A.: T h e solubility of lead salts in hum an gastric juice, and its bearing on the hygiene of the lead industries. J. Amor. M ed. Assn. HI : 181. 1913. 33. M eillere, G.: Fe Satnrnism e. T hese p o u r le D o c to ra t en mf'dicine, Paris. )>. 05. 1903. 34. C arlson and Woelfel. loc. cit. p. 181. 35. M inot, A. S.: T h e d istrib u tio n of lead in th e organism a fte r absorption by resp irato ry tra c t and sub cu tan eo u s tissue. J. Ind. H yg. 6 : 137. 1929 30. Hloomfield, J. J., an d D allaV allc, J. M.: T h e d eterm in atio n and control of in d u strial dust. Public H ealth B ulletin No. 217. p. 50. 37. T e n ta tiv e d eterm in atio n m ade in th is lab o rato ry . 38. Pleissner, M.: t)b e r die F oslichkeit einiger llleiv erb in d u n g en in W asser. Arb. K aiser Ges. Amt. 26 : 405. 1907. 39. A uerback, F., and Pick, IF : D as V erhalten von H leichrom at und basischem H leichrom at in w asserigen Fosungen k ohlensaurer Alkalien. Arb. K aiser Ges. A m t. 4 6 : 107. 1913. 10. G lasstone, S.: Physical chem istry of th e oxides of lead. P a rt I. T he solubility of lead m onoxide. Jo u r. C hem . Soc. I l f ) : 1089, 1914. 1921 11. G lasstone, S.: Physical ch em istry of th e oxides of lead. P a rt IV. Rial lead und load sesquioxide. Jour. C hem . Soc. 121 : 1450. 1922. 42. G lasstone, R.: Physical chem istry of th e oxides of lead. P a rt V. T h e electrom otive behavior of lead dioxide. Jour. Chem . Roc. 121 : 1409. 1922. 43. B iittger, W.: Fosliehkoitstudien an seliwer loslichen RtofTen. le ip z ig . 1903. 14. F an d o lt-B o rn stein . Physikalisch-C hem ische T abollen. 5 th ed. J. Springer, Berlin. ]>. 521. 1935. 4n Clowes, F.: T h e action of distilled w ater upon lead. Chem . News. 66 : 108 1902. 40. C ushny, A. R.: A textbook of pharm acology and th erap eu tics l e a A Febiger, Philadelphia, p. 20. 1930. 17 M eillere, G.: I e S atnrnism e. T hese, Paris, p. 75. 1903 32 48. Legge, 1'. M., an d G oadby, K. W. loc. cit. 118 : 28-29. 49. W eller, C. V.: T olerance in respect to th e m eningo-cerebral m an ifestations of acute and subacute lead poisoning. Archives of Internal Medicine. 39 : 45. 1927. 50. M inot, A. S.: T h e physiological effects of sm all am o u n ts of lead: An e v a lu a tion of th e lead hazard of the average individual. Physiological Rev. I S : 554. No. 4. 1938. A CK N OW LEDG M EN TS The author wishes to thank Junior Chemist Frances L. Hyslop for the blood examinations, Laboratory Assistants Norman E. Sharpless and Robert G. Keenan, Junior Physical Aid F. L. Weaver, Jr., for analytical and technical aid, and Bacteriological Technician Ralph Donald Read for the photomicrography. PATHOLOGIC CHANGES INDUCED BY LEAD AND BY SOM E OF ITS COM POUNDS By J. W. M iller, Pathologist The pathological material described was obtained from the animals used in determining the relative toxicity of lead and some of its compounds, reported in the first portion of this bulletin. Not all of the guinea pigs used in that work were available for histological ex amination because a number were completely utilized for quantita tive chemical examination. In some of the animals one or more organs were used for chemical tests so that in estimating the relative amount of pathological damage, consideration was given to the num ber of individual organs showing similar changes as well os to the total number of animals. An estimate was also obtained of the changes produced in each organ by various factors that might have had an influence on the changes that took place. These factors were grouped as follows: (1) The several compounds, (2) the various dosages of each compound to which the animals were exposed, (3) the different methods of administration, and (4) the time elements, including the duration of test, the length of exposure period, and the interval between the end of exposure and death. A total of 2,800 histological sections from 230 guinea pigs was studied. Paraffin sections were made from the heart, lungs, liver, spleen, kidneys, adrenals, ami pancreas arid were routinely stained by L illie's (1) m odification of the eosin-polychrom e m eth y len e blue m ethod. Many spleen sections were stained by ferrocyanide to demonstrate the presence or absence of iron-bearing pigm ent. Kidney sections showing p ath ology in the eosin -m eth ylen e blue-stained m aterial were treated w ith L illie's (2) current m odification of G allcgo's elastic and connective tissue stain, which effectively dem onstrated the degree of interstitial nephritis. The following table presents the salient features regarding com pounds, numbers of animals, am ounts administered, and durations of experim ents considered in this report. (33) Ingestion serif Inhalation series 34 2 g}2s?ia?saec i 5 g S? 4*^ 4; d --sb 4; 5 CO o N C U 1 ! ! c aNiO^OifOvOQOO^f)lN" I:s3oW'r3 'r32 : pO.Ii- . Hsi-g Do Os afIsOsHsOgQ 5 S ? i s 4.4.J. -i "CMC *0>C i g -fO00^ --1-rf" XX XX X SSS SCCOO C0O0 C0O3 0CO3 C*O1*C3O0 E 7 *-- or1, *-- i' - ~Tr c*r < S g ` s s s ;ss0n3 0--5>Cr~O0-*0 Cr-O. I- c? w 03 o i *o or c-i n o <ci C! Cl Ol Ol lO N ?>0 1^lO Cl -H D f-*01 Q C C IQ 031 000 5`-0r C--Sr.r--o*!<c4o<-HCS^'Tl^^dfc3-Od'3tT'dC3NOCc>-o^O.c-cti^cn!c- -h o C*5 co -n *r >o oci >o c i >o o Tf Tf C l -> O r--< ^ C C IN C IC c-o oi oi ci <n cc t a CCOdOOCO o o c Intraperitoneal injection series jt>'2Saji'S ;- : SI.2i'Sg1^3'5?0-T-5Sg3!$T^o3!e.'S0gS'rg^'^cBTES3^?Z-_|IegX_'a-S3'_=J03clo_SxS0J3gt-O; HISTOPATHOLOGIC-AL FIN D IN G S Liver.- F at was found in the cytoplasm of tlio liver colls in ab o u t 40 percent of all th e anim als on test, exclusive of controls. Fine and medium-sized fat droplets were usually found in cells scattered throughout the lobule, rather than about the central vein. Capillary congestion was infrequent. These findings would seem to indicate th a t the fatty m etam orphosis was the result of toxic action on the cells rather than the result of circulatory disturbances. Significant am ounts of fat were found m ore frequently in the anim als dying on test than in those which were killed. T he greatest am ounts of fat were found in the injection-series anim als th a t died during test. The difference between am ounts of fat found in the anim als th a t died and those th a t were sacrificed was g reater than the variation in am ounts of fat noted among the anim als to which lead compounds had been adm inistered in various ways. F a t was conspicuously present in the anim als injected with red lead. I t occurred in five of the six anim als killed 355 days after injection. Areas of necrosis, usually small b u t sometimes fairly large, were found in the livers of ab o u t 1.5 percent of the exposed anim als. This observation had been m ade by others (3). The necrosis was of the coagulation v ariety and occurred in various portions of the lobule. C apillary congestion occurred in less than 2 percent of the animals, and usually was more m arked about the central veins, although in q u ite a n um ber of the. anim als th e whole lobule was involved. Congestion also was generally slight. Increase in peri portal connective tissue was found in ordv four anim als. T hese find ings cannot be regarded as significant. Spleen.-- T h e principal pathological change noted in the spleen was the co n sistent increase in extra- and intra-cellular pigm ent which for the most p art gave the iron reaction with acidulated potassium ferrocyanide. T he presence of hemosiderin and hem orrhages in the splenic pulp in anim al experim ents h as previously been reported (3). Some p ig m entation was found in a few of the controls b u t it was by no m eans as frequent or as m arked as th a t found in the anim als exposed to the lead compounds. T he presence of pigm ent (hemosiderin) in th e spleen was most, frequently noted in the inhalation-series, less in the ingestion-series, and least in the injection-series. T he average q u a n tity of pigm ent for all anim als followed this sam e order, and was irrespective of w hether the anim als died or were killed. H em osiderin was found in ab o u t tw o-thirds of the anim als dying on test and in a very few more of those sacrificed, regardless of the route bv which the lead compounds were adm inistered. 36 The inhalation-series anim als th a t were allowed to survive 3 to 4 m onths after com pletion of exposure sliowed less hemosiderin in the spleen th a n did those killed in the first week following conclusion of exposure. Among the anim als dying on test the most pigm entation was found in those in which death occurred in less than 5 days. The am ount of pigm ent in the spleen of ingestion-series anim als th a t were allowed to survive from 3 to 4 m onths following completion of exposure was com parable with th a t found in the inhalation-series anim als of the sam e period. No anim als in the ingestion group were killed in the first week following exposure. In the intraperitoneal-injection-series the time interval between injection and examination appeared to make no difference in the distribution of pigm ent over this interval or the quantity produced in the animals sacrificed. Less pigm ent was noted in the anim als th a t died on test b u t the distribution was similar. T he am ount of splenic pigm ent found in animals injected with the various lead compounds was notably greater in those treated with lead monoxide, lead carbonate, and lead hydroxide, respectively. T h e am ount of pigm ent was low or absent in the, anim als injected with lead sulfide and with lend silicate. T here was less variation in the am ount of splenic pigm entation produced by the various lead compounds when administered by the inhalation m ethod. Lead monoxide, red lead, and lead phosphate caused the largest am ounts, but these values did not vary greatly from th e am ount produced by the oth er com pounds used in this series. Ingested m etallic lead, lead carbonate, lead phosphate, lead monoxide, and lead sulphate caused production of approxim ately equal q uanti ties of hemosiderin while lead sulfide, lead dioxide, and lead sulphate caused a slightly lesser am ount. From the am ount of splenic hemosiderosis it appears th at with the injection m ethod the various lead compounds acted as individual entities, since considerable variation was shown. In the inhalation and ingestion tests very little variation in the am ount of pigment produced by th e various com pounds used was noted. I t is possible th at in the inhalation series there was some com plication by ingestion. T he lead salts in these two groups m ay have undergone chemical changes before absorption and consequently showed quite similar effects. T h ere appeared to be no relation between the am ount of pigm ent in the spleen and the am o u n t of the lead com pound ingested. Congestion of the cavernous veins and small hem orrhages in the pulp, either alone or together, was found in more than half of the ani mals. The am ount of hem orrhage and the extent of the congestion varied considerably. An increase in the reticuloendothelial tissue was noted in a sim ilar num ber of anim als. T h e M alpighian corpuscles and the splenic pulp were of usual appearance. F ig u r e 12.-- A rea of m ark ed ly d isten d ed tu b u les, an d in te rstitia l n ep h ritis w ith m uch fibrosis in kidney of guinea pig killed 3f>(> d ay s a fte r a single intrap eritoncul injection of 0.1 gin. of red lead. F iguhk in .-- K alty iih-Iainorphosis an d areas of necrosis in liver of guinea pig fed 7.0 mg. of red lead over a period of 51 days. 37 Lungs.-- M ost of the lung tissue examined was obtained from in halation and ingestion-series anim als. Pneum onia was found in 36 of 134 anim als studied. T he frequency was essentially the sam e in both the inhalation and the ingestion-series. The frequency was greater in the injection-series b u t the total num ber of anim als examined in this group was far less than in the o th er two groups. Pneum onia was more frequent in the sacrificed anim als than in those th a t died on test. S ubacute bronchopneum onia was found in 22 anim als. In 0 of these proliferative changes in the bronchial epithelium had taken place giving the consolidated areas an adenom atoid appearance. This type of pneum onia appeared in both the sacrificed anim als and those dying on test and was ap p aren tly unrelated to the com pound used, mode of exposure, dose, or length of time following beginning of exposure. Acute diffuse pneum onitis was found in the rem aining 14 of the 36 animals with pneumonia. Tw o-thirds of the animals with this con dition had died on test. No pneum onia was found in 16 controls. T he acute pneum onitis, which was found m ost frequently in the inhalation-series, could have been the result of local irritation by the inhaled compounds. The occurrence of the subacute bronchopneu monia appeared to have no direct hearing on the absorption of the lead compounds unless it was an indication of general physiological debility. The presence or absence of pneum onia had no appreciable influence on the occurrence of n ep h ritis in either the anim als th a t died or those th at were killed. The same was true of presence of the splenic pig m ent in relation to pneum onia. Pneumonia occurred as frequently with hepatic fat as without in the animals killed. Among the animals that died on test, pneumonia was about half as frequent in those with fatty livers as in those without demonstrable hepatic fat. Pulmonary congestion and edema did not appear to be a significant finding. In the inhalation-series the presence of appreciable quantities of dust was noted in over half of the animals studied. The dust particles were found in macrophages and connective tissue cells in the alveolar walls and in the connective tissue about small bronchi and blood vessels. It is interesting to note that repeated examinations failed to show the presence of dust in the lungs of the animals that had in haled lead carbonate. No similar dust was noted in the lung tissue from the controls or in the animals which had been fed lead compounds. K idneys.-- A su b acu te in terstitia l nephritis was found in 42 of 214 animals. The change consisted of a cellular infiltration of wedgeshaped areas in the cortex, usually in the pyram ids. T he affected areas varied in size from only a few cells to large portions involving 38 several glomeruli, the capsules of which were often thickened. In some affected regions a scant to considerable fibrosis was noted and was best identified with picroindigocarmine. The nephritis usually occurred unilaterally, infrequently bilaterally. In a number of the kidneys only a single small area of the cortex was affected. I t was found m ost frequently in the injected anim als, less frequently in those which h ad been fed lead com pounds, and least frequently in the inhalation experim ents. In the injection series the frequency of nephritis was about 1 to 3; in the ingestion series 1 to 4; and in the inhalation series 1 to 7. In a control group of anim als the frequency was 1 to Hi. R egardless of the m ethod of adm inistration, nephritis occurred som ewhat less frequently in the animals dying on test than in those which were killed. Slight to m oderate congestion of the interstitial capillaries of both the cortex and m edulla was noted in about one-third of the animals. The glomerular tufts appeared uninvolved and the renal epithelium showed no changes of note. T he presence of pigm ents in the spleen bore no relation to the presence of nephritis. Adrenals.- Slight to m oderate congestion of the capsular capillaries was found in a very few of the anim als. T he am ount of fat in the cortex varied from none to a very large am ount, a usual finding in any sim ilar series of guinea pigs. Heart. No changes of note were found. Pancreas.-- No changes of note were found. Tissues from o th er organs were not examined as t hey were used in making quantitative chemical determ inations. Peritoneum. When injected into the peritoneal cavity, lead and its compounds form nodules th a t are somewhat similar to those pro duced in the sam e tissue by inorganic m ineral dusts th a t produce an inert type of reaction (4). The nodules studied, which consisted of large masses and num erous small clum ps of d u st more or less uniform in size, occurred in anim als receiving a single injection 104 to 405 days prior to exam ination. These dust, clum ps had the appearance of having been taken up by m acrophages b u t were so num erous th a t they obscured the outline of the cell. Isolated particles were found in the ad jacen t subperitoneal connective tissue and throughout the m atrix of the nodules and some occurred in ad u lt connective tissue cells or in m acrophages. A scant amount of fibrous tissue, with a predominance of collagen and very few nuclei, was found about, and interwoven betw een, the clum ps of the lend com pounds. The collagen strands stained blue with eosinm ethylene blue and appeared to have undergone some retrograde change. Occasional small, irregular deep-blue-staining masses were found in some of the nodules, generally near the m argins. No fine, K-'inti: II. Area of mi Iki hi Ic inter.-t it ini n e p h r i t i s in k i d n e y of guinea. pig exposed to lead >ilicale hy i nhalation. Average lead c o n t e n t of air 2.SI mg. per c u b i c loot. rl ot a I ex posui e. dN1- hours. An i mal k illed 2.`>I d a y s aft er i nit.ial exposure. 3!) granular, necrotic m aterial (a characteristic finding with some of the mineral dusts) was observed. The fibrous tissue capsule about the masses of lead compound was rather scant, especially that portion i adjacent to the peritoneal epithelium. Because of the peculiar and i consistent staining properties of the collagen and the presence of the u n d ifferen tiated small blue m asses in the nodules it appears that some solution or chem ical change in the lead com pounds had taken place. This local change m ay have been the deposition of lead or calcium salts in th e tissues. We have not y et found a reliable I histological staining m ethod, or a microchemical procedure, for differentiating lead and calcium salts in tissues. SU M M A RY | Pathological changes produced in guinea pigs by m etallic lead and i eleven lead com pounds were studied. These chemicals were adm inis tered by intraperitoneal injection, inhalation, and ingestion. The intervals of exposure varied from 1 to 407 days. ! The principal pathological findings were: Liver.-- F a tty m etam orphosis and, to a much lesser degree, coagula' (ion necrosis and capillary congestion. Spleen.--H em osiderosis and in terstitial hem orrhages in the pulp ' and congestion of cavernous veins. L ungs.-- S u b acu te bronchopneum onia and acute, diffuse pneui monitis. An excessive am ount of d u st was found in the lungs of animals exposed to the m aterials by inhalation. K id n eys.-- S u bacute in terstitial n ephritis in ab o u t 20 percent of the j animals. j Adrenals, heart, and pancreas. No changes of note. Peritoneum. The nodules formed bv injected lead compounds showed evidence of slow absorption of the m aterial. CONCLUSIONS T he fa tty m etam orphosis found in the liver appears to be the result of the toxic action of the lead com pounds on the cells rather than the result of circulatory disturbances. T he splenic hem osiderosis is p robably an indication of destruction of red blood colls and appears to be a fair pathological index of the relative toxicity of lead and ifs com pounds. It is interesting to note th at th e am o u n t of hem osiderosis in the spleen produced by the various lead com pounds closely follows the relative toxicity described in the first part of this report. I A cute diffuse pneum onitis was m ost frequent in the anim als exposed to th e lead com pounds by inhalation and m ight be the result of irrita tion of the lung tissue. S ubacute bronchopneum onia appeared to 40 no direct relation to the method of exposure or the compound .<1 b u t could possibly be an indication of general physiological debility. There appeared no particular relation between the presence of penumonia and other significant pathological findings. The subacute interstitial nephritis occurred with sufficient frequency to be considered as im p o rtan t and could be. interpreted as an indication of a low-grade damage caused by the action of lead on the kidneys. The behavior of the dust in the peritoneal tissue indicates th a t the com pounds are slowly absorbed b u t (hiring the process the nodules have the appearance, of those produced by the inert group of mineral dusts. BIBLIOGRAPHY ( 1) Lillie, R. I t . and Pasternack, J. G.: R om anow sky staining w ith buffered solutions II. C urrent modification. Bull. Internntl. Assn. Med. M useum s. 15: 65 70. 1936. P ersonal com m unications from au th o r regarding recent modifications. (2) Personal com m unications from D r. It. D. Lillie. (3) P etri, E.: P athologische A natom ic und Histologic dor V ergiftungen, H enke und Lubarsch. Ju liu s Springer. 1930. (4) Miller, J. W. , and Sayers, R. R.: M icroscopic appearance of experim entally produced d u st nodules in th e p eritoneum . P ublic H e a lth R ep o rts 50: 1619-1628. 1935. O t