Document DMLrKMwE4vwJxaNw55NKb0RNO

y- (I- Reprinted from Th e Jo u r n a l or Ih x t o STOia l Ht g ib x b a n d To x t c o l o g t Vo!. 22, No. 9, November, 1940 EXPERIMENTAL STUDIES ON THE INGESTION OF LEAD COMPOUNDS* Ro b e r t A. Ke h o e, Ja c o b Ciio b a k , Do n a b d M. Hu b b a r d , Ka r r Ba mb ac h , Ro b er t R. Mc Na r t a n d Ro be r t V. St o r t Kettering Laboratory of Applied Physiology, University of Cincinnati, Cincinnati, Ohio. HREE general modes of study excretions of exposed persons, has Thave been employed by experi been correlated with clinical data. mental and hygienic workers in For many reasons the third method the determination of the degree aonf dapproach appeared to us to offer the significance of human exposure to lead best means for the practical solution compounds. First, the results of ani of a problem which presented itself a mal experimentation have been ap number of years ago. Accordingly the plied more or less directly, and, in rates of urinary lead excretion of some instances, much too literally, to groups of workmen in a variety of lead man. Second, the extent of an exist trades, determined under conditions ing lead exposure, expressed on the of exposure and after discontinuance basis of the measured lead content of exposure, were correlated with the of food materials or drinking water, or, presence or absence of symptoms and in industry, in terms of the quantities signs of lead intoxication so as to of particulate lead compounds in the furnish a practical basis of differenti atmosphere of work-rooms, has been ation between safe and dangerous set against clinical observations on occupational conditions (1, 2). The exposed persons. Third, the relative expression "practical basis of differen magnitude of human lead absorption, tiation," is used advisedly here, in as revealed by the determination of that our initial definition of degree of the lead content of certain tissues and hazard in terms of lead excretion (1) * Received for publication June 12,1940. Read before the American Association of Industrial Hygiene, New York, June 4-5, 1940. was tentative and frankly pragmatic, as are all other available definitions of lead hazard. The method of study, 381 N 24997 fl' 382 JOURNAL OF INDUSTRIAL HYGIENE AND TOXICOLOGY hot. M, no. 9 however, based on the functional responses of exposed persons, gave promise of revealing the broader pic ture of lead metabolism in the human organism and thereby providing wholly valid and permanent criteria for clinical and hygienic practice. In any final sense, safety in relation to lead exposure, whether occupational or otherwise, must depend on a com prehensive knowledge of the physio logical behavior of lead, and the mani fest need for precise quantitative standards based on such knowledge has prompted us to increase our efforts in both intensity and scope. When it became evident that men in general were absorbing and excreting appreciable quantities of lead (3, 4, 5, 6, 7, 8), that this was not a recent or localized phenomenon, and that lead in some small quantity entered into the normal physiological processes of the human organism, it followed that some level of lead absorption must be regarded as wholly devoid of any injurious effect upon man. As the logical outgrowth of this concept we sought to obtain fairly compre hensive information as to the natural sources of the lead which occurs in food, as well as the incidental and acci dental means of contamination of foods and beverages (3, 7, 9), and to deter mine the quantities of lead regularly met with in the American dietary (4, 5, 6, 7). The ultimate disposition of the lead present in food and drink wa3 also studied in a series of experi ments in which the dietary lead intake as well as the lead output in the feces of normal human subjects was fol lowed over a period of months (4, 5,7). The facts obtained from these studies have been amplified and confirmed by further studies, implemented by more precise means for the collection of samples for analysis, and by recently developed analytical methods of great sensitivity and accuracy (10, 11, 12, 13, 14). Certain of the data of these later investigations are shown below, in illustration of the magnitude and variability of the lead content of the present freely chosen diet of healthy North American adults. Our chief attention, however, will be given to the presentation of some of the more ob vious results of experiments designed primarily to determine the relation ships obtaining between lead intake and output at various levels of inges tion over long periods of time and ulti mately to establish the threshold of intoxication for lead when ingested by man. It may be advisable to recall the fact that lead is taken in by the in dustrial worker largely through inhala tion of particulate lead compounds, and that severity of exposure is ex pressed commonly in terms either of the measured lead content of the air, or of the estimated lead intake per day by inhalation. Neither of these ex pressions has any meaning when ap plied to lead taken orally, and at tempts to identify the toxic level of daily ingestion with that of daily inhalation are ill conceived. The studies described here are concerned chiefly and most immediately, there fore, with the safe limits of lead inges tion in food and beverages on the part of the general population. On the other hand, the ingestion of lead by industrial workers is not without hygienic significance, however slight may be its relative importance. From the viewpoint of a physiological ap- DUP050312604 Nov. mo] INGESTION OF LEAD COMPOUNDS 388 proach to the problem of lead intoxi cation, moreover, the rate of lead ab sorption as compared with the rate of lead excretion is of primary impor tance regardless of the avenue of entrance of lead into the body. Th e Le a d Co n t e n t o f t h e Pr e s e n t Ame r ic a n Die t Table 1 gives examples, from recent observations, of the range of varia- wide variability of tlie results on the feces of Subject M. R. and from the occurrence of an unusual number of high values within a short period of time, as shown by the large probable error and the Standard Deviation in relation to the mean value.) The similarity of the results for food and feces implies that the lead in food is not effectively absorbed by the ali mentary tract, and this is verified by TABLE 1 Da il y Oc c u r r e n c e o f Le a d in t h e Fo o d a n d t h e Co r r e s p o n d in g Fe c es o f Th r e e No r ma l Ame r ic a n Ad u l t s IilllAD IN MG. FEB 24 BBS. irBEQUBHCIBSOr OCOtJERBNCE Or tWAXTHTEB OF ISAS INDICATED M. R. In food* E. B. H.D. M.R. In feces E. B. H.D. 0-0.099 4 0.10 12 ii 59 0.20 7 13 42 0.30 8 13 10 0.40 1 12 3 0.50 141 0.60 and over 2 3 2 738 1 11 37 6 12 42 5 13 25 264 461 654 Totals................ 31 56 121 31 56 121 Mean..................... P.E......................... S.D......................... 0.270 0.017 0.137 0.352 0.016 0.181 0.220 0.007 0.110 0.377 0.037 0.301 0.338 0.016 0.172 0.247 0.007 0.121 * Lead in food includes that in. all beverages for subject E. B. but excludes that in water for subjects M. R. and H. D. The latter amounts to 0.02 mg. daily. bility of the daily intake of lead in the food of three normal healthy young adults who provided duplicate sam ples of all food and beverages con sumed. The daily alimentary lead output for the corresponding period is also shown for each subject, and may be seen to be of the same order of magnitude as the intake, inconformity with our previous observations on other subjects (4, 5, 6). (The one appreciable discrepancy arose from the the significant positive correlation between the individual daily food samples and the corresponding indi vidual fecal samples, with respect to their lead content. In the caselffMt Subject H. D., for example, the com parison of 121 daily food samples with 121 twenty-four-hour samples of the feces for the following day, yielded a correlation coefficient of 0.31 =fc 0.06; when all the samples were combined to represent forty-eight-hour periods, DUP050312605 384 JOURNAL 05 INDUSTRIAL HYGIENE AND TOXICOLOGY [ol; 22, no, 9 thereby reducing the effect of varia tion in alimentary emptying time, their correlation coefficient was 0.63 =fc 0.06. TABLE 2 Le a d In t a k e a n d Ou t p u t in a No b ma l Hu man Su bj e c t (H. D.)* tdBADIN FOOD AND WBHKLTPSBZOSB DBXNKf IN MQ LaAD BXCBETED IN 1CG Total In feces In urine April 1st............. 2nd............ 3rd............. 4th............. 2.03 1.52 2.08 1.50 2.87 1.78 1.84 1.75 2.67J 0.20 1.68 0.10 1.67 0.17 1.61 0.14 December 35th........... 36th........... 37th........... 38th........... 1.35 1.24 1.17 1.78 1.67 1.50 1.44 2.62 1.35 1.28 1.28 2.37 0.32 0.22 0.16 0.25 April 51st........... 52nd.......... 53rd........... 54th........... 1.65 1.38 2.24 1.42 2.43 1.65 2.24 1.79 2.16 1.46 2.07 1.57 0.27 0.19 0.17 0.22 August 68th........... 69th........... 70th........... 71st............ 1.63 1.77 1.41 1.69 1.88 2.07 1.85 1.76 1.67 1.74 1.58 1.43 0.21 0.33 0.27 0.33 16 25.76 31.14 27.59 3.55 * Lead taken in by inhalation and ex creted in perspiration ignored. t Duplicate water samples were analyzed daily during last 4 periods. To all other weekly totals were added 0.14 mg. At An aberrant result of 4.00 mg. obtained on one day of this week and not represented in the duplicate food sample was reduced to the mean value of 0.22 before the sum mation. Representative data on the rela tionship between gross daily intake of lead (exclusive of that inhaled) and gross daily output of lead (exclusive of that lost through perspiration, ex pectoration, insensible desquamation of the skin and loss of hair and nails), are given in tables 2 and 3. It is apparent that there is an approximate balance, and considering the quanti ties of lead involved, as well as the obvious smallness of the factors on both sides whichhave been disregarded TABLE 3 Le a d In t a k e a n d Ou t p u t in a No b ma l Hu man Su bj e c t (E. B.)* BUCCXSSIVS TTOBRL? FSBXODS LEAD IN FOOD AND DRINK IN ucu LEAD BXCHETBD IN MG. Total In feces In urine January 1st............. 2nd............ 3rd............. 4th............. 1.24 3.36 2.12 1.90 2.11 2.33 1.58 2.73 1.85 2.04 1.32 2.47 0.26 0.29 0.26 0.26 February 5th............. 6th............. 7th............. 8th............. 2.73 2.53 2.69 2.94 2.50 2.35 3.37 3.60 2.26 2.03 3.14 3.41 0.24 0.32 0.23 0.19 19.51 20.58 18.52 2.06 "`Lead taken in by inhalation and ex creted in perspiration ignored. The latter at minimum during this period. through present technical necessity, the balance is so nearly exact as to be quite convincing. In this connec tion Calvery's (15) comments on our previous and similar data may not be ignored. This investigator failed to note our reference to the lead content of the drinking water of our experi mental subjects and suggested errone ously that this factor had been ig nored. He also by implication gave undue weight to the factor of lead inhalation and suggested that our DUP050312606  Nov. 1940] INGESTION OP LEAD COMPOUNDS 38S failure to provide a constant environ ment in this respect tended to vitiate our conclusions. There are adequate reasons for disregarding the factor of normal lead inhalation in experiments of this type at the present time,-- first, because this factor is not unpre dictable in its minute general magni tude under the conditions of our obser vations,* and second, because it is not feasible to carry out prolonged obser vations on the normal metabolism, of human beings under artificial experi mental conditions which impose undue restraints on normal life and behavior. The lead content of food and bever ages is clearly the predominant source of normal human lead intake, and the foregoing data partially illustrate the opportunities presented to 3 experi mental subjects for lead ingestion from this source. Table 4 extends the pic ture by giving the results of similar observations on 12 subjects whose diets were somewhat variable both in quantity and quality. The whole range of variability and the frequen cies of occurrence of various quantities of lead in the daily diet are here por- * Our subjects spent their working day in a laboratory building from which, leadbearing materials have been excluded, so far as possible, and in which such lead com pounds as are required are segregated and handled with rigid precautions against their dissemination. Atmospheric dusts are eliminated to a considerable degree by filtration of in-coming air. The subjeotB lived in suburban Cincinnati. The data of Bloomfield and iBbell (16) give some direct basis for evaluating the extent of general atmospheric lead content, and our unpub lished results on the correlation between certain low concentrations of lead in the air of industrial plants and the rate of uri nary lead excretion indicate that the inhala tion of lead by normal persons under our experimental conditions is less than 0.1 mg. per day, of which only some unpre dictable but doubtless small proportion is available for absorption, the remainder escaping with the expired air. trayed. These data relate to the commonly available food in Cincin nati and its environs, but they repre sent the facts as to the daily lead intake of men generally in the United States of America, since they are in agreement with our observations on persons in all parts of the country. Table 5 gives the results of some recent TABLE 4 Da il y Oc c u r r en c e o p Le a d in t h e Fo o d a n d Fe c es o p Tw e l v e No r ma l Ame r ic a n Ad u l t s Wit h o u t Co r r e s p o n d e n c e a s t o Time LEAP IN 116. FEN 24 h o u r s FREQUENCIES OF OCCURRENCE OF QUANTITIES OF LEAD INDICATED In food In feces 0-0.199 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.80 2.00 and over 259 583 143 43 12 9 2 3 1 8* 248 414 95 30 5 2 3 1 5* Totals........... 1063 803 * Mean and prob- able error ... 0.315 0.004 0.288 0.004 Standard devi ation............. 0.199 0.180 * Omitted from calculations. observations made in 10 cities in the United States. The variation among individuals with respect to the mean lead content of their daily diet, as illustrated in table 1, has been found to be due chiefly to differences in the quality of their food, (i.e. to their choice of food, or to differences in the preparation of food in various households and restau rants), rather than to gross differences DUP050312607 386 JOURNAL OF INDUSTRIAL HYGIENE AN& TOXICOLOGY [nol. 22, no. 9 in the quantities of food consumed. Nevertheless it seems profitable to examine the facts as to the gross quantities of food likely to be con sumed daily by adults, and to note the actual concentration of lead in such food aB a whole. We have assembled our data on the food of 12 healthy subjects in table 6, in which attention is called to the theoretically permis sible limit of lead concentration in terms of the present tolerance estab- TABLE 5 Le a d Co n t e n t o f Ra n d o m Sa mp l e s o f Fe c es o f No r mal Pb b s o n s f r o m Te n Wid e l y Sc a t t e r e d Ame r ic a n Cit ie s avoidance of any general increase in the lead content of a large number of food materials with a present minimal lead concentration is desirable. Our observations over the past 15 years have given us no reason for believing that any appreciable or pro gressive change in the lead content of TABLE 6 We ig h t o f Fo o d Co n s u me d Da il y No r ma l He a l t h y Ame r ic a n Me n En g a g e d in Ph y s ic a l La b o r by (Including all beverages except water and large volumes of beer and wine) (Combined data on 12 men varying from 120 to 240 pounds weight) XJ3AU XN MG. SAUPLS OP FECES FBUHTTUNCraa OP OCCURRENCE 07 QUANTOIES OF DEAD INDICATED WEIGHT OF FOOD IN GH. FBEQ'UaSCXBS 07 OCCURSSNOB OF WEIGHTS 07 POOD INDICATED 0-0.199 0.20 0.40 0.60 0.80 1.00 1.20 2.00 and over 26 43 17 7 2 4 2 1 800-999 1200 1600 2000 2400 2800 3200 3600 ll 92 271 409 232 35 4 1 Total....... ...................... 102 Total................................. 1115 Mean and probable error... 0.398 0.021 Standard deviation............ 0.310 Mean and probable error.. 2135.16 7.87 Standard deviation............ 389.53 lished by federal authority in the United States. We do not imply that the regular occurrence of 7.62 mg. of lead in the daily adult diet is believed by government authorities to be safe, nor that there is a twenty fold factor of safety in the present situation as we have presented it. We merely call attention to the facts as they now exist and as they would be found to exist if all foods were as high in lead as certain items are now permitted to be. Obviously the Permissible, mean lead content = 7.62 mg. = 3.57 p.p.m. Actual' mean lead content = 0.32 mg. = 0.15 p.p.m. American food, or in the magnitude of the lead intake of the average Amer ican citizen has occurred during that period. It scemB probable, however, that human ingestion of lead has decreased rather than increased in reeent generations. Such evidence as we have, although inadequate to prove the point, also indicates that the gen eral level of lead content of the Amer- DUP050312608 Nov. 1940J INGESTION OF LEAD COMPOUNDS 387 ican dietary is somewhat less than that current in France or England. There is thus little occasion for anxiety over the lead content of American food materials as they are now handled, processed, and distributed. From time to time, however, there are ex amples of foods contaminated with lead to an unusual and even danger ous degree, and greater care can be exercised in avoiding the introduction of lead into food at little or no cost or inconvenience except that asso ciated with the recognition ofnumerous unnecessary contacts of foodstuffs with lead-bearing materials. For these reasons there is need of responsible and effective public and private agen cies for the detection of such occur rences and for the correction of the conditions which bring them about. Le a d In g e s t io n a n d Le a d Ex c r e t io n a t Ab n o r ma l Le v e l s The results of studies carried out over a period of years in industrial plants, the details of which have been reported in part elsewhere (1, 2), have indicated that an approximate balance exists between the lead ab sorption and the lead excretion of men who are subject to occupational lead exposure within certain limits. Such a balanced metabolism is appar ently differentiated from that of the normal individual only in that it is maintained at a higher level after a period of absorption of lead into the tissues to a point determined largely by the magnitude of the regular lead intake. Expressed somewhat differ ently, these observations have shown that if the lead exposure of an indi vidual be increased within certain limits above the normal or commonly observed levels, the increasing quan tity of lead absorbed into the tissues results in a progressively increasing rate of elimination until eventually excretion approximates absorption. The determination of the limits within which such a balance can be achieved without injury or illness would, obvi ously, be of the utmost practical usefulness not only in the field of in dustrial hygiene but also in relation to the health and safety of the public. We, therefore, entered upon a series of experiments in which lead in known quantities was administered regularly to normal subjects over extended peri ods of time during which their daily lead intake in food and beverages and their daily output in the feces and urine were followed. (The intake and output of calcium and phosphorus have been determined simultaneously over the greater portion of the period of study.) In order to maintain the induced lead exposure of our subjects within safe limits, the dosage of lead was kept below that previously found to be toxic for experimental animals, and also below that necessary to pro duce concentrations of lead in the blood and urine which, from industrial experience, could be regarded as dangerous. After a preliminary period of 28 days of clinical and experimental study which demonstrated the exist ence of a satisfactory state of health and physiologic reactivity, one young man was started on 1.00 mg. of lead acetate per day administered in solu tion in a dose of 0.333 mg. with each of three meals. This experiment has been continued over a period of almost 3.) years up to the present (May 1940). A second subject began taking a daily DUP050312609 388 JOURNAL OP INDUSTRIAL HYGIENE AND TOXICOLOGY [ml. 9$, no. 9 dosage of 2.00 rag. in a similar manner at the end of February 1939, after 56 days of preliminary clinical and metabolic study, and has continued sinee on that dosage. At weekly in tervals, each subject was given a phys ical examination, a complete micro scopic examination of his blood, and an analysis of his blood for its lead content. Blood smears have been made daily for counts of stippled erythrocytes and reticulocytes. Aside from certain inconveniences associated with residence in or near the labora tory, and the necessity of collecting duplicate samples of all food and bev erages as well as all excreta, these sub jects have led normal and adequately active lives, and despite a somewhat detailed supervision of their daily activities, they have maintained a satisfactory attitude of mind, going about their daily duties with regu larity and without apprehension or undue introspection. During the course of the observa tions up to the present time, no clin ical evidence of the slightest harmful effect has been recognized by either subject or has been detected by our examinations. No loss of appetite or weight, and no decrease in general health or in the sense of well-being has occurred. Both subjects have had intercurrent respiratory infections of minor severity which ran their normal course. One (M. R.) had a typical attack of food poisoning with diar rhea and vomiting, with recovery in 3 days. He was not too ill to collect all the materials evacuated by diar rhea and emesis, so that experimental observations were not interrupted. No appreciable changes in the lead metabolism occurred at this time. The same subject developed a faint but definitely punctate blue deposit at the margin of a central incisor in a localized area overlying a small pocket of purulent material. This was seen first on February 1st, 1938 and was allowed to remain untreated until October 24, 1939. During this in terval the deposit varied somewhat TABLE 7 OCCURRENCE OP STIPPLED ERYTHROCYTES in t h e Bl o o d o p No r ma l Hu ma n Su b j e c t s a t Va r y in g Le v e l s o p Le a d In g e s t io n BRJ$VXJBT> BBYTHBOCTTHS PBB SO FIELDS FREQUENCIES OF OCCURRENCE OF STIPPLED BBTTHROCTTZa IN NUMBERS INDICATED Normal subjects Normal subjects (control) (test) H. D. C.H. M. R. E. B. 0-1.9 2 4 0 8 10 12 14 16 18-19.9 363 97 690 183 42 25 162 93 7 15 67 51 2 7 24 24 1 4 15 15 679 333 333 11 11 Totals............. 415 161 962 383 Mean.................. 1.01 3.10 2.02 3.23 Probable error.. 0.04 0.19 0.05 0.11 Standard devi- ation............... 1.10 3.54 2.14 3.07 in intensity, almost but not quite dis appearing on two occasions when the gum seemed on the way to spontane ous recovery. When the lesion was evacuated and treated the deposit disappeared promptly and entirely, remaining absent during the healing process, which was complete, and fail ing to return. The other subject (E. B.) has not developed any gingival DUP050312610 Nov. mo] INGESTION OF LEAD COMPOUNDS 389 discoloration despite the presence of a considerable importance to note that slight gingivitis which has varied in the variability in all the subjects as degree in accord with his persistence in indicated by the Standard Deviations, the care of his teeth. is of the same degree. The absorption No microscopic blood changes indi of lead evidently failed to induce cative of the effects of lead absorption either a constant or an intermittent have been observed in either subject. stimulatory effect upon hematopoesis. Table 7 groups the data of the daily- Certain other factors, however, had I observations on the' stippling of the a well defined influence upon the num ' i erythrocytes of the two subjects, in ber of stippled'erythrocytes. Atten- BASOPHILIC STIPPLING OF SUBJECTS M.R. AND E. B. DURING ORAL ADMINISTRATION OF LEAD Fig . 1. Seasonal variation in occurrence of stippled erythrocytes in blood of human adults. The points on the curve represent the summation of the numbers of- stippled erythrocytes found during the indicated periods in the examination of 50 microscopic fields (approx. 12500 erythrocytes) on a number of days, divided by the number of days on which observations were made, and multiplied by 30 so as to express them in terms of months. comparison with results obtained over comparable periods of time on 2 normal healthy young men who were not ingesting or absorbing abnormal quantities of lead during the period of the observations and who had no history of abnormal exposure to lead in any prior period. No evidence of any effect of the lead absorption is seen in comparisons of the mean values or the distributions of the frequencies under the different rubrics. It is of tion is called to the wide fluctuations which occur on a seasonal basis, roughly, in the two subjects M. R. and E. B., as shown in figure 1. These fluctuations occurred at approximately corresponding times and to a similar extent in the control subjects C. H. and H. D., taking place in the one (H. D.) at a low level of this hematopoetie response, and in the other (C. H.) at a relatively higher level, as indi cated by the averages for a year. DUP050312611 ji 390 JOURNAL OF INDUSTRIAL HYGIENE AND TOXICOLOGY [ml. 22, no. 9 (There is some question as to the most data on the frequencies in table 7 valid and illustrative means of ex would seem to portray all the facts \H pressing the results of blood examina faithfully.) The results on Subject tions of this type. It is not clear M. R. by months vary above and be whether the frequency of occurrence low the average yearly values obtained of stippled erythrocytes in the blood in control subjects C. H. and H. D., is a measure of intensity of this remaining within the normal limits as hematopoetic reaction or whether defined by these subjects. The aver the actual number of such forms found age of the results on Subject E. B. i, is the better criterion. It seems prob- for the year is considerably higher TABLE 8 Le a d In t a k e a n d Ou t p u t or No r mal Su b j e c t (M. R.) Du r in g Or a l Le a d Ad min is t r a t io n DEAD IN MO. INGESTED IK FOOD LEAD IN MG. EXCRETED SUCCESSIVE PERIODS AND BEVERAGES LEAD IN HQ. ;i OF 12 \PHBXS AND EXPERIMEN TALLY ADMINIS TERED Total la feces In urine RETAINED 1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th 11th 12th 13th 112.84 118.74 107.22 118.79 110.18 116.22 109.44 125.45 117.11 105.48 107.57 114.24 113.14 101.48 108.95 105.51 111.57 111.22 121.59 93.41 118.86 114.81 98.86 109.19 102.06 100.68 98.32 103.74 100.15 105.57 104.66 114.15 86.57 112.47 108.97 91.22 102.25 94.03 94.02 3.16 5.21 5.36 6.00 6.56 7.44 6.84 6.39 5.84 7.64 6.94 8.03 6.66 11.36 9.79 1.71 7.22 -1.04 -6.37 16.03 6.59 2.30 6.62 r-1.62 12.18 12.46 1476.42* 1398.19 1316.12 82.07 78.23 * Approximately 22.00 mg. in drinking water, 372.32 mg. in food and other beverageB, and 1082.10 administered in solution. 'i:l i1! able that both the frequency and the than that of Subject M. R. and some- numerical extent of the occurrence what above that for control subject !;! are important. We have chosen to C. H., but the difference cannot be express our results on a monthly attributed with any degree of eonfi! basis, by adding the total number of dence to the factor of lead absorption | stippled erythrocytes found on the in view of the comparison of the dis- several days of the month on which tribution of the frequencies and the observations were made, dividing by mean values as shown in table 7. the number of days represented, and Reticulocytes in the peripheral blood multiplying by 30. This mode of of both subjects varied within narexpression, when combined with the row limits without any definite sea- DUP050312612 New. 1940] INGESTION OF LEAD COMPOUNDS 391 sonal or other trends exeept some slight tendency toward increase in Septem ber, October and November. No parallelism between stippled erythro cytes and reticulocytes was apparent. The facts of greatest and most im mediate significance as derived from the study of analytical results on the. two subjects are illustrated in a series of tables and charts. Table 8 gives a both cases approximately 95% of the ingested lead was eliminated during the period of the observations, and about 94% of the eliminated lead was found in the feces. The output of lead in the feces increased greatly and abruptly above the preliminary level for these subjects (cf. tables 1 and 3), as soon as the administration of lead was initiated. (The fecal lead was TABLE 9 Le a d In t a k e a n d Ou t p u t o p No r mal Su b j e c t (E. B.) Dv r in o Or a l Le a d Ad min is t r a t io n BUCCE8SIVK F2JU0DS OF 4 `WEEKS LEAD INUG. 1K01BTBD IN FOOD AND RBVBBAGBS AND EXPERIMEN TAL!/? a d u t n t s * TEBBD DEAD CM MS. EXCRETED Total In fecee In urine DEAD IN MG. RETAINED 1st 2nd 3rd 4th 5th 6th . 7th 8th 9th 10th llth 12th 13th 60.94 59.76 65.34 63.01 63.61 64.77 64.46 63.81 63.34 62.87 62.09 58.89 57.30 55.55 54.85 57.16 62.87 61.79 63.52 64.93 60.34 55.40 56.11 55.65 56.62 53.87 53.20 52.27 54.32 60.14 58.71 60.45 61.67 56.78 51.96 52.43 52.36 53.31 51.14 2.35 2.58 2.84 2.73 3.08 3.07 3.26 3.56 3.44 3.68 3.29 3.31 2.73 5.39 4.91 8.18 0.14 1.72 1.25 -0.47 3.47 7.94 6.76 6.44 2.27 3.43 810.09* 758.68 718.74 39.92 51.43 * Approximately 7.00 mg. in drinking water, 76.76 mg. in food and beverages, and 727.33 administered in solution. greatly condensed summary of the results obtained on Subject M. R. over 39 lunar months, while table 9 shows the corresponding data on Subject E. B. in a more extended form made possible by the much shorter duration of the observations on this subject. Attention is called in these tables to the large and prompt loss of the greater portion of the ingested lead in the fecal evacuations. In greatly elevated on the first day after the beginning of the administration, and reached almost its maximal level within a few days, these facts point ing clearly to the passage of most of the ingested lead through the ali mentary tract without absorption.) The urinary output, on the contrary, increased slowly and gradually in response to the administration of 1.00 mg. daily (Subject M. R.), and some DUP050312613 392 JOURNAL OF INDUSTRIAL HYGIENE AND TOXICOLOGY [sol., no. 0 what more rapidly in the case of Sub ject E. B. on the higherdosage. Thus, the urinary lead output of Subject M. It. practically doubled in about 12 weeks, but continued to increasefor three times that length of time, while that of E. B. had doubled in less than 2 weeks, arriving at a substantially urinary lead concentration of the two subjects as calculated for each period of 28 days. The initial slope of the curve for E. B. is both steeper and shorter than that for M. It. In figure 2 are also plotted the blood concentrations as averaged for periods of 4 weeks from the results of the Fig . 2. The mean urinary lead eoneentrations were calculated from 60 daily analytical results, and the average lead concentrations in the blood were calculated from 4 weekly pairs of duplicate analyses. The dotted line in the lower section is the longer line in the upper section, superimposed upon the curve of urinary Concentrations, for purposes of comparison. maximal level in about 10 weeks. Apparently, an important point of difference in the reaction to these two. dosages consisted in the speed with which the rate of urinary lead excre tion increased to a practically maximal level. Figure 2 provides a graphic illustration of the foregoing differences in terms of the mean values of the weekly analyses. One notes here what we have found regularly in studying the results of occupational lead ex posure, that the lead concentration in the blood shows less proportional change for a given increase in lead absorption than does that in the urine. Two significant features of the ef- DUP050312614 Nov. INGESTION OP LEAD COMPOUNDS 393 feets of prolonged lead absorption on the lead concentration of the urine and blood are revealed by the considera tion of the facts in tables 8 and 9, and figure 2. First, it is apparent that the levels of lead concentration in the urine and blood in Subject E. B. are significantly higher than the corresponding levels in Subject M. R. in accordance with the higher dosage of administered lead. After making every allowance for individual varia tion in the metabolic responses of the two subjects, the conclusion is ines capable that the rate of daily lead absorption, regardless of the ultimate disposition of the lead in the body, is a significant factor in determining the concentration of lead in the mine and the blood. The second point of importance re lates to the length of time required for the development of changes in the lead concentration of the urine and blood comparable with the increases in the intake of lead. The lag here may be explained in part, no doubt, by the smallness of the quantities of lead absorbed from the alimentary tract, so that time was required to produce an appreciable effect. On the other hand, considering the fact that the ingestion and presumably the general rate of alimentary lead absorp tion over any considerable period of time were substantially constant for each subject during the entire period of the observations, the explanation of the gradual increase in these con centrations would appear to lie in a gradually increasing quantity of lead in the tissues of the subject. Obvi ously, as indicated above, the quantity of lead distributed in the tissues was not the only factor responsible for the elevation of the lead concentration in the urine and blood of the subjects. If it had been, the position of the sub jects with respect to these concentra tions should have been reversed at the point of termination of the urinary and blood curves of figure 2, since at this time, M. R. had retained more lead in his tissues as the result of the experimental regime than had E. B. (cf. tables 8 and 9). That retained lead was one factor, however, seems reasonably assured, and because of this assurance, coupled with the facts demonstrated by the study of the elimination of lead by persons whose lead exposure had been discontinued, the prediction is justified that when the administration of lead to these subjects is brought to an end, the lead concentration in their urine and blood will decrease rapidly to levels deter mined by the lead distributed, in the tissues of the entire body, after which they will continue at lower and slowly decreasing levels until equilibrium between intake and output has been restored. This prediction, in due time, will be put to experimental test. For present purposes, we shall omit a detailed examination and discussion of a number of factors which have influenced the absorption and excre tion of lead on the part of these sub jects. The practical significance of one matter, however, necessitates brief consideration. The daily variations in the concentration and output of lead in the urine were considerably greater than might be expected from the con densed results in tables 8 and 9 and from the curves in figure 2. The most cursory examination of data of daily observations revealed the importance of a factor which we have demon strated elsewhere (4, 6), i.e. that the volume of water available for excre DUP050312615 394 JOURNAL OF INDUSTRIAL HYGIENE AND TOXICOLOGY [vol. 22, no. 9 tion by the kidneys had a highly sig nificant relation not only to the con centration of lead in the urine but also to the quantity of lead eliminated daily. The effects of other factors were not so obvious. It seemed ad visable, therefore, to study the blood and urine at frequent intervals during the day to determine, if possible, the relationships obtaining between lead concentrations in the blood and urine, and the changes which occurred in both in response to the three doses of stage.) The urinary lead concentra tion, on the contrary, varied over a wide range during each 24 hour period, and the plotted curves of these concen trations are almost exactly opposite, throughout their course, to the cor responding curves of urinary volume. It is apparent, especially in the first group of observations, that no factor other than the volume of available water exerted any important role in the induction of variations in urinary lead concentration during these 24 VARIATIONS IN CONCENTRATION OF LEAD IN URINE AND BLOOD DURINO 24 HOUR PERIODS. [SUBJECT M.R.) Fig . 3. The analytical results appearing as points on the lower section were obtained on the urine voided at 2 hour intervals, and on blood samples taken in duplicate at 2 hour intervals. lead with the meals. Figure 3 shows hour periods. The absorption of lead the results of three sets of observations from the alimentary tract was too of this type on Subject M. R. at vari slight in its periodicity to cause de ous stages of the experiment. The monstrable variations in the concen blood showed little variation in its tration of lead in either blood or urine. lead concentration throughout any Figures 4 and 5 give graphic repre one 24 hour period, no change beyond sentations of the periodic and total the limits of analytical variation being quantities of the lead taken orally and demonstrable. Lead concentration of excreted in the feces and urine of the the blood increased progressively, two subjects over the entire period of however, from stage to stage of the ex the observations reported herein. periment. (There was no appreciable The abruptness of the gross increase change in the partition of lead between in the alimentary output of lead, the II plasma and formed elements at any gradual increase in the urinary output, i ': * j; il DUP050312616 Nm.lSffi] INGESTION OF LEAD COMPOUNDS 395 and the minuteness of the discrepan cies between intake and output, are brought into their proper perspective. There was a surprisingly small reten tion of lead in the tissues of these subjects from the beginning of the observation. Indeed, in the case of Subject M. R., the intake and output as measured, with the exception of three periods, one at the beginning, another about midway, and the other sponds closely to that in the urine. On this basis, our subjects probably excreted as much lead in this way during warm weather as in the urine, and the inclusion of such quantities on the elimination side would result in a virtually balanced metabolic picture. It seems probable, therefore, that the actual retention of lead on the part of these subjects wan considerably less than that indicated by our figures, LEAD INTAKE AND OUTPUT OFA NORMAL SUBJECT CMFO DURING ORAL ADMINISTRATION OF LEAD near the end of the record, were so nearly equivalent as to be only doubt fully beyond the limits of the com bined opportunities for error in the collection and analysis of the samples. Moreover, the excretory loss of lead from the body by way of the perspira tion does not enter into the recorded total output. Recent observations, which are in process of extension and verification, have indicated that the concentration of lead in the perspira tion of a normal individual corre- and that, in fact, it was of almost insig nificant proportions. Despite the practical conclusion drawn above, critical interpretation of certain facts necessitates the opinion that some degree of lead retention oc curred in both subjects. First, there is the fact, in accord with expectations, that the discrepancy between lead intake and output of Subject E. B. as measured, was greater for compar able periods of time than that in the case of M. R. Second, the concentra DUP050312617 396 JOURNAL OF INDUSTRIAL HYGIENE AND TOXICOLOGY [vol, Si, no. 9 tion of lead in the blood and also the urine of M. R. showed a slight but definitely progressive increase at vari ous stages in the observations (fig. LEAD INTAKE AND OUTPUT OF A NORMAL SUBJECT DURING ORAL ADMINISTRATION OF LEAD Fro. 6. Lead intake in food and drink, and lead output in feces and urine of a human adult on a dosage of 2 mg. Pb daily as a solution of lead acetate, in addition to that occurring in the diet. amination of the curves in figure 6, in which the cumulative totals of the differences between intake and output of lead as measured for the two sub jects were plotted against the succes sive periods of time, reveals a remark able fact. The cumulative retention of lead was slight in both cases, and also apparently irregular,- but if the attempt is made to fit a curve to the plotted points, the result is a straight line. It would be presumptuous at this time to draw a definite conclusion from this faet. The magnitude of the lead excretion in the perspiration must be accounted for, and certainly further study in the case of Subject E. B. is required. Doubtless other factors will have to be fitted into their proper place for the elucidation of the com plete picture. Until such time, the evidence for or against the existence of a completely balanced lead metabolism under these conditions is not wholly convincing. 3), with some tendency, perhaps tem The emphasis placed by the fore porary, toward increase in the most going remarks on lead retention under recent period (fig. 2). Third, an ex- the conditions of our experiment seems DUP050312618 Nov. 1940} INGESTION OE LEAD COMPOUNDS 397 likely to give undue prominence to this point. However great may be the physiologic significance of the concept of a balanced lead metabolism under conditions of normal or moder ately increased lead absorption, cer tain practical aspects of these experi mental observations merit the greater present consideration. It is apparent, for all practical purposes, that the healthy human organism responds to such limited increases in lead inges tion by a greatly increased and sub stantially equivalent elimination. It is also clear that the largest proportion of ingested lead, even when taken in the form of a solution most favorable for absorption, is not absorbed in the alimentary tract, but is carried through the body and eliminated in the feces. Dis c u s s io n The facts briefly outlined in the foregoing pages have a direct and important bearing upon the hygienic problem of lead as a contaminant of food materials. They do not justify the conclusion that the lead content of the food of the community may, with safety, be permitted to increase four-fold or more, but they do signify, we believe, that there is a reasonable public security against the dangers of insidious intoxication and gradual physical degeneration, which many have feared from the use of foods, especially fruits, generally available in this country. Such present secu rity should not be permitted to lull us into an attitude of indifference toward potential hazards in food production and processing, but should promote a somewhat greater intellectual poise in the investigation of the facts with respect to such hazards. It should also alleviate a somewhat hypochon driacal tendency on the part of a seg ment of our population, and allow attention to be foeused on the more serious problem of community health in relation to lead compounds, namely, the elimination of dangerous and dis abling occupational lead exposure. Perhaps an additional step toward the solution of the latter problem has been made in our attempt, as yet in complete, to define the limits of safety for one type of lead exposure in physi ological terms. The extension of sim ilar studies to controlled conditions of respiratory lead exposure may go far toward the ultimate goal. SuMMABT AND CONCLUSIONS The mean values for the daily in take of lead in the food and drink of three normal American adults, as determined by recent and precise analytical methods, were 0.24, 0.29 and 0.35 mg., as compared with values of 0.25, 0.34, and 0.38 mg., respec tively, for their daily fecal lead output during the same period. The total quantities of lead in gested with their freely chosen food and beverages, by two American adults during periods of 16 weeks and 8 weeks, respectively, were 25.76 mg. and 19.51 mg.; the corresponding quantities of the combined output of lead in the feces and urine of the two were 31.14 mg. and 20.58 mg., re spectively. A highly significant degree of cor relation existed between the lead con tent of the food for each daily period and that of the feces for the day fol lowing. In observations which extended DUP050312619 ,398 JOURNAL OF INDUSTRIAL HYGIENE AND TOXICOLOGY [vol. US,.no. 8 over a period of a year, 12 normal American adults, engaged in occupa tions involving a moderate degree of physical effort, consumed a mean daily weight of 2135 gm. of food and bever ages per person (excluding occasional large volumes of beer and wine), the mean lead content of which was 0.32 mg., equivalent to 0.15 p.p.m. Experimental studies were carried out on 2 healthy adults to whom 1 mg. and 2 mg. of lead, respectively, as a solution of lead acetate, were administered dailyin three doses taken with the meals, for periods of 39 lunar months, in the former case, and 13 lunar months in the latter. Clinical data were collected at daily and weekly intervals, while the daily oral intake and the daily alimentary and urinary output of. lead were deter, mined over the entire period of the study. The results may be sum marized as follows: (1) no definite or suggestive evidence of lead intoxica tion developed in either subject; (2) no microscopic evidence of any hematopoetic response to lead absorption was found in daily examinations of the blood; (3) fecal output of lead in each '.case increased abruptly with the initi: ation of the experiment, promptly reached a maximal level, and ac counted for the evacuation of about 90% of. the ingested lead from the body; (4) urinary lead concentration .and output increased gradually in each ease, (more rapidly and to a higher level in the subject on the higher dosage), reached a substantially constant level in somewhat less than .a year in the subject on the lower dosage, and in about 10 weeks in the other subject, and resulted in both eases in the elimination of about 5% of the ingested lead; (5) lead concentra tion in the blood of the two subjects increased in general correspondence with the increase in urinary concen tration, but to a somewhat lesser degree; (6) in the case of the subject to whom 1 mg. of lead was administered daily, the mean level of lead concen tration in the urine during the last 4 months of the observations was 0.068 mg. per liter, and the average concen tration in the blood for this period was 0.059 mg. per 100 gm.; (7) the subject on 2 mg. daily, had corresponding values of 0.087 mg. per liter of urine and 0.063 mg. per 100 gm. of blood during the same period; (8) lead con centration in the blood of the subjects was subject to insignificant variation during the course of a day, and to only minor variations from day to day, while the urinary lead concen tration, as well as output per unit of time, varied widely in accordance with the volume of water excreted by the kidnpys; (9) the total amount of lead retained in the tissues of both sub jects over the period of study was quite small, and with due regard for an additional and undertermined loss by way of the perspiration, as well as the effects of certain factors which could not be controlled entirely, it was almost negligible; (10) the dis crepancy between total oral lead in take and output in the feces and urine of the subject on 1 mg. daily of ad ministered lead for a period of 39 lunar months was 78.23 mg. of lead, while the corresponding discrepancy in the case of subject on the 2 mg. dosage for 13 lunar months was 51.43 mg., each of these quantities repre senting about 5% of the lead ingested during the respective periods; (11) DUP050312620 Nov. 1940] INGESTION OF LEAD COMPOUNDS 399 the combined evidence indicates that some actual retention of lead occurred in both subjects, and that the rate of retention was substantially constant for each subject over the period of the observations, being greater in the subject to whom the larger dosage of lead was administered. The following conclusions are justi fied by these results: 1. The largest proportion of the lead which is ingested by normal persons, with food or otherwise, whether in soluble or insoluble form, is eliminated in the feces without absorption; 2. Daily ingestion of lead in quan tities somewhat in excess of 1 mg. by healthy adults, over long periods of time (3| years) is not only compatible with the maintenance of normal health and well-being, but is also associated with such an increase in the elimina tion of lead as to result only in a slight and almost negligible retention of lead in the body; 3. Daily ingestion of slightly more than 2 mg. of lead, for a period of more than 1 year, failed to result in any demonstrable effect upon the health or well-being of a healthy adult, but induced a level of lead elimination higher than that caused by the inges tion of 1 mg. daily, and a slightly greater rate of lead retention in the tissues; 4. Maintained concentrations of 0.06 to 0.07 mg. of lead per 100 gm. of blood over periods of months are entirely compatible with normal health and well-being of human adults; 5. Concentrations of lead in the urine which vary from 0.05 to 0.15 mg. per liter and slightly more, with mean concentrations as high as 0.09 mg. per liter over long periods of time, correspond with lead concentrations in the blood of 0.06 to 0.07 mg. per 100 gm., and likewise, are compatible with normal health and well being of human adults; 6. The quantities of lead ingested in food and beverages by adult citizens in various parts of the United States vary from somewhat less than 0.10 mg. to somewhat more than 2.00 mg. with a mean value of approximately 0.32 mg., per day; 7. From the results of the foregoing studies, it would appear that there is a sufficient factor of safety in relation to the lead content of the general food materials in the United States. BIBLIOGRAPHY 1. Ke h o e, R. A., Th ama n n , F. a n d Ch o d a k , J.: Lead absorption and excretion in certainlead trades. Th is 3., IS, 306 (1933). 2. ---------: Discussion of recent studies .. . and suggested methods of plant con trol. Report of Industrial Hygiene Sessions, 16th Annual Convention, National Battery Manufacturers Assn. Part II, p. 61 (1939). 3. ---------, Th a ma n n , F. a n d Ch o ia x , J.: On the normal absorption and excre tion of lead. I. Lead absorption and excretion in primitive life. Th is J., 16, 2S7 (1933). 4.---------, Th ama n n , F. a n d Ch o l ak , J.: On the normal absorption and excre tion of lead. II. Lead absorption and excretion in modem American life. Ibid., IS, 273 (1933). 6. ---------, Th ama n n , F. a n d Ch o i.a x , J.: Normal absorption and excretion of' lead. J. A. M. A., 104, 90 (1935). 8. ---------, Th a man n , F. a n d Ch o l ak , J.: An appraisal of the lead hazards associated with, the distribution and DU P050312621 400 JOURNAL OF INDUSTRIAL HYGIENE AND TOXICOLOGY [vol. Sg, no. 9 use of gasoline containing tetraethyl lead. II. The occupational lead ex posure of filling station attendants and garage mechanics. Th is J., 18, 42 (1936). 7. ---------, Ch o l a k , J. a md St o r y , R. V.: A spectrochemical study of the nor mal ranges of concentration of certain trace metals in biological materials. J. Nu t r it io n , 19, 579 (1940). 8. --:--, Ch o l a k , J. a n d St o r y , R. V.: Manganese, lead, tin, aluminum, copper, silver in normal biological material. Ibid., SO, 85 (1940). 9. ---------, Th ama n n , F. a md Ch o l ak , J.: On the normal absorption and excre tion of lead. III. The sources of normal lead absorption. Th is J., IS, 290 (1933). 10. Ch o l a k , J.: Quantitative spectrographic determination of lead in biological material. Ind. Eng.Chem., Anal. Ed., 7, 287 (1935). 11. Hu b b a r d , D. M.: Determination of lead. A photometric dithizone method as applied to certain bio logical material. Ibid., 9, 493(1937). 12. Ch o l a k , J., Hu b ba r d , D. M., Mc Na r y , R. R., a n d St o r y , R. V.: Determina tion of lead in biological materials. Comparison of spectrographie, dithi zone, and s-diphenylcarbazide meth ods. Ibid., 9, 488 (1937). 13. --------- a n d St o r y , R. V.: Spectrographic analysis of biological material III. Lead, tin, aluminum, copper and silver. Ibid, 10, 619 (1938). 14. Ba mba c h , K.: Determination of lead by dithizone. Ibid., 11, 400(1939). 15. Ca l v er y , H. O.: Chronic effects of ingested lead and arsenic. A review and correlation. J. A. M. A., Ill, 1722 (1938). 16. Bl o o mf iel d , J. J., a n d Is b el l , H. S.: The presence of lead dust and fumes in the air of streets, automobile repair shops, and industrial establishments of large cities. 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