Document R22VgakvKGVXdmOjy9gqXgvNk

1 STUDIES ON THE AESORPTION OP LE'AD FROM ETHYL GASOLINE by Root. A. Kehoe, M. D., Frederick Thamnn and Lester Sanders Dated, November 18, 1926. Printed in U.S.A. STUDIES ON THE AESORPTION OF LEAD FROM ETHYL GASOLINE by Robt. A. Kehoe, M.D., Frederick Thamann and Lester Sanders -0 OO0 - INTRODUCTION In anticipation of the general distribution of Ethyl Gasoline for use in automobiles, this laboratory began a series of investigations in NOTE: Attention is called to an error on page 18 of tils report. Monkeys No. 3, No. 4 and No. 5 are called Ringtail Monkeys, they having been so designated by the dealer from whom "they were obtained. Investiga tion shows that they are Macacus Monkeys, of the type ordinarily called "Common Macacus." This is a matter of some importance, since cross breeding of Macacus Rhesus and Ringtail Monkeys may be regarded as a practical impossibility. elsewhere, in so far as animal experimentation might be capable of yield ing information. Thus there was time and opportunity for two types of in vestigation, without duplication of effort. 1. Observations could be made on men actually exposed to all the practical conditions resultant upon the distribution and use of Ethyl Gasoline. And, 2, animal experi mentation dealing with the various types of exposure to the leaded gaso line under the various conditions associated with the commercial handling of such gasoline could be carried out. The first of these has been made the object of one study by this laboratory, on which report was made in June 1925.* This study was check ed and verified in detail by an independent study carried out by a com * Report of First Field Investigation made to Board of Directors of The Ethyl Gasoline Corp. Kehoe and Edgar, 1925. STUDIES ON THE ABSORPTION OF LEAD FROM-ETHYL GASOLINE by Robt. A. Kehoe, M.D., Frederick Tharoann and Lester Sanders 1 -0 OO0 - I ' I J. INTRODUCTION . In anticipation of the general distribution of Ethyl Gasoline for use in automobiles, this laboratory began a series of investigations in 1924, with the idea of determining to what extent certain dangers which suggested themselves as possibilities in connection with this commerce might be of actual importance. Evidence that no acute danger existed was to be had at the outset, by reason of the fact that the gasoline had been on the market for some months, without the occurrence of untoward results. The problem of possible dangers in connection with finely divided lead . dusts in exhaust gases, was being carefully and adequately investigated elsewhere, in so far as animal experimentation might be capable of yield ing information. Thus there was time and opportunity for two types of in vestigation, without duplication of effort. 1. Observations could be made on men actually exposed to all the practical conditions resultant upon the distribution and use of Ethyl Gasoline. And, 2, animal experi mentation dealing with the various types of exposure to the leaded gaso line under the various conditions associated with the commercial handling of such gasoline could be carried out. The first of these has been made the object of one study by this laboratory, on which report was made in June 1925.* This study was check ed and verified in detail by a n independent study carried out by a com * Report of First Field Investigation made to Board of Directors of The Ethyl Gasoline Corp. Ifehoe and Edgar, 1925. -2- mittoe appointed in 1925 by the Surgeon General for Public Health.* A second study on our part is nov in progress, on which report will he made at a later data. Since this problem is concerned principally with accumulation of lead, time is the important factor, so that it may not be completed until several years have elapsed. The second type of study has been carried on continually and has been interwoven with the first to the extent that the observation of human exposure has given a more complete understanding of the details of experimental conditions which must needs be duplicated in animal experi mentation. In the light of careful observation of the methods in gener al use for handling gasolines the hazards which may exi'st in connection with the handling and use of Ethyl Gasoline are capable of much more ac curate statement than was possible at the time the experimental program was undertaken. The only health hazard which might arise from the com mercial usage of Ethyl Gasoline is that, after long exposure, persons might eventually accumulate in their bodies sufficient lead to produce the symptoms and signs of lead poisoning of a ciironic type. Exposure may occur in several ways, and may best be illustrated by examples from prac tical life. The garage mechanic may have his hands bathed in gasoline for considerable periods of time in the course of repairing and adjusting carburetors and various other parts of a motor. He may use the gasoline for cleaning tools, etc., as well. If such a thing is possible he may ab sorb some lead through his skin out of the gasoline. In addition he may handle tobacco or other articles that go into his mouth, with unwashed hands, and so ingest lead. He may allow the gasoline to dry on his hands and may thus be exposed to small quantities of more concentrated Ethyl * Investigation of Health Hazard from Tetraethyl Lead Gasoline - J. P. Leake - U.S.Public. Health Service, 1926. U.S.Treasury Dept. -3- Fluid, ivhich being less volatile than the gasoline will remain on his skin longer than the gasoline. The gasoline may spill on the floor of the gar age, and if allowed to remain will evaporate, leaving behind some of the less volatile tetraethyl lead. This will vaporize to some extent, and will be decomposed in part to tri-ethyl lead compounds which will remain to make up part of the dust of the garage. In the above situations the mechanic may inhale vapors arising from the gasoline, and if the vapor contains lead he may absorb it through the lungs. After the gasoline has evaporated, he may still be exposed to some vapor of the residual tetra ethyl lead, and, finally, he may inhale lead dust from the air as it is picked up by eddies and stirred up by movements in the garage. In tearing down motors the mechanic comes in contact with lubricating oil containing minute quantities of tetraethyl lead and a greater or lesser quantity of decomposition and combustion products of tetraethyl lead. He will scarce ly be exposed to any important danger of skin absorption here, but he may ingest lead as a result of eating, chewing, and drinking materials contam inated by unclean hands. The above constitutes the entire picture of the possibilities for the absorption of lead as a result of the use of leaded gasoline, with the exception of the exhaust gas exposure. The latter is completely omitted from this discussion because the experimental work herein presented has no application to this matter. . The above methods of exposure are present to a greater or lesser extent in the case of all handlers and users of Ethyl Gasoline. In dif ferent situations, however, one or the other type is of importance while the others are less important or negligible. _ The service station employee is exposed to vapors arising from-- gasoline, to skin contact with gasoline, to vapors and dusts from the street or area around his station, which may contain lead, or to skin con tact with oil in draining crank cases. Of these, however, the skin con tact with gasoline is undoubtedly the most important. However, the. pos sibility of ingestion of lead because of unclean hands must not be for gotten, in the case of this man. The loading rack handler of gasoline is exposed to vaporizing gasoline, to skin contact with it in filling cars, sampling, and in drain ing the cars by removing the drain cap. There may be occasional bad. spills here in which a large body surface is covered with gasoline. There may be vaporizing tetraethyl lead and lead dusts as a result of frequent spillage over the same area of ground or loading platform.' Lead vapors may be inhaled in sweeping and cleaning cars which have carried Ethyl Gasoline and lead may be ingested because of unclean hands. Here again probably the most important exposure, at present, lies in the skin contact with gasoline. The tank wagon and bulk gasoline handler is exposed to exactly the same situations, and in case of bad fittings, careless, handling or in case bucketing is done, his skin contact with gasoline may be considerable The owner and handler of a car is presented with the same possi bility of exposure, to just the extent of his taking over the work carried out generally by the above 'workmen. Questions arise, therefore, as to the relative importance of these types of exposure, and as to whether any of them are sufficient in quantity to cause trouble. Experience, only, will fully answer these questions, but experimentation may give some predic tions, which when taken with the proper reservations, are of considerable importance. The facts which are of the utmost importance to determine are the -5- answers to the following questions. 1. Under conditions which allow for spillage, evaporation of spilled gasoline leaving residual tetraethyl lead and dust of decomposed lead, shin contact, and ingestion from contaminated hands, is the quantity of absorption sufficient to produce accumulation of lead and lead poison ing? 2. Can tetraethyl lead be absorbed out of gasoline by the skin, as long as the concentrations of lead and gasoline remain at their normal commercial level? . 3. If so, can it be absorbed in sufficient quantity to accumulate in toxic amounts in the tissues? 4. Can tetraethyl lead be inhaled in the vapor arising from Ethyl Gasoline, in sufficient quantity to produce accumulation of lead? The following experiments undertake to answer some of the above questions. Some are answered completely and decisively. Some are answer ed tentatively, because of the care which must be taken against a too literal interpretation of animal experimentation in terms of human beings. (Note: Animal experiments dealing with the above subject were reported at a Conference called by the U. S. Public Health service May 20, 1925. The following studies are a continuation of experiments reported at that time, and in some cases the same animals have been used. In as much as the results in certain aspects of the problem are not conclusive as yet, some of the experiments are being continued at the present time. The data included herein, therefore, r-epreaent observations up to the present, inclusive of those previously reported.) -6- EXPERIMEHTAL METHODS Guinea Pigs, Rabbits, and Monkeys vrere treated with Ethyl Gasoline in a variety of ways, so as to study the possible means of absorption of lead contained in gasoline in various concentrations. Exposure to the gasoline was arranged in such a way as to duplicate as nearly as possible the conditions of human exposure. I. In order to provide a situation in which animals might be exposed simultaneously to skin contact with Ethyl Gasoline, inhalation of gasoline vapor, inhalation of residual tetraethyl lead vapor, and dust of decompo sition products, and to ingestion of lead from contaminated skin and food, rabbits were treated with Ethyl Gasoline in the following way:- The hair was clipped from a skin area without injury to the skin, and an accurate ly measured quantity of gasoline containing tetraethyl lead was dropped on and allowed to remain until absorbed or evaporated, lio attempt was made to prevent inhalation of volatile materials after treatment. No precau tion was taken to prevent the animal from licking himself or his compan ions and thus ingesting the lead. It was observed, however, that there was very little tendency shown to lick off the gasoline. In this way all the tetraethyl lead put on a given animal remained on him, or near him, in the form of vapor or dust. It even contaminated his food to some ex tent. In case he excreted lead his excretion remained for a time to con taminate his food and the air he breathed. In this way it was hoped to reproduce the worst conditions of commercial handling. Observations were made, and at various intervals animals were killed for analysis. II. In order to determine whether or not tetraethyl lead might >e ab- -7- sorbed out of gasoline the following methods were used. The hair was clipped off the fore leg and first one and then the other, on alternate days of treatment, was dipped in the gasoline and kept so immersed for a period of half an hour, after which the treated leg was carefully washed with kerosene, soap and water and then coated with cold cream (to prevent drying and cracking of the skin). In this way absorption was confined to the period of actual exposure to the gasoline. After considerable per iods of exposure the animals were killed and analysis for lead was made. III. In order to determine whether tetraethyl lead may be inhaled along with gasoline vapor from exposed surfaces of Ethyl Gasoline, experi ments were arranged as follows: Air was .bubbled through gasoline' contain ing tetraethyl lead at a rate sufficient to volatilize 30 to 50$6 of the gasoline in six hours. The gasoline vapor was mixed with air sufficient to maintain a concentration of gasoline vapor just endurable by rabbits over a period of six hours. Preliminary experiments with the gasoline base determined what concentration could be borne and this concentration was maintained. Rabbits were exposed to these conditions daily over a period of several months. The residual gasoline after the vaporization of the 30 to 50/6 fraction was analyzed for its lead content, in order to determine just what amount, if any, of lead was given off in the vapor passing through the cage. The animals were killed after a period of ex posure, and analyses made to determine what quantity of lead, if any, had been absorbed and accumulated. 8- EXPERIMEHTS I. Skin Application of Gasoline Containing Tetraethyl Lead: 1. A. Guinea Pigs: Healthy full grown guinea pigs were selected and divided into four pens. At first they were kept in hoard and wire pens in a lferge storage room at the Dayton Plant. The pens were separated by tight board parti tions so as to avoid any possibility of contamination of one pen by fecal material or dust and vapor from an adjoining pen. The- front and top sides vrere of wire to allow ventilation and sunlight. On April 2nd the animals were moved to the Laboratory in Cincinnati where they were put in cages of concrete and galvanized iron grating, the partitions being of solid concrete. All the guinea pigs were fed on a mixed diet which has been found to be quite satisfactory after use for a long time. The Ethyl Gasoline used for treatment was made by mixing three parts of tetraethyl lead with two parts of ethylene dibromide, and mixing this .in gasoline in the propor tion of 0.25 cc. to 200 cc. gasoline as needed. This corresponds to the use of the 3 E Mix in gasoline representing approximately 1 part tetra ethyl lead to 1300 parts gasoline. The same gasoline base was used on the control animals. Observations on reproductive activity were desired, so that males and females were kept together, except in the case of Pen 2. Here a male from Pen 4 was introduced periodically. It will be observed that this animal was not being treated with any lead, so that he had no influence upon this factor. The experiment was continued over a period of eight months, during which time the oldest of the test animals received 69 treatments, at the - K 0018011 -9 rate of tivo per week. Three animals each from Pen 1, 2 and 3 were killed for analysis March 3rd, and one each from Pen 1 and 2 were killed for analysis March 31st. The animals killed on these dates had received 22 doses and 28 doses respectively. Two pigs were stolen from the cages just prior to the time of moving to Cincinnati, one each from Pen 1 and 2. One pig from Pen 2 was badly injured in a fight, dying two days later. The remainder of the animals were killed at the end of 8 months of experimen tation, on September 1st, and analyses were made then. Ho illness was observable at any time during the experiment in any of the animals, and no deaths occurred from any cause except in the case of one animal in the control group, whose death was accidental. The following tables indicate the experimental findings and the analytical results. -10- TABLE I Experimental History Pen One Treatment Males Females Deaths 1 cc. Ethyl Gasoline 7 4 None Young born 17 Young died 4 Premature births 2 Illness None Blood changes N o n~e <D o 2$ | Pen Two 0 11 1 (Injured in fight) 57 9 3 None None Pen Three Pen Four 0.2 cc.Ethyl Gasoline 5 6 None 1 cc.Ordinary Gasoline 1 2 None 31 9 4 None None 12 2 1 None None Date . Mar. 3 Mar. 31 *Sept. 2 TABLE II Analytical Results Total Pb . found in Mgs. Pen 1 Pen 2 Pen 3 2.10 2.10 2.55 2.55 2.00 2.00 2.10 2.10 1.60 2.00 Lost *0.90 0.90 0.80 0.85 1.20 i.oc5 *1.00 0.82 0.70 0.90 0.80 *0. 70 0.63 1.30 1.05 1.15 2.62 1.56 1.03 Pen 4 *2.50 0.50 0,67 * All animals above the double line were exposed to lead dusts in Dayton plant, because of storage of lead containers in experimental quarters. All below that line were kept after such exposure over a period of five months in the Cincinnati Laboratory, where there was no exposure to lead beyond that brought about by the experimental conditions, the experiment continuing as before up to Aug. 28, 1925. -li lt is observed that all the animals in the group contain lead in their carcasses. Much of this lead is due, undoubtedly, to the inhala tion and ingestion of lead dust in the room at the Dayton plant. (Note reference to this in Public Health Bulletin, No. 158.) The significant feature of the results is seen in that with two exceptions, the results of analyses show a lead content at the end of eight months treatment, which is decidedly lower than that at the end of three months. Obvious ly, during the last five months of experimental study in Cincinnati, these animals were not accumulating more lead, but were losing some which they had previously taken in. It is plain, therefore, that absorption, if any occurred, did not keep pace with secretion. This is especially significant when the test group Pen 1, is compared with control groups 2 and 4. In the case of the latter two groups there is no possibility of further lead intake, except in so far as the food and water may have con tained a normal minute amount. There was no dust, nor were there any compounds of lead in the rooms occupied by the animal cages. In spite of this fact these animals did not lose lead from their bodies at a rate in excess of that shown by the test group. The only conclusion one can ar rive at, therefore, is that the test group were not absorbing appreciable quantities of lead. B . Guinea Pigs: __ Because of the exposure of the guinea pigs to extra-experimental sources of lead in the previous experiment, and because information was desired on the possibility of lead absorption from lower concentrations of tetraethyl lead in gasoline, a further study was made in the following manner. -12- A fresh supply of full grown guinea pigs was obtained. These were divided into two groups, Pens No. 5 and No. 6. The animals were fed in the standard manner and were kept carefully separated from each other, using all the precautions previously described to prevent contam ination of control animals by dusts and vapors from the pen containing test animals. The animals in the control pen (No. 5) received no treat ment and were handled only in weighing and feeding. The animals in the test pen (No. 6) were treated three times weekly with 1 cc. of gasoline containing half the standard quantity of Ethyl Fluid. This was prepared by mixing three parts of tetraethyl lead with two parts of ethylene di bromide and mixing this in gasoline in the proportion of 0.25 cc. to 400 cc. gasoline. The same type of observations were made as in the case of the animals previously studied. The experiment began in August, 1925 and continued without interruption until the latter part of January, 1926. During this time the test animals received sixty-two treatments, with the exception of one female which died after the seventeenth treatment. This animal received an injury from a fall, resulting in a ruptured liver and death from haemorrhage into the peritoneal cavity. In the control pen three of the four animals died during the five months, one female on Oc tober 20, after the birth of two young, one female on October 27 of pneu monia, and one male on November 19 of abscesses of the liver. All of these animals were analyzed for lead content. The other survived and was killed for analysis at the end of the experiment. The following table summarizes the results of the experiment. ' 0018015 -13- TABLE III Pen Treatment Animal Date of Young Young Premature Pb.Content Ho. Type No. No . Sex Death Eorn Died Birth in Mgs. 46 F. Oct. 20 2 2 No ne Young of 46 Analyzed 5 Hone None 48 F. Oct. 27 3 0 None 49 M. Nov. 19 50 M. Jan. 26 (killed) 0.32 0.13 0.24 0.08 0.35 1 cc. 17 tj cone. Ethyl 62 6 Gaso- line 62 3x v/eek 62 45 F. Oct. 3 * 0 0 Unborn young of ITo. 45 Analyzed 51 M. Jan. 26 (killed) 52 M. Jan. 26 (killed) 53 F. Jan. 26 6 0 (killed) None None 0,64 0.32 0.28 0.38 0.45 * Pregnant - 4 young almost at term. A study of the above data indicates that there is little evidence of the accumulation of lead in the animals treated with half concentration Ethyl Gasoline. The amount of lead found in the entire carcasses of the treated animals never exceeds a fraction of one milligram, and although the average quantity found is slightly higher in test animals than in controls, there is not a sufficient difference to be significant. It is important to note that all the animals show some small quantity of lead in their bodies. However, there is no relationship between the amounts so found and either the period of time spent by the animal in our laboratory, or the period of actual treatments. For this reason one must conclude that either the ani mals are not absorbing appreciable quantities of lead, or that they are excreting it as rapidly as they are absorbing it. -14- 2. A. Rabbits: Full grown rabbits were obtained from stock raised outsido the laboratory, so as to avoid using any of the offspring of leaded animals, which might by chance contain abnormal quantities of lead. Six animals were used, of which two were used as controls. The controls were weighed once a week and observed. The four test animals were treated on the skin of the belly from which the hair was clipped, with 2 cc. of one half of that concentration of Ethyl Gasoline which had been the standard experi mental gasoline previously. This was prepared as previously described for the treatment of guinea pigs in Pens No. 5 and No. 6. Test animals were weighed weekly and observed. Controls and test animals were kept separated but were fed on a standard diet in the same m'anner. The experiment was begun August 19, 1925, and concluded January 28, 1926. During this time no illnesses or deaths resulted from the treatment, but three of the animals including one of the controls sickened, and were either killed or died. The carcasses of all were analyzed for total lead content, exclusive of the skin. Rabbit No. 107 developed a paralysis and was killed October 14th. A necropsy showed large haemorr hages into the musculature of both sides of the body, both inside and outside the peritoneum, which were obviously the result of injury on the animal board, (it occasionally happens, in handling a considerable number of animals, that an animal becomes frightened and begins struggling vio lently. At such times an injury is very likely to result in the case of delicate animals as rabbits and guinea pigs.) Rabbit No. 110 developed a parasitic infection of the ear (common among rabbits) which extended into the middle ear with abscess formation. It vras considered humane to kill the animal. This was done December 8th. Rabbit $112, a control, developed a pneumonia and died January 10th. 0018017 -15- TABLE IV Lead H o . of Sex No. of Time Weight in Lbs. Offspring Date of content Rabbit Treatments Per io d Initial Final Living Dead Death in Mgs. 107 108 109 110 111 112 __________ F. F. F. F. F. F. 20 58 55 39 Control Control * Killed for analysis. 2 mo. 5 mo. 5 no. 3 mo. 5 mo. 5 mo. 3-3/4 5-7/16 5-9/16 5-3/16 6-1/4 5-3/4 3-1/2 4-7/8 5-3/4 4 -I/ 4 6 -I/ 4 5-1/4 0 4 0 2 6 0 0 Oct.14 1.40 0 Jan. 28* 0.11 0 Jan.28* 0.35 2 Dec. 8* 3.30 1 Jan.28* 0.29 0 Jan.10 0.20 It will be noted that all of these animals contained small quanti ties of lead in their carcasses. However, there are wide variations in the lead content. Two of the treated animals contain .considerably larger amounts than either of the controls. On the other hand the other two test animals, treated for longer periods of time, show no greater quantity than is found in the control animals. There is, therefore, no relation ship between quantity of lead found and the period and quantity of treat ment. This fact alone-makes one doubt if the treatment was responsible for the relatively high lead content of Rabbits No. 107 and 110. It sug gests one of two possibilities. Either the relatively high lead values are accidental, i.e., they represent variations such as may be found in control animals; or, the rate of excretion in these two animals did not keep pace with that shown by normal animals, because of their complicat ing illnesses. The latter can hardly be true in the case of Rabbit No. 107, for his injury occurred abruptly and he was killed soon after the injury. This illness could hardly have had any influence either on the rate of >sorption or the rate of excretion of lead. Furthermore, this animal was killed early in the experiment having been exposed to only twenty treatments. In the case of Rabbit No. 110 a lingering infection KE 0018018 -16- night be responsible for a somewhat decreased rate of secretion, for it produces general injurious effects to all the physiological processes of the cnimal. However, it has been our experience previously, that such infections have a tendency to increase the rate of excretion of lead. The evidence, therefore, points to the incidental character of the high lead content of these two animals, rather than the resultant effect of the experimental treatment. That this is likely is further in dicated by the analysis for lead of two animals taken at random from the hutches of the Physiological Laboratory. One of these Plt contained 1.10 mgs. Pb., and the other P 1X contained 0.50 mgs. Pb. This indicates that animals kept under standard laboratory conditions are exposed to sources of lead, and normally show lead in their tissues. In any case the results of these experiments are not. fully con clusive except in so far as they point out that lead poisoning did not result from the treatment, and that rabbits do not, of necessity, accum ulate lead in their bodies as a result of prolonged skin contact with gasoline containing the experimental concentration of tetraethyl lead. 3. Monkeys: Seven monkeys have been used in this study. Two Rhesus monkeys originally intended for controls have died, one of pneumonia, shortly after being received, and the other of exposure, he having escaped during cold weather. The first experiments were conducted on two Rhesus monkeys, one male and one female, without controls. Some months later three Ringtail monkeys, one male, and two females, were procured, one of these being used since as a control. After being obtained the monkeys were kept under observation for some weeks in order to insure their good health at the outset. A diet k 0018019 -17- of oranges, bananas, carrots, apples, eggs, nuts and milk have served to maintain an excellent state of nourishment. Observation has been made of temperature, weight and blood find ings over the entire period of pre-experimental and experimental period, for test and control animals. These observations are shown in the charts below. The following experimental treatment was carried out. Monkey Jo. 1, a Rhesus male, has been treated daily (excepting Sundays and holidays), since January 19, 1925, with 2 cc. Gasoline prepared by adding 0.4 cc. Tetraethyl lead to 500 cc. gasoline. An area on the back was clipped free of hair and kept so, the gasoline being dropped on with a pipette, without any further procedure. . Up to November 18, 1926, this animal has had 515 such treatments. The chart below shows the observations as to weight, etc. This animal has never been ill at any time. He is small, and has been so since being obtained, and in addition is restless and excitable, having never been tamed to the extent shown by the other monkeys, especially the females. His sexual development is apparently not normal, since at no time does he regard the females with any interest, other than to betray a loneli ness and anxiety if left alone. In as much as he was apparently of adult age* when the experiment began these things may be disregarded in all probability, so far as any experimental- significance is concerned. Monkey No. 2, a Rhesus female, weighing 6^ lbs. at the beginning of the experiment was treated just as Monkey Ho. 1, up to May 20, 1926, she having received 375 treatments of Ethyl Gasoline, 2 cc. at a dose, * The age of these animals is problematical, since there is so little information available on matters of this kind. X 0018020 -18- since January 19, 1925. On the former date, somewhat unexpectedly, this monkey gave birth to a strong, fine young male. Treatments were discon tinued at once in order to avoid any possibility of injury to the off spring from gasoline and lead which might be taken into the mouth from the skin of the mother. The young one has thrived and is healthy. He is very active. His sire is a large, strong Ringtail male, Monkey Ho. 5. The mother has not been ill at any time, though she was in a condition of poor nourishment when obtained. At the time of the birth of the young one, the mother showed no apparent illness, and she has continued through the suckling period up to the present to have every appearance of excellent health, with sleek hair, bright eyes, and generally good appearance. Three new monkeys, healthy examples of the Ringtail type, were obtained in September, 1925. Monkey ITo. 3, a Ringtail female, was kept as a control, observations being made as in the case of the test monkeys. This animal has been healthy and has done well until recent months. She has always had a tendency to stay array from the other monkeys, and is apparently sterile. Her occupa tion consists in picking ceaselessly at objects real and imaginary in her surroundings, so that she keeps her nails worn away. She looks old and has lost some weight recently. She was apparently the healthiest of the females when the experiment began, and for this reason was chosen as the control. p^ ( ii.-f*'Ci^ Monkey No. 4, a J^^gt-kil female, was treated with 4 cc. Ethyl Gasoline daily, from November 5, 1925 to February 13, 1926, 75 treatments in all. At this time it was noted that the animal was pregnant. Not knowing the time at which the pregnancy would be terminated, but thinking it to be near at hand, the treatments were discontinued in order not to take even ~19< the slightest chance of injuring the young through ingesting lead from the skin of the mother while suckling. The animal delivered a normal, healthy male young one on April 3rd. The mother was not ill and both she and the young one have contin ued to thrive. Treatments of the mother are now about to begin, since the young one is apparently able to care for himself. uw/-. fu* .&{*> Monkey No. 5, a large male has been treated with 4 cc. of Ethyl Gasoline daily, since February 25, 1926. Up to November 18, 1926 he has received 214 such treatments. At no time has he shown any evidence of illness. This animal is the sire of the two baby monkeys now in the laboratory. K 0018022 -20- TABLE VI Chart of Experimental Observations MONKEY SO. 1 Date ''/eight Lbs. Thousand Temp.F . Erythrocytes Leucocytes Reading Eo. (Dare) Stippling per 50 fields Jan. 19, 1925 Jan. 19, 19 25 Feb. 7 13 20 27 liar. 31 Apr. 21 28 May 5 14 21 30 June 5 11 18 25 July 2 9 16 23 30 Aug. 7 14 22 29 Sept. 5 12 19 26 Oct. 3- 5-7 10 17 24 31 Nov. 7 14 21 28 Dec. 5 12 26 Jan. 2, 1926 9 16 23 30 Treatment vi th 2 cc. 5-l/8 102.8 4-9/l 6 102.4 4-14/l 6 102.2 2-11/16 102.8 4-6/16 103.0 4-ll/l 6 - 102.6 -- 4-12/l 6 4-13/l 6 4-ll/l 6 102.0 102.6 102.4 4-13/l 6 - 4-3/4 5 c? 102.3 - 103.4 102.4 102.8 5 -1 / 1 6 5 -1 / 8 5-1/16 4- 14/l 6 - 102.6 103.2 103 102.8 4-14/16 5-5/16 5-3/l 6 5-1/2 5 -I/I6 5-1/2 - 102.5 102 102.6 102.4 102.1 102.0 - 5-13/16 102.0 5-11/16 5-7/l5 5-6/16 6 5-13/16 5-13/16' 6-l/l 6 5-1/2 5-ll/l 6 5 -II/ 1 6 5-V2 5-lO/l 6 102.0 102.2 102.8 101.8 101 102.5 102.2 102.4 102.0 102.0 102.1 102.4 - 5 -II/ 1 6 5 -9/I0 5-9/l 6 - 5-11/16 - 102.9 102.4 102.8 102.1 Ethyl Gasoline b egun. 4,144 13,800 - - - 4,437 - 4,9 32 5,252 5,368 5,512 5,360 5,844 5,768 5,584 - 5,088 5,672 5,440 5,728 4,653 5, 840 6,024 5,886 5,928 6,104 5,552 6,036 6,232 5,072 4,946 5,616 5, 340 - 5,436 6,083 5,008 5,260 4,960 5, 300 5,080 5,208 5,048 5,184 5,260 ' - - 16,050 - 16,700 10,500 11,700 12,400 13,400 11,700 11,050 11,200 - 12,500 11,150 12,000 16,350 12,500 11,450 9,800 14,600 13,100 12,000 13,050 11,500 13,.500 22,200 10,400 20,050 10,150 11,400 13,500 13,500 9,400 8, 850 11,000 11,800 9,300 9, 800 13,850 10,100 14,160 ' 70 _ 65 60 60 65 50 50 52 50 -- 55 53 55 55 55 54 57 54 57 58 57 60 50 55 58 63 55 58 56 56 57 58 60 60 58 58 60 60 0 - - 0 0 0 0 0 0 0 3 0 2 3 0 2 0 7 0 1 1 2 8 '0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 m o n k e y n o . i - (Cont *d) -21- Date 'Yeight Lbs. Tenp.P. Erythrocytes Leucocytes lib.(Dare) Stippling Feb. 6, 1926 13 26 Mar. 8 13 20 29 Apr. 7 19 May 1 4 14 20 28 June 5 11 17 24 July 1 8 15 22 30 Aug. 5 11 20 26 Sent. 2 25 Oct. 2 7 14 22 29 Nov. 4 11 18 5-3/4 5-3/4 5-14/16 5-3/4 5-13/16 5-3/4 5-lO/l 6 5-ll/l 6 5-14/l 6 5-3/l 6 5-13/l 6 6- 2/l 6 5-15/l 6 5-14/l 6 5-15/l 6 5-15/16 S-1 / 4 5-14/16 6 - 1/I6 6 6 6-2/16 6-2/16 6-3/16 6 -I/ 6 6-1/2 6- 2/l 6 6-2 /1 6 5-6/16 6-5/15 6-1/4 6-6/ 1 6 6 -I/ 4 6-9/l 6 6-3/4 6- H / I 6 6-3 / 4 102.5 102.1 102.1 102.4 102.0 101.8 101.6 101.0 102.4 102.1 102.1 102.2 102.0* 102.2 102.1 102.4 102.1 102.0 102.4 101.8 102.0 101.6 102.6 102.0 102.0 101.0 102.2 102.6 101.7 102 101.8 102.0 102.6 101.9 101.6 102.4 102,1 5,392 6,032 - ` '5,456 5,240 4,896 5,192 4,720 4,976 5,288 4,816 4,984 4,3524,688 4,680 4,672 4,552 4,160 5,280 5,504 5.208 8,048 5,464 ' 6,024 - . 6,201 6,460 6,090 5,920 6,590 13,750 16,600 - - - - - 14,050 13,900 14,450 10,800 14,300 9,400 12,350 13,300 10,150 9,050 11,500 7.850 8, 650 6,900 12,400 11,800 12,450 13,000 12,450 9,300 12,600 9,600 14,800 11,100 16,550 9,050 9,350 58 0 60 0 - -' --- -- .-- -- 55 0 55 1 65 0 - .0 50 2 55 1 55 0 50 0 50 0 50 0 50 0 50 0 50 1 50 1 50 1 55 0 52 0 60 0 50 1 50 0 50 0 55 0 50 0 53 1 50 0 50 0 57 0 Experiment being continued. -22- TAELE VII Chart of Experimental Observation MONKEY NO. 2 Date Weight Lbs . Temp.F . Erythrocytes Leucocytes b1.(Dare) Stippling Jan. 1926 Treatment with 2 cc. Ethyl Gasoline, daily, begun. Jan. 19, 1925 Feb. 7 13 20 27 Liar . 31 Apr. 21 Apr. 28 May 5 14 21 30 June 5 11 18 25 July 2 oKi 15 23 30 Aug. 7 14 22 29 Sept. 5 * 12 19 26 Oct. 3 10 ' 17 26 31 Nov. 7 14 21 28 Dec. 5 12 19 26 Jan. 2, 1926 9 16 6-1/4 6-6/l 6 6-1/2 6-ll/l6 6-4/l6 S-7/16 - 7-3/l 6 7-6/l 6 8-2/16 8 3 -I/ 4 7-3/4 7- 1/16 8- 6/l 6 8- 1/ 2 8-9/l 6 8- 9/16 9- 2/l 6 9 8- 3/4 9- l/lS 9-3/16 9-3/8 8-13/l 6 8-3/ 4 8- ll/l 6 9- 2/l6 10 9-14/l 6 10 9-13/16 9-13/16 9-14/l 6 9 -9 / 1 6 9-ll/l 6 10 9-9/16 9-6/16 9-3 /16 9-1/2 102.3 102.2 102.2 102.2 102.0 103.0 - 102.0 102.0 101.4 102.3 - 103.2 1 0 1 .2 102.6 1 0 2 .2 1 0 2 .2 10 2.6 102.4 1 0 2 .0 1 0 2 .6 102.4 1 0 2 .0 1 0 2 .0 103.0 102.8 1 0 2 .0 1 0 2 .0 1 0 2 .0 1 0 1 .6 103.0 1 0 2 .0 102.5 102.4 102.0 101*9 102.0 102.1 1 0 2 .6 102.4 101.4 101.0 5,020 - - - - - - 5,284 5,080 5,712 4, 816 4, 816 4,888 5.576 5.344 4,944 4,112 5,280 4.240 4.344 4,696 5,792 5,416 5,056 5,304 5,512 5,256 5,976 6,128 5,448 5,384 5,232 5,904 5,024 5.576 5,952 5.240 5,184 5,568 5,346 4,624 4,824 4,224 16,400 - 75 - 0 - - -- -- - -- - -- -- - 13,500 70 . 0 12,350 70 0 8,700 70 0 10,150 75 0 14,900 70 0 11.350 72 0 11.250 70 0 11.050 0 1 2 ,0 0 0 0 0 13.000 75 0 11.450 75 0 11.700 12.050 75 75 0 0 13.700 75 0 11.050 70 0 11.000 73 0 10.750 73 0 11.450 80 0 15.300 75 0 14.200 78 0 17.050 76 0 11,650 78 0 15.350 80 0 10,100 85 0 19.100 85 0 1 2 .2 0 0 78 0 13.250 75 0 14.300 80 0 12.050 80 0 13.300 80 0 15.750 84 0 14.100 83 0 13.500 14.500 16.750 16,850 80 82 85 84 0 0 0 0 liONKSY NO. 2 - (Cont'd) -23- D a t e______ Weight Lbs. Temp.F . Erythrocytes Leucocytes Hb.(Dare) Stippling OOI I I I I I t OOOO Jan. 23, 1926 30 F eh. 6 13 26 Uar. 8 13 20 27 Apr. 7 19 Hay 3 14 20 - - 4,504 15,250 83 9-3/4 102.1 4,584 12,950 78 9-13/16 101.2 4,832 13,900 78 10 101.0 5,464 11,250 80 10-3/4 101.2 - - 10-3/4 101.4 - _ 10-14/13 101.4 - _ 11 101.0 11 102.1 _ 11 102.5 _ 11-o/l 6 101.1 - ^' H - 7/ 17 100.6 5,052 11,600 60 H - 3/4 100.0 3,280 12,250 54 Young born. Treatments and handling discontinued on account of anxiety and rage! of mother at; approach of workers in laboratory. RE 0018026 -24. TABLE Vili Chart o f Experimental Observations _____ MONKEY N O . '3 (COHTROL)_______ Date Weight Lbs. Temp.F . Erythrocytes Leucocytes Hb.(Dare) Stippling Sept.17, 1925 25 Oct. 2 9 16 23 31 Nov. 7 14 21 28 Dec. 5 12 26 Jan. 2, 1926 9 16 23 30 Feb. 6 13 26 Mar. 8 .20 29 Apr. 7 19 May 4 14 20 28 June 4 11 16 24 July 1 8 15 22 29 Aug. 6 13 26 Sept. 2 23 Oct. 1 7 14 22 _ - 6-10/16 6-11/16 6-5/16 6-6/l 6 7 7 7 7 7-1/16 7-1/2 7-9/l 6 - 7-9/l 6 7-5/I6 7-6/16 7-1/2 7-9/16 7-ll/l 6 7-7/l 6 7-3/4 8 8-1/4 8-3/l 6 8-1/4 8-3/l 6 8 - 7/l 6 8 -IO/I6 8-9/l6 8-8/16 8-6/l 6 8-1/28-6/16 7-9/16 7-15/16 7 -9 / 1 6 7-1/2 7-5/16 7-1/2 7-1/2 7-5/16 7-3/l 6 7-1/2 7 7-1/4 6-3/4 6-l5/l 6 7 _ - 101.2 101.5 101.8 102.1 101.6 101.6 101.8 100.8 102.0 101.0 102.0 - 101.4 101.4 101.6 101.8 102.0 102.1 101.5 - 102.0 100.1 100.3 101.4 102.0 101.6 102.2 101.2 101.0 101.0 101.0 102.0 102.0 102.4 101.0 101.4 100.2 101.6 102.4 101.8 101.6 102.4 102.6 102.2 100.0 101.6 101.1 5,560 4,904 4,752 5,110 4,480 5,276 5,024 4,664 4,392 4,416 5,750 4,936 5,280 5,400 5,488 6,088 5,336 5,120 4,900 5,272 4,944 - - 4,112 4,600 4,576 4, 664 5,040 4, 504 4,440 4,112 4,289 4,288 4,664 4,296 4,960' 4,280 5,040 5,280 5,680 6,960 6,048 6,018 6,040 22,400 20,600 26,250 23,600 21,000 24,900' 21,850 16,600 21,000 24,350 19,650 16,700 18,500 17,800 19,700 13,700 15,400 17,620 17,400 22,150 18,500 - 17,300 19,350 19,500 18,900 19,000 15,700 15,100 17,700 19,500 13,200 15,800 16,550 13,000 15,250 18,500 20,900 17,000 13,650 15,400 14,700 17,200 16,500 80 0 75 0 75 0 75 0 70 0 70 0 73 0 70 0 70 0 80 0 80 0 84 0 78 0 80 80 -- 90 84 -- 85 -- 83 -- 85 80 - _ 90 90 80 80 90 30 75 65 60 70 75 80 70 80 67 75 75 69 74 70 70 0 0 3 0 0 26 22 3 0 0 2 0 14 60 72 .. 153 94 26 35 231 -29, MONKEY NO. 3 (CONTROL) - (Cont'd) Date 'weight Los. Temp.F . Erythrocytes Leucocytes Hb.(Dare) Stippling Oct. 29, 1926 Nov. 9 12 18 7 7 7 7 102.0 102.0 101.0 101.6 6,430 6,630 6,630 6,740 15,300 15,400 18,200 15,159 75 70 80 . 70 60 31 *26 TABLE IX Chart of Experimental Observations MONKEY NO. 4 Date YTeight Lbs. Temp.F. Erythrocytes Leucocytes Hb .(Dare) Stippling Sept.17, 1925 25 Oct. 2 9 16 23 30 Nov. 5 7 14 21 28 Dec. 5 12 19 26 Jan. 2, 1926 9 16 23 30 Feb. 6 13 _ 5,872 17,250 70 - - 5,432 15,050 85 5-15/16 101.6 -,5,424 17,700 80 5-3/4 102.6 5,320 13,900 80 6-1/16 101.2 5,408 20,200 83 - - 5,492 18,300 80 7 100.6 5,496 20,000 80 Treatment Trith 4 cc. Ethyl Gasoline , daily, begun 7-2/l 6 101.6 5,496 16,500 ' 85 6-3/4 100.8 5,424 15,150 83 6 -1 5 /lS 100.0 5,360 16,350 85 7 100.7 5,528 21,950 86 7-2/l 6 101.0 5,032 14,800 84 7-lo/l 6 101.4 5,544 18,650 86 7-H/I6 - - - - - 5,384 17,300 84 7-13/l 6 101.3 5,304 15,800 80 7 -I4/ I 6 - 5,360 15,450 85 8-3/l 6 101.8 4,568 14,800 80 8-3/l 6 101.8 5,088 10,200 80 8-3/4 101.1 5,288 10,400 80 8-ll/l 6 100.4 4,944 10,700 80 8-3/4 101.5 5, 712 11,950 82 ' . . 0 0 0 0 0 0 0 0 0 0 0 0 - 0 0 0 0 0 0 0 0 Treatment and handling discontinued because of pregnancy. -27- TABLE X ' CHART OF EXPERIMENTAL OBSERVATIONS' MONKEY NO. 5 Date Weight Lbs . Temp.F. Erythrocytes Leucocytes Hb.(Dare) Stippling Sent.17, 1925 25 Oct. 2 9 17 24 31 Nov. 7 14 21 28 Dec. 5 12 25 Jan. 2, 1926 9 16 23 30 Feb. 6 13 25 Mar. 8 13 20 29 Apr. 7 19 May 4 14 20 28 June 3 11 17 24 July 1 8 15 22 29 Aug. 5 13 21 27 Sept . 2 >%0 _ 5,312 14,100 87 - - 5,440 19,900 87 7 101.4 5,704 2 0 ,1 0 0 98 7-5/16 1 0 2 .1 5,000 25,450 95 7-3/4 1 0 2 .1 5,392 22,500 98 7-3/l 6 103.0 5,484 17,300 95 7-ll/l 6 1 0 1 .2 5,400 15,600 90 7-ll/l 6 1 0 2 .6 5,379 13,800 90 7-lo/l 6 1 0 2 .6 5,280 16,000 95 7-14/16 120.7 5,648 22,600 95 7-15/16 102.4 5,136 18,850 86 7-14/16 1 0 1 .0 5,336 18,550 90 7-ll/l 6 - 1 0 1 .8 - 5,344 5,400 14,510 16,400 95 96 8-0/I6 102.3 5,400 12,450 98 8-1/ 2 - 5,944 18,800 95 8 -l/l6 - 101.4 - 5,432 5,294 13,950 16,300 95 90 8 -l/l6 1 0 2 .0 5,384 18,550 92 8 102.4 5,170 16,450 85 8-1/4 1 0 2 .2 5,200 20,500 88 Treatments with 4 cc;. Ethyl Gasoline, daily, begun. 8 -2 /l6 101.4 -- _ 8 -I/ 2 102.3 8-7/l 6 102.0 - w 8-2/l 6 102.1 - -- 8 -I/ 1 6 102.5 - _ 1 8-1/4 101.4 - * 8 101.4 5,728 14,200 80 8-1/1d 8 8-6/16 100.0 101.4 100.8 4,488 5,056 5,504 11,250 15,050 16,200 80 90 90 8*1/16 98.6 5,096 17,050 76 8-3/l 6 101.0 4, 744 11,300 85 8-6/16 101.1 5,024 14,650 90 8-1/4 101 4,320 18,850 85 8-1/ 16 101.0 5,072 14,200 80 8-3/16 101.7 5,336 12,250 90 8-1/4 101.6 4,848 11,750 85 8-6/16 102.0 4,928 . 11,250 90 8 -I/ 2 101.6 4,816 15,600 80 8 -I/ 4 102.0 4,848 17,100 80 8-6/l 6 101.8 4,976 12,500 80 8-6/l 6 8-6/16 101.4 101.2 5,856 6,184 17,250 19,550 94 92 8-1/2 102.0 6,120 12,200 95 8-1 5/l 6 100.0 6,136 18,300 80 . . 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 - 2 4 3 1 2 1 3 0 1 0 21 0 0 0 12 0 6 2 0 K 0018.030 -28- MONKEY HO. 5 - (Cont'd) Date Height Lbs. Temp.?. Erythrocytes Leucocytes Hb.(Dare) Stippling Oct. 1 ,1926 7 14 22 29 Nov. 4 11 18 8-13/16 8-10/16 8-15/16 8-10/16 8-11/16 8-3A 8-ll/l 6 8-3/4 102.0 101.0 101.4 101.4 101.5 102.1 101.2 101.6 5,816 5,768 - 6,369 6,060 6,310 6,800 6,810 14,050 18,350 16,800 15,050 13,050 19,500 17,850 19,050 80 80 85 85 85 80 85 80 co ro oi o o 22 8 Experiment being continued at present. HE 0018031 -29- A study of the a'oove charts shows no evidence of the development of lead poisoning in these monkeys. The appearance of stippling in the blood of the animals, however, introduces at once the question of the ab sorption of small amounts of lead. Since the control monkey Ho. 3, shows as much as any test animal, it is fair to conclude either that the stippling is not due to lead, or that there is some source of lead absorp tion, other than that intentionally provided. Such a source has been found to exist, during a portion of the experimental period. Konkeys Ho. 1 and Ho. 2 were quartered in the Dayton Plant during the period from January 19, 1925 to April 2, 1925, and were exposed consequently to such amounts of lead as were found to produce some absorption in the other ex perimental animals during that time. After being transferred to Cincin nati, this source of lead was brought to an end. This, therefore, never influenced monkeys Ho. 3, 4, and 5. But another source of possible lead absorption arises, out of the curiosity, destructive propensities and peculiar appetites of these animals. The monkeys have been housed at night in cages of concrete with metal doors. During the day time they have been allowed to run on the tops of their cages, being separated from each other as to prevent inhalation of vapors as a result of the treatments, and inhalation and ingestion of dusts from excreta, etc. The wall and ceiling of the room were of plaster covered with paint, and there were as bestos, covered pipes running through. The animals played on the pipes and tore off the asbestos, and then began to tear off the plaster from the wall. It was noted after the lapse of some time that they were eating the plaster, and with it some of the paint, this being a lead paint. Since the means of housing than was the only one available, an attempt was made to prevent the eating of the plaster by providing clean earth for their consumption. This has served to prevent the destruction and consumption -30- of the '.mil, out not until a considerable quantity of lead covered plaster ims consumed. This may account for the stippling in the blood of the con trol monkey. So far as the testing of the possibility of lead poisoning from skin exposure to Ethyl Gasoline is concerned, the above variable only adds to the severity of the test conditions, and in as much as the experiment is being continued nothing of practical value has been lost. However, any analytical results which might be done at a later date in order to deter mine what quantity of lead may have been absorbed and stored by the monkeys under test conditions, will be valueless. The experiment is being continued under conditions which do not allow of further extraneous lead consumption. The walls of the space in which the monkeys are permitted to run about is covered with a hard sheet asbestos. Further observations are being carried on on all of the above monkeys. The young have been separated from their mothers, allowing for the further handling of both mothers and young. The young, too, are being used now for continuing the experiment. One of them, the offspring of Monkey No. 2, is beirg treated daily with 2 cc. Ethyl Gasoline, while the other, the offspring of Monkey No. 4, is remaining as a control. 31- II. On the Absorption of Tetraethyl Lead out of Gasoline: The question of whether or not tetraethyl lead may be selectively absorbed out of gasoline, in the concentrations dealt with commercially, is an important one. It is known that both gasoline and tetraethyl lead may be absorbed by the skin. The question is whether there is a selective absorption of tetraethyl lead, i.e., whether in a system composed of normal skin, gasoline and Pb(Et)^, the conditions favor a partition of tetraethyl in the skin, rather than in the gasoline. Upon this factor the possibility of lead poisoning from skin contact with Ethyl Gasoline may depend, because of the fact that excess gasoline which is spilled or otherwise spread upon the skin in commercial handling is likely to be wiped off. In this way most of the exposure to the lead would exist only during that period of time in which the skin is being bathed in the gasoline* For this reason experiments were carried out in which rabbits were exposed to various concentrations of tetraethyl lead in gasoline by dipping the fore foot and leg in the leaded gasoline for half an hour at a time, over various periods of time. The animals were so treated three times week ly alternating the fore feet on successive days of exposure. Precautions as previously described were taken against allowing any Ethyl Gasoline to remain on the skin, and also against excessive removal of oil from the skin. Control animals of two kinds were provided. One group were treated with commercial gasoline containing no tetraethyl lead, in exactly the same manner as the test animals were treated with Ethyl Gasoline. M o t h e r group received no treatment of any kind. All the animals were housed in the same manner, but separately, and all were fed and cared for in the same way. The table below indicates the experimental procedure and the results. TABLF XI KE" 0018035 N o . of Rabbit Source Sex Treatm 3nt Type Frequency No. Test period Fate Weight in Lbs. Pb Initial Final 86 Old stock* M l/2 hr.1:10 Daily 3 3 days 87 same ' 88 same 89 same 90 same 91 . same 92 same 93 same 94 same 95 same 96 same 98 same Con same trol-1 Con same ` trol-2 Con New stock trol P-, "T O O same 101 same 102 same M same Daily 0 3 days F same Daily 3 3 days M 1/2 hr.gaso 2 x wk. 29 5 months line only M l/2 hr.1:100 Daily 6 6 days F 1/2 hr.1:100 2 x wk. 32 5 months M l/2 hr.1:100 2 x wk. 28 5 months M 1/2 hr.gaso 2 x wk. 23 5 months line only M 1/2 hr.1:100 2 x w k . 16 2 months M l/2 hr.1:100 2 x wk. 26 5 months M 1/2 hr.1:100 2 x wk. 25 5 months M l/2 hr.gaso 2 x wk. 28 5 months line only M None M None F None M IfZ Hr.1:1000 3 x wk. bB9~ 8 months M 1/2 Hr.1-1000 3 x wk. 90 8 months M l/2 Hr.1:1000 3 x wk. 89 8 months Paralysis and Death same same Survived 4-1/2 4 4-3A 4-1/16 - - 5-7/l 6 33 No No 1 Survived ~4-i72 "41172 Survived 3-3/4 lrf/1 Survivd** 4-13/16 5-7/16 Survived 4-5/t6 5-3/16 5 0 1 1 Died *** Survived Survived Survived 4-5/16. 4-3/l 6 " 5-3/16 3-3/l 6 4-9/16 3-10/16 6-l/2 3-3/4 7 1 1 1 1 1 1 Survived Survived Survived 4-3/4 3-3/4 5 -I/ 2 4-l/S 4-2/l6 5 1 1 1 * "Old stock" refers to animals raised in our laboratory. "New Stock" to animals raised else ** This animal became paralyzed probably frcm injury on the animal board, near the end of the *** Died 2 hours after last treatment. Cause not discovered. -33- From the above data several important conclusions may be drawn. It may be seen at once that tetraethyl lead may be absorbed from gasoline in immediately lethal quantities, provided the concentration of tetraethyl lead in the solution is sufficiently high. Rabbits Nos. 86, 87 and 88 exposed to a 1:10 dilution of tetraethyl lead in gasoline died after the third treatment, undoubtedly as a result of lead absorption, one of these animals containing 36.46 mgs. Pb in his carcass. . There is no evidence of a selective absorption, however, for if the concentration of tetraethyl lead is reduced to 1:100 in gasoline, the rate of absorption is even lower than the proportionality existing in the various concentrations. For example, Rabbit No. 90, exposed twice as long as Rabbit Ho. 86 to one tenth the concentration of tetraethyl lead in gasoline, contains only about one-tenth the quantity of lead, while Rabbits Nos. 100, 101 and 102 exposed thirty times as long to one hundredth of the concentration, show at most about one thirtieth the quantity of lead. In the case of the latter three animals, however, it must be pointed out at once, that there is no evidence that they have absorbed any quantity of lead, for they show no more lead in their carcasses than is found in un treated control from the same source. In fact, the same tiling is true of the animals exposed to 1:100 tetraethyl lead in gasoline, with two ex ceptions, cf. Rabbit Ho. SO and Ho. 94. The conclusion of most importance to be drawn from the series of experiments is that rabbits do not accumulate lead in their bodies when ex posed to tetraethyl lead in gasoline in concentrations which do not exceed one part in one thousand parts by volume in gasoline. 34- Ili . On the Inhalation of Tetraethyl Lead out of Gasolijae; Five full grown healthy rabbits were obtained for this experiment from stock animals reared outside the laboratory. One of these, No. 121, was kept in the laboratory during the entire course of the experiment, with no handling or treatment of any kind, as fet bontfol on the lead con tent 0 ^ ahimals from the source. Two othets; N6 . 119 and No. 120 'were used for preliminary experimentation in order to determine vrhat concentra tion of gasoline vapor could be borne by rabbits. They were exposed to gasoline only. No. 119 was kept as an additional control as to lead con tent, and No. 120 was used for other experiments. Rabbits No. 117 and No. 118 were used in the study of Ethyl Gasoline. The animals were enclosed in a tight metal cage with a glass front, which was supplied with an iniet tube at the top* and an outlet tube at the top on the opposite side. They were kept there for six hours daily, dur ing which time they received neither food hor water. At first two streams of air were supplied, one of which was bubbled through gasoline. These streams were measured by carefully calibrated flowmeters, and were mixed before being led into the cage by means of a !!Y"' tube. The quantity of air and gasoline vapor were regulated by adjustments of the flowmeters. Pre liminary experiments determined that rabbits could endure without suffer ing more than a mild vertigo a mixture of air and gasoline vapor contain ing 4.5 liters of air, together with 2 liters of air saturated with high test gasoline vapor. At this rate 160 to 170 cc. of gasoline were vapor ized out of the original 400 cc, in six hours. When a low test standard commercial gasoline was used, it was necessary to bubble the entire volume of air through the gasoline, in order to vaporize the same quantity in unit time. Accordingly the standard conditions for the experiment were US' 0018037 -35- established by passing 5.6 liters of air per minute through the gasoline, beginning with 400 cc. and vaporizing from 160 to 200 cc. of the gasoline in the six hour period. (Some variation occurs because of variability of room tanperature.) Under these conditions the animals occasionally showed some slight intoxication from the gasoline. The air volume was in consider able excess over their respiratory requirements. The experiment with Ethyl Gasoline was begun Sept. 2, 1925. The gasoline was prepared by adding 1.5 cc. of purified tetraethyl lead to 3785 cc. of the gasoline base. 400 cc. of this gasoline was introduced daily into a closed jar through which air was bubbled into the cage. At the end of each six hour period the residual gasoline was transferred into a gallon bottle, and notation was made of the quantity used. Two samples of the original gasoline were removed for analysis from the stock bottle each time the above quantity was prepared, and when the 3785 cc. were used up, two samples of the mixed residues were taken. Thus two analyses (as a check on each other) were made on the lead content of each quantity of stock gasoline, and on each batch of the resid ual gasoline from that stock. In this manner a check was maintained on the quantity of lead, if any, which actually passed over into the respiration cage. Analysis of the lead in the gasoline was made by a standard method suggested by Graham Edgar. At the end of five months of exposure daily (except Sunday and holi days) Rabbit Ho. 118 began to show indication of injury from the gasoline. (Thus it may be seen that the animals were exposed continually to concentra tions just bordering on immediate toxicity.) This animal lost weight and collapsed in the cage after a few hours of exposure on several successive days. He was killed, therefore, and analysis of his carcass was made in the usual way for lead. Rabbit No. 117 showed no evidence of injury, and was -33- continued under the same experimental conditions for three months longer. At this time, April 28, 1926, the experiment was concluded, and Iio. 117 was killed and analyzed. The following tables illustrate the experimental results obtained. It is scarcely worth while to present all of the data on the daily evapora tion of gasoline, and the analyses of all the samples, since they are essen tially the same. For this reason the tables present sufficient data to il lustrate the various points involved. TABLE XII Typical Experiment and Calculations Date Gasoline at start Gasoline remainof experiment ing after 6 hrs. Dec.16 17 18 19 21 29 Jan.12 13 14 15 '18 19 25 26 27 400 11 rr k u it M if II ft I! ?r >i ii 200 cc. 215 215 200 300 315 205 200 215 200 215 200 200 205 Z5 Totals or Averages 6000 3300 Pb.content per 100 cc. gasoline Original Residual 0.0400 0.0754 0.0405 0.0408 0.0404 0.0404 0.0748 0.0751 Total lead at start (6000 X0.0404) 2.424 Total 1ead at end (3300 X0.0751) - 2.478 Total 1ead in vaporized gasoline (calculated from above) 11 i 1 - 0018039 -37- TABLE XIII Typical Experiment and Calculations Date Gasoline at start Gasoline remain Pb. content per 100 cc. gas of experiment ing after 6 hrs. Original Residual Jan. 28 29 30 Feb. 1 2 3 4 5 8 9 10 11 12 15 16 ` 400 cc fl 11 11 !l # If 1 U 11 ri ' 11 11 ii U It n ' 200 205 270 210 200 200 200 200 205 200 20C 220 205 205 230 220 .0400 0.0787 .0402 .0408 .0404 0.0780 Total and averages 6400 ce 3370 0.0403 0.0783 Total lead at start (6400 x .0403) 2.58 Total at end (3370 x .0783) 2.64 Total lead vaporized with gasoline (calc, from above) Hil TABLE XIV Animal Ho. Analytical Results on Animals Mgs. rb in Treatment Period of time Weight Lbs. Carcass 121 ' Ifone. Control 119 Gasoline inhalation. Control. 117 Ethyl Gasoline in halt ion 118 Same 8 Mo. 8 Mo. 8 mo. 5 mo. 6-3/4 5-1/4 6-1/2 4-10/l 6 2.60 0.45 0.77 1.00 The temperature of the gasoline was taken by having a thermometer in serted into the jar through which the stream of air was passing. It was ob served at the beginning of the experiment and at the end. Variations of from 21 C. to 30 C. were observed over the period of experimentation. K 001-8040 -38- A study of the data above demonstrates in two ways that under the experimental conditions the animals were not exposed to inhalation of lead vaporized out of the gasoline. In the first case Tables XII and XIII, which are typical of the analytical results obtained throughout the experi ment, show that all of the lead in- the gasoline remained behind in the un volatilized gasoline. Tab]e XIV shows the same fact in a different manner, in that the test animals fail to show evidence of having absorbed more lead than may be found in control animals that have had no exposure to gasoline containing lead. On either of these bases it may bo stated def initely that at the temperatures prevailing throughout the months of these experiments, no lead volatilizes out of gasoline when the concentration f tetraethyl lead in gasoline does not exceed 0.0787 grams per 100 cc. gaso line. This is equivalent to approximately 3 cc, of tetraethyl lead per gallon of gasoline. . CONCLUSTOUS ' In as much as each experimental result has been discussed in its proper place it is scarcely necessary to elaborate on discussion and conclu sions. A consideration of the questions originally proposed for answer may be of some benefit, however. 1. It has been shown that under conditions which allow for spillage and evaporation of spilled gasoline leaving tetraethyl lead and dust of decomposition products of tetraethyl lead, and which provide for regular skin contact and some ingestion of lead, such experimental animals as guinea pigs and rabbits do not give evidence of accumulation of lead in their bodies. Further, these animals as well as monkeys, show no evidence -39- whatever of injury to any of the functions normal to such animals, including reproductive functions, as a result of such treatment. It may he objected that the experimental conditions were not severe enough to duplicate the conditions of human exposure. It has been our purpose, however, to make the conditions more severe than those which occur in the handling of Ethyl Gasoline and in our judgment we have done so. It is worthy of note that the above conclusions, as are the exper imental data, are in complete contradiction of those of Frederick B. Flinn,* on the same problem. 2. Tetraethyl lead is absorbed out of gasoline in immediately toxic quantities when the concentration of tetraethyl lead in gasoline <is of the order of magnitude of 1 part to 10 of gasoline. 3. In concentrations not exceeding 1 part of tetraethyl lead to 1000 parts gasoline, there is no evidence that rabbits absorb sufficient lead to cause accumulation of lead in their tissues. There is no selective absorp tion of the lead under these conditions. 4. Tetraethyl lead is not given off in the vapor of Ethyl gasoline at the temperatures normally prevailing during the seasons of the temperate zone. It cannot give rise to accumulation of lead in the tissues of per sons who are exposed to volatilization of normally concentrated Ethyl gasoline. Dated, November 18, 1926.- * Frederick B. Flinn - Some of the Potential Health Hazards from the Use of Ethyl Gasoline - The Journal of Industrial Hygiene VIII 51-53 Feb. 1926. .