Document gb1MarmweN6vgZ1LjkbjNLZJV

REPORT TO THE KQHSAHTO CHEMICAL CCO'AIJY by Cecil K. Drinker, 1S.D, Dean and Professor of Physiology Harvard School of Public Health, Boston, tlass. Septeeber 15, 1933 DSW 346074 STLCOPCB4084393 Face I. Experiments to determine the possible toxicity of the following substances: . ................................................................................................................ 2 1. Chlorcosone .............................................................................................. 2 2. Diphenyl phthalate................................................................................ 3 3. Chlorinated diphenyl#1268 ................................................................. 4 v 4. Mixture of chlorinated diphenyl and chlorinated diphenyl benzene #5460 ....................................................................... 7 II. Some general considerations:................................... 9 1. Evidence for the destruction of a mixture of penta and hexachlornaphthalenes (#1006) in the body.................. 9 2. The effect of increasing the sodium chloride in the diet upon animals receiving toxic -doses of =-1006 .... 10 3. The effect of high and low calcium intake on animals inhaling high concentrations of #1006 ........................... 10 4. The effect of injections of xanthine on animals inhaling high concentrations of #1006 ................................ . 11 DS\N 346075 STLCOPCB4084394 I. IXFERIKEKT5 TO DETSRI.?IKE IKE POSSIBLE TOXICITY 0? THE FOLLOhTlIG SUBSTANCES 1. CHLORCOSANE On July 14, 1937, we were informed by F. D. Smith that each 6 ounce bottle of a soft drink might contain 60 to 70 mgm. of chlorcosane. There Was no pharmacological reason to believe the compound would be irritating or toxic in any way, and therefore it was decided to dose rats with it very heavily, the argument being that if they survived heavy dosage without any evidence of damage a verdict of no toxicity might bo given and much time saved. If, on the other hand, the large doses proved poisonous it would be necessary to reduce dosage until a non-toxic level was reached. The first of these alternatives proved correct. Experiments. -- August 10, 1937. 20 adult white rats ranging in weight from 162 to 210 0. were each fed 70 mgm. of chlorcosane in 1 cc, of olive oil daily by stomach tube. These animals showed no evidence of damage in any way attributable to chlorcosane. They were examined microscopically from the 35th to tho 92nd day. The only constant change observed was a possible slight increase in the granularity of the cells of the liver cords, hut the pathologist in charge felt this to be within normal limits. This finding is doubly reassuring since many of the animals developed a chronic suppurative lung condition very prevalent in Boston stock rats at the time. But even with this added handicap there was no evidence of damage and we thus conclude that chlorcosane even in enormous dosage is an inert material in the body and in all probability passes through the intestine unchanged. DSW 346076 STLCOPCB4084395 2. DIPHENYL PHTHALATE On August 23, 1937, wo wore informed by Dr. R. E. Kelly that diphenyl ' phthalate might be absorbed through the skin or might be inhaled and swallowed during* spraying operations. After consultation it was decided to feod heavily, and if there were indications of toxicity to project other experiments. Experiments. -- September 20, 1937 . 20 adult white rats ranging in weight from 242 to 301 gm. were each fed 0.5 mgm. of diphenyl phthalate suspended in 1 cc. of water by stomach tube daily. The animals were from new stock and with one exception remained clear of the lung condition which existed in the chlorcosane group. During the period of experiment, which lasted 92 duys, practically all the animals gained weight. Examination of 8 animals killed at intervals during this period of test resulted as follows: The organs were normal grossly, and on microscopic examination the liver alone was of possible interest. The changes in the liver varied from those in which the cells of the liver cords were almost normal, having only a slight swelling and granularity of their cytoplasm, to those in which these changes were pronounced. In the latter there was a moderate degree of vacuolization and a rare hyaline body. These alterations it must be understood were the result of very certain dosage, since the compound was given by stomach tube and the animals had no possible way of avoiding at. Neither the pathologist, nor myself was able to consider them of enough moment, to cause us to make further experiments. I am confident that with the ordinary precautions .accompanying spray lacquering no possible ham could be done, even if the concentration of diphenyl phthalate in the lacquer wus far above the 5 per cent figure given us by R. E. Kelly. ` DSW 346077 STLCOPCB4084396 3. CfflflHIKAEZD DIFHINYL -- COMPOUND #1268 . s` " This material was furnished by the Monsanto Chemical Company. It was said to have a chlorine content of 68 per cent, the highest chlorine figure of any compound tested by us during the past three years. It had been our impression that toxicity and chlorine content followed one another closely, but this has not turned out to be the case for #1268.XEdxinxaaiai8atfiai;ocxji Further discussion of this point will follow the description of the experimental observations and their results. Experiments. -- August 2, 1937. The compound #1268 was administered by inhalation, the technique being that described in the paper entitled The Problem of Possible Systemic Effects from Certain Chlorinated Hydrocarbons, Journal of Industrial Hygiene, 1937, 19, 283. For the first observations 80 adult white rats were used. They v;ere exposed ns follows: Group 1. -- 80 animals. Temperature to which the compound was heated to introduce fume into air line 140-190 C. Average concentration in air breathed by rats 0.53 mgm. per cubic meter. Average daily exposure 16 hours. The experiment was continued for 119 days, animals being sacrificed at intervals for pathological examination and for the carbon tetrachloride and alcohol test (see paper previously cited). The conditions described above caused swelling end increase of granularity of the liver cells. Hyaline inclusions were rare. These changes were quite uniform in all animals examined after the 31st day, but were not certainly progressive. The rats were very.healthy throughout the period and there was an 'almost uniform gain in weight. The carbon tetrachloride-alcohol test was positive after 52 days, v.hich indicates that though liver damage vjqs apparently . slight some degree of harm had been done the organ. There vans no evidence of DSW 346078 STLCOPCB4084397 damage in any part of the body except the liver. At the end of 93 days 10 rats, apparently in excellent condition, were removed from the experiment and set aside for observations upon liver recovery. These recovery animals showed no clinical changes of any sort. When sacrificed 72 and 141 days after removal from exposure it was observed that the swelling of the liver cells had disappeared, but the granular and hyaline material remained in the liver cells and had apparently become permanent. There was absolutely no progression of damage after removal from exposure. These changes may be compared v/ith those produced in rats by inhalation of chlorinated diphenyl #4465, administered in a similar manner in concentra tions of 0.57 to 0.93 ngm. per cubic meter over similar periods of time. In the case of this compound the conspicuous difference was the far greater incidence of hyalinization of the liver cells, which in our opinion was the characteristic lesion caused by #4405 and mixtures containing it. One can therefore conclude that #1268 in low concentrations is definitely less toxic than #4465. Group 2. -- After 119 days, the low concentration of #1268 having proved but slightly poisonous, it was decided to increase the concentration in the air breathed by the 54 rats then remaining. Temperature and other conditions v/ere maintained ns during the first 119 days, but by the use of 4 vaporisers instead of 1 the average corccr.tration of #12.68 in the air was brought to 6.23 mgru. per cubic, meter of air. Exposure to this very high eoncentrati on was continued for 87 days. Tinder these extreme conditions the animals again remained in perfect health. The carbon tetrachloride or.4 alcohol test was positive but there v.as ' -4 . absolutely no other indication of liver damage and no evidence of disturbance to oilier organs. DSW 346079 ' STLCOPCB4084398 -6- T/hen examined at autopsy at the end of 87 days -- the animals had experienced a total exposure of 206 days -- there were no gross evidences of abnormality in any part. On microscopic examination no organ showed chenges except the liver. The number of cells containing hyaline and the amount of hyaline in the cells involved increased during this period of exposure to the higher concentration of #1268. The hyaline was most abundant in the portal zone of the lobule with very little in the central, area. After 42 days of exposure 10 rats were set aside and were sacrificed 73 days later. They were in excellent condition when removed and did not change. Grossly, at autopsy, they showed nothing abnormal, and on microscopic examination the liver cells had lost their swelling but retained increased granularity and hyaline inclusions. The experiments on inhalations of high concentrations of #1268 reenforce the conclusion that this compound is of low toxicity as compared with #4465 or with chlorinated naphthalenes above trichlornsphthalene. The question as to why #1268, the most highly chlorinated compound tested, proved but slightly harmful cannot be answered with any definiteness. It has been suggested that the toxicity of all these chlorinated compounds, even though of varied composition, may depend on the ability of tbe animal to decompose them efter lodgment in the tissues, and that this decomposition might be shown by an increase in the chlorine in the`urine in suitably conducted feeding experiments. In experiments upon this point we have shown a definite increase in the urinary chlorides when dogs and rats were fed a mixture of penta and .hexachlornaphthalenes (#1006), a'compound highly injurious to the liver and containing 62.6 per cent of chlorine, but similar observations have not been made with any of the chlorinated diphenyls or allied compounds. It may, however, be that when about 65 per cent chlorination is reached tho substances formed ere quite stable in the body and so cause h. minimum of damage. ' DSW 346080 STLCOPCB4084399 -7- In conclusion, #1268, If handled with ordinary precautions as to ventila tion should be entirely harmless to workmen. While it cannot be given an absolutely clean bill as to health, it is preferable to #4465 and #5460. ' 4. MIXTURE OF CHLORINATED DIPHENYL AND CHL0R3HATED DIPHENYL BENZENE -- COMPOUND #5460 This substance was furnished by the Monsanto Chemical Company end was said to have a chlorine content of 60 per cent, being in this respect below #4465 and far below #1268. Experiments. -- The inhalation technique was used as in the case of #1268 and #4465. August 2, 1937. 80 adult white rats vjere the subjects. Temperature to which the compound ivas heated to introduce fume into the air line 140-195 C. Average concentration in sir breathed by rats 0.085 mgm. per cubic meter. Average daily exposure 16 hours. The'experiment lasted 119 days, a certain number of animals were sacrificed for pathological examination, others were used for the carbon tetrachloride and alcohol test, and still others set aside for observations as to recovery from possible damage. In spite of the fact that the concentration in the sir breathed by the mis averaged about 1/6 that obtained at comparable tempera tv'.-os from -#1260, t nur her of these animals became sick and lost weight towards the end of the second month of exposure. When killed, such individuals showed gross mottling of the liver but no changes in other organs. On microscopic examination swelling ol1 the liver cells, increased granularity ur.d hyaline inclusions wore note.1 in ; . Nr killed as early as the 16th day of exposure. These changes obviously occurred rapidly. Hyaline deposits were never as numerous us with #1268. Animals DSW 346081 STLCOPCB4084400 -3- removed for recovery after 51 and 119 days of exposure did not gain markedly in weight, and one died for no obvious cause. Uhen examined at autopsy the swelling of tho liver cells had subsided, but abnormal granularity and hyalinization remained. Needless to say the carbon tetrachloride and alcohol test was positive whenever used. In view of the fact that #5460 in such loxv concentration proved so definitely toxic, no higher concentrations were tested. It seems imperative that whenever this compound is used in industry, great care be taken to keep concentrations in the air at an extremely low level. No liberties can be taken with it, as with :/12G8. 0S\N 346082 STLCOPCB4084401 II. SOIS GENERAL CONSIDERATIONS In addition to these tests of compounds, certain experiments were done which are of interest to those manufacturing or using chlorinated hydrocarbons. Details as to these experiments are of course available but are not included in this report since they are not of direct industrial interest. 1. EVIDENCE 3'0R THE DESTRUCTION OF A MDCTURE OF PEHTA AND HSXACIIDiaJAHrniALKIES (#1006) IN THE BODY It has always been a question as to 'Whether the chlorinated hydrocarbons which have been examined by ourselves and by others do harm per se or whether toxicity depends on their breakdown in the body with the liberation of some thing harmful to the liver. A partial answer has been obtained by feeding #1006 to rats and dogs which were on a low chloride diet with uniform excretion of chlorides in the urine. V/hen these animals received the chlorinated hydrocarbon (#1006) the urinary chlorides rose. This indicates that the body certainly has power to detach chlorine from this compound, and it is probable that the same condition is true for allied toxic compounds. It vjould be most interesting to see whether ingestion of #1268 results in similar findings or whether in the case of this relatively non-toxic compound there is practically no splitting off of chlorine. Neither time nor our.financial resources permitted such tests. t DSW 346083 STLCOPCB4084402 -10- 2. THE, EFFECT OF INCREASING THE SODIUM CHLORIEE IN THE DIET UPON ANItlAIS RECEIVING TOXIC DOSES OF #1006 On the ground that chlorides night be fundamentally associated with toxicity, a group of 15 rats was fed a low toxic dose of #1006 and compared with a similar group on the same dosage of #1006 plus a marked increase in chloride intake secured by giving 5 cc. per kilogram of body weight of 4 per cent NaCl solution daily. No differences were found between the two groups, and it may be concluded that chlorine increase secured through the diet does not enhar.ce toxicity. This experiment was done in order to find out whether increase in chloride intake during hot weather might be harmful. A particular phase of the problem, the possible enhancement cf typical skin lesions by increasing chloride intake, cannot be decided by experiments on fur-bearing animals with no sebaceous glands. All that can be said at the moment is that increased chloride intake does not increase systemic toxicity. DSW 346084 3. THE EFFECT OF HIGH Aim L0H CALCIUli OH AKB'ALS IwIlAHKG iulO;. Cvc :C--u. H TUta C 1( lOOo It is well known that a diet rich in calcium is markedly effective in preventing the acute yellow atrophy of the liver produced by carbon tetra chloride (The Prevention and Treatment of Carbon Tetrachloride Intoxication. By P. D. Lamson, K.B., S. Minot, Ph.D., and B. II. Bobbins, i:.S. Journal of the American Medical .association, 1920, volume 90, page 345). To discover whether calcium, in the diet would prou^ct against liver damage STLCOPCB4084403 -11- from a toxic chlorinated hydrocarbon, 30 adult white rats were placed upon a diet of lean horsemeat, starch and. lard, a combination adequate for maintenance but very low in calcium. Another group was given a diet consisting of dog chow, milk, lettuce and eggs, with added calcium lactate -- a ration very high in calcium. Both groups were exposed simultaneously to inhalation of high concentra tions of #1006, an average of 11.21 mgm. per cubic meter for 16 hours a day. After 16 days, 12 high calcium diet rats were alive and 7 of the low calcium group. At intervals animals were killed for examination. In both groups the liver was abnormal grossly and microscopically, and in both groups animals died from liver damage. It was impossible to consider that the high calcium diet was in the least degree protective. It may, therefore, be concluded that adding calcium to the diet of workers either in the form of extra milk or of calcium lactate vjill not prevent liver damage. t 4. THE EFFECT OF INJECTIONS OF XANTHINE ON ANH.1AIS INHALING HIGH C0NCHNTRATI01S OF #1006 In 1937, R. C. Neale published a brief paper (The Protective Action of Certain Purilies against Liver Necrosis Produced by Carbon Tetrachloride and Chloroform. Science, 1937, volume 86, page 83). He claimed that rats injected with sodium xanthine became markedly resistant to carbon tetrachloride. This suggested that xanthine might have similar protective power over liver damage from the chlorinated hydrocarbons on examination in this laboratory. .accordingly 38 adult white rats v.ere caused to inhale #1006 in concentra tions averaging 15 mgrn. por cubic meter for 16 hours daily. One group of 20 rats was given 20 irigm. of xanthine subcutaneously every other day and 4.0 ngm. DSW 346085 STLCOPCB4084404 of xanthine by stomach tube on the alternate days. The second group of 18 rats had the same exposure to #1006 without xanthine treatment. No differences were noted and one cannot expect any efficacy from xanthine either in the prevention or treatment of liver disease due to this chlorinated hydrocarbon, and in all probability the same negative result would be encountered in connection with allied toxic compounds. DSW 346086 STLCOPCB4084405