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THE JOURNAL OF INDUSTRIAL HYGIENE AND TOXICOLOGY Volume 21 MAY, 1939 Number 5 FURTHER OBSERVATIONS ON THE POSSIBLE SYSTEMIC TOX ICITY OF CERTAIN OF THE CHLORINATED HYDROCARBONS WITH SUGGESTIONS FOR PERMISSIBLE CONCENTRA TIONS IN THE AIR OF WORKROOMS* > Cecil K. Drinker Department of Physiology, Harvard School of Public Health, Boston, Mass. INCE 1937, several papers have The Effect of High and Low S appeared dealing with the sys temic effects of certain of the Calcium Intake on Animals In haling High Concentrations of a Chlorinated hydrocarbons. These are Mixture of Penta and Hexa- listed in the bibliography in the order of their appearance (1, 2, 3, 4, 5). chlornaphthalenes 62.6%). (Chlorine The first of these papers included recommendations as to safe concentra tions in the air of workrooms, that is, concentrations that would cause no ill effects to individuals who breathed them throughout a working day. It also mentioned certain other observa tions, such as the effects of diet on toxicity, which were being made at the time but which had not progressed far enough to merit description. It is now possible to report upon these matters and to list a fairly large num ber of chlorinated hydrocarbons to gether with permissible concentrations in air. * Received for publication February 10, It is well known that a diet rich in calcium is markedly effective in pre venting the acute yellow atrophy of the liver produced by carbon tetra chloride (Lamson et al. (6)). To dis cover whether calcium in the diet would protect against liver damage from a toxic chlorinated hydrocarbon, 30 adult white rats were placed upon a diet of lean horsemeat, starch and lard, a combination adequate for main tenance but very low in calcium. Another group was given a diet con sisting of dog chow, milk, lettuce and eggs, with added calcium lactate,--a ration very high in calcium. Both groups were exposed simultaneously 155 156 JOURNAL OF INDUSTRIAL HYGIENE AND TOXICOLOGY [vol. SI, no. 5 to inhalation of high concentrations of a mixture of penta and hexachlornaphthalenes (chlorine 62.6%), an average of 11.21 mg. per cu.m, for 16 hours a day. After 16 days, 12 high calcium diet rats were alive and 7 of the low cal cium 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 dam age. 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 will not prevent liver damage. The Effect of Injections of Xan thine on Animals Inhaling High Concentrations of a Mixture of Penta and Hexachlornaphthalenes (Chlorine 62.6%). In 1937, R. C. Neale (7) published a brief paper in which 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 examina tion in this laboratory. Accordingly 38 adult white rats were caused to inhale a mixture of penta and hexachlornaphthalenes (chlorine 62.6%) in concentrations averaging 15 mg. per cu.m, for 16 hours daily. One group of 20 rats was given 20 mg. of xanthine subcutaneously every other day and 40 mg. of xanthine by stomach tube on the alternate days. The second group of 18 rats had the same exposure to the compound 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 con nection with allied toxic compounds. Evidence for the Destruction of a Mixture of Penta and Hexachlornaphthalenes (Chlorine 62.6%) in the Body. It has always been a question as to whether the chlorinated hydrocarbons which have been examined by our selves and by others do harm per se or whether toxicity depends on their breakdown in the body with the liberation of something harmful to the liver. A partial answer has been obtained by feeding the above com pound to rats and dogs which were on a low chloride diet with uniform excretion of chlorides in the urine. When these animals received the chlorinated hydrocarbon the urinary chlorides rose. This indicates that the body certainly has power to de tach chlorine from this compound, and it is probable that the same condition is true for allied toxic compounds. The Effect of Increasing the Sodium Chloride in the Diet upon Animals Receiving Toxic Doses of a Mixture of Penta and Hexachlornaphthalenes (Chlorine 62.6%). On the ground that chlorides might be fundamentally associated with tox icity, a group of 15 rats was fed a low toxic dose of this compound and com pared with a similar group on the same dosage plus a marked increase in TOXICOLOGY [vol. SI, no. 5 t. No differences were noted cannot expect any efficacy thine either in the prevention aent of liver disease due to rinated hydrocarbon, and in ability the same negative >uld be encountered in convith allied toxic compounds. e for the Destruction of a RE OF PENTA AND HeXINAPHTHALENES (CHLORINE i in the Body. always been a question as to the chlorinated hydrocarbons we been examined by ourid by others do harm per se ier toxicity depends on their m in the body with the 1 of something harmful to the partial answer has been by feeding the above comrats and dogs which were on chloride diet with uniform of chlorides in the urine, hese animals received the ed hydrocarbon the urinary rose. This indicates that ' certainly has power to derine from this compound, and able that the same condition ar allied toxic compounds. fect of Increasing the i Chloride in the Diet Animals Receiving Toxic of a Mixture of Penta Iexachlornaphthalenes sine 62.6%). : ground that chlorides might mentally associated with toxroup of 15 rats was fed a low e of this compound and com* ith a similar group on the age plus a marked increase in Map, MS61 TOXICITY OF CHLORINATED HYDROCARBONS 157 chloride intake secured by giving 5 cc. per kg. of body weight of 4% sodium chloride solution daily. No differ ences were found between the two sebaceous glands. All that can be said at the moment is that increased chloride intake does not increase systemic toxicity. - TABLE I A List of 14 Chlorinated Hydrocarbons, with Chlorine Contents and Permissible Limits (in mg./cu.m.) for the Air in Workrooms* COMPOUND CHLO PERMIS RINE SIBLE CONTENT LIMIT % mgj cu.m. 1. Trichlornaphthalene plus a trace of tetrachlornaphthalene. Tested upon rats by inhalation and by feeding............................................ 49.9 2. Tetra and pentachlornaphthalenes. Tested upon rats by inhalation and by feeding.................................................................................... S6.4 3. Penta and hexachlornaphthalenes. Tested upon rats by inhalation and by feeding, and upon dogs by feeding alone.................................... 62.6 4. Tetra and pentachlornaphthalenes plus refined chlorinated diphenyl. ; Tested upon rats by feeding............................................................... 43.5 5. 90% penta and hexachlornaphthalenes plus 10% chlorinated diphenyl 10.0 1.0 0.5 0.5 benzene. Tested upon rats by inhalation and by feeding................. 63.0 0. Chlorinated diphenyl plus chlorinated diphenyl benzene. Tested upon 0.5 rats by inhalation and by feeding......................................................... 65.0 .7 Chlorinated diphenyl oxide. Tested upon rats by inhalation............. 54.0 .8 Chlorinated diphenyl oxide. Tested upon rats by inhalation.............. 57.0 .9. Chlorinated diphenyl. Tested upon rats by inhalation......................... 50-55 10 Hexachlor diphenyl oxide plus 5% trichlornaphthalene. Tested upon 0.5 0.5 0.5 0.5 .' rats by inhalation............................................................................... 50-55 11 Hexachlornaphthalene and crude chlorinated diphenyl. Tested upon rats by inhalation............................................................................... Un 0.5 0.5 12. Special chlorinated naphthalene. Tested upon rats by inhalation. 13. Chlorinated diphenyl. Tested upon rats by inhalation................. 14. Chlorinated diphenyl benzene. Tested upon rats by inhalation... known 50-56 68 60 0.5 10.0 0.5 *The analytical method and apparatus used routinely for field determinations is that described by Tebbens (This J., 19, 204 (1937)) and by Drinker et al. (ibid., p. 283). groups, and it may be concluded that chlorine increase secured through the diet does not enhance 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 of typical skin lesions by increasing chloride in take, cannot be decided by experi ments on fur-bearing animals with no The Systemic Effects of 14 Chlo rinated Hydrocarbons with Sug gested Limits for Concentra tions in the Air of Workrooms. The technic for inhalation and feeding experiments upon rats has been fully described (Drinker et al. (1)). It has been uniform for all the compounds listed in table 1, and for each group of experiments 24 to 48 158 JOURNAL OF INDUSTRIAL HYGIENE AND TOXICOLOGY \vol. SI, no. 6 animals have been sacrificed and toxicity because it is not broken down examined grossly and microscopically so as to gain very complete post mortem reports. in the body, but that would seem the probable explanation. Compound number 12 was prepared especially in The first 6 substances in table 1 order to have a substance for test have been dealt with in previous pub which should fall in chlorination be lished reports (cf. 1 and 3 in bibliog tween compounds 1 and 2. It proved raphy). In connection with them it somewhat toxic and, in addition to was recommended that in the case of changes in the liver, caused eye ir trichlornaphthalene and compounds of ritation and in one instance cata lower chlorination a limit of 10 mg. per racts. The substance is not in com cu.m, be permitted, the idea being mercial use and evidently at the that the essential feature of the action temperatures used for volatilization of these compounds, namely damage gave off some sort of irritant, possibly to the liver, did not become prominent hydrochloric acid. until a chlorine content of 50% was With the exception of this isolated reached. So far as trichlornaphtha effect upon the eyes, we have seen no lene is concerned the limit of 10 mg. lesions from any of the 14 compounds should be allowed to stand, since listed except in the liver. The char nothing has occurred which indicates acter of these lesions has been thor the necessity for changing it. The oughly described by Bennett et ol. second compound, a mixture of tetra (3). These additional data and the and pentachlornaphthalenes is in com tolerances recommended deal spe mon industrial use. A tolerance of cifically with systemic effects since one 0.5 mg. per cu.m, was formerly pro can get no information as to chloracne posed for it, but on the basis of labora in the skin from observations upon tory and field experience it is apparently entirely safe to increase this to 1.0 mg. per cu.m. The sixth compound has been listed previously as chlorinated diphenyl. It contained 65% of chlorine and proved very destructive to the liver. Later experiments with compound 13, fur-bearing animals. But it may be pointed out that where industrial workers have been careful as to clean liness and where air concentrations have been kept within the limits sug gested, skin lesions apparently cease to occur. which contained 68% of chlorine and which was also labelled chlorinated diphenyl, were a surprise to us since this second compound was almost non-toxic. On inquiry it was found that substance 6 was in reality a mixture of chlorinated diphenyl and chlorinated diphenyl benzene and that number 13 was actual chlorinated Summary Certain experiments carried out upon large groups of white rats indi cate the following: 1. A high calcium diet has no pro tective effect against the characteristic liver lesions caused by these com pounds. diphenyl. We have no information 2. Administration of xanthine does as to whether this last compound lacks not prevent liver injury. I TOXICOLOGY m. 21, no, 5 because it is not broken down jody, but that would seem ible explanation. Compound 12 was prepared especially in have a substance for test lould fall in chlorination bempounds 1 and 2. It proved it toxic and, in addition to in the liver, caused eye irand in one instance cataPhe substance is not in coin use and evidently at the tures used for volatilization some sort of irritant, possibly toric acid. the exception of this isolated >on the eyes, we have seen no rom any of the 14 compounds :cept in the liver. The charthese lesions has been thordescribed by Bennett et al. ise additional data and the es recommended deal spewith systemic effects since one no information as to chloracne skin from observations upon ing animals. But it may be out that where industrial have been careful as to cleannd where air concentrations m kept within the limits sugskin lesions apparently cease Summary n experiments carried out -ge groups of white rats indi following: ugh calcium diet has no proffect against the characteristic ions caused by these com- ninistration of xanthine does ent liver injury. May, J939l TOXICITY OF CHLORINATED HYDROCARBONS 159 3. When a mixture of penta and hexachlornaphthalene is given to dogs by mouth, the urinary chlorides rise, indicating that in some way the body cells detach chlorine from this com pound. 4. A high chlorine intake does not enhance toxicity. 5. A tabulation can now be pre sented covering 14 chlorinated hydro carbons with permissible safe concen trations in the air of workrooms. BIBLIOGRAPHY 1, Dbinker, C. K., Warren, M. F., and Bennett, G. A.: The problem of possi ble systemic effects from certain chlorinated hydrocarbons. This J., 19, 283 (1937). 2. Flinn, F. B., and Jarvik, N. E.: Liver lesions caused by chlorinated naphtha lene. Am. J. Hyg., 27, 19 (1938). 3, Bennett, G. A., Drinker, C. K., and Warren, M. F.: Morphological changes in the livers of rats resulting ' from exposure to certain chlorinated hydrocarbons. This J., 20, 97 (1938). 4. Cleary, R. V., Maier, J., and Hitchings, G. H.: The metabolism of chlorinated naphthalenes. J. Biol. Chem., 127, 403 (1939). 5. Greenburg, L., Mayers, M. R., and Smith, A. R.: The systemic effects re sulting from exposure to certain chlorinated hydrocarbons. This J., 21, 29 (1939). 6. Lamson, P. D., Minot, A. S., and Robbins, B. H.: The prevention and treatment of carbon tetrachloride in toxication. J.A.M.A., 90, 345 (1929). 7. Neale, R. C.: The protective action of certain purines against liver necrosis produced by carbon tetrachloride and chloroform. Science, 86, 83 (1937).