PUBLIC HEALTH REPORTS VOL 45 OCTOBER 17, 1930 NO. 42 THE PHARMACOLOGICAL ACTION OF CERTAIN PHENOL ESTERS, WITH SPECIAL REFERENCE TO THE ETIOLOGY OF SO-CALLED GINGER PARALYSIS (Second Report) By MAURICZ I. SMITH, Senior Pharmacologist, National Institute of Health, United States Public Health Service, with the cooperation of E. ELVOVE, Chemist, National Institute of Health, and W. H. FRAZIER, Chemist, Bureau of Industrial Akohol In a preliminary communication (1) on the probable cause of the recent widespread epidemic of the condition clinically described as peripheral multiple neuritis, believed to have resulted from drinking adulterated fluid extract of Jamaica ginger, certain pharmacological and chemical evidence was presented to show that a phenol, firmly bound chemically with phosphoric acid, appeared to be the specific etiologic factor. The precise chemical nature of the phenolic com- pound was not revealed owing to the technical difficulties involved in its isolation and identification, though on the basis of the evidence presented it was suspected to be a phosphoric acid ester of one or more of the cresols. Conldusive evidence on the pharmacological side of this problem was equally lacking, on account of the difficulties experienced in faithfully reproducing the human disease in the usual laboratory animals. After much indirect evidence had been obtained pointing to the cresol ester as the probable immediate cause of the paralysis in question, a successful experiment was conducted upon calves. This experiment showed that the human disease could be faithfully reproduced in the calf, as distinguished from the usual laboratory animals, and that the offending agent must have been contained in a technical grade of tricresyl phosphate, the peculiar action of which was either due to an impurity, to the substance itself, or to a combination thereof with some ginger constituent. The obvious procedure to carry the solution of this problem to a satisfactory conclusion was to ascertain (a) whether an impurity in the technical tricresyl phosphate had anything to do with the pa- ralysis, (b) which of the three isomers of tricresyl phosphate had this specific action on the motor nerve mechanism, (c) what the probable 11777°-30-.1 (2509) October 17. 1930 2510 reason was for the wide differences in species susceptibility, and lastly (d) what is the probable manner of action of the specific ester. The present experiments are given in an attempt to answer at least in part some or all of these questions. Some of the results of this study indicate that the suggestion made in the first report to account for differences in species susceptibility solely on the ground of differences in hydrolytic cleavage of the ester in different animals is no longer tenable; and while this suggestion may still be in part correct, it will have to be modified to harmonize with the new facts. EXPERIMENTAL It will be recalled from the evidence presented in the first report (1) that the most important single lead to the solution of this problem was the observation that the suspected adulterated gingers presented uniformly and characteristically a toxicity in rabbits on oral adninis- tration which could not be accounted for by the ginger, the alcohol, or any of the well-known poisons. The chemical finding of phenols on saponification, acidification, and steam distiration directed our attention to stable phenolic compoumds for two reasons: First because the phenols could only be obtained upon drastic hydrolysis; and, second, what is perhaps more important, because the symptom com- plex produced by the suspected ginger in rabbits resembled that of the systemic action of phenol or the cresols, yet differed from them in oe.tain essential details. In particular the manifestations of the adulterated ginger poisoning in the rabbit were characterized by a long delay in the onset of the symptoms, sometimes by an interval of several days, and by evidence of very pronounced stimulation of the spinal cord, sometimes lasting over a period of several days before the condi- tion finally terminated in respiratory paralysis. Furthermore, the toxicity of the suspected ginger extracts was far greater than could be accounted for by the phenols or cresols recoverable from them. These early observations indicated, therefore, a plan of investiga- tion aiming at a comparative qualitative and quantitative study of the toxicity and cumulative effects in the rabbit of as many phenolic esters as were procurable, for which there was no or only a limited amount of data in the literature. Four phosphoric acid esters were procured for this work: 1. Triphenyl phosphate, technical grade; and 2. Tri-ortho cresyl phosphate, technical-both supplied by Celluloid Corporation, Newark, N. J. 3. Tri-ortho cresyl phosphate, C. P.; and 4. Tri-para cresyl phosphate, C. P.-both supplied by Eastman Kodak Co., Rochester, N. Y. There is no reliable information available as to the degree of purity of the technical triphenyl plhosphate, or the technical tri-ortho cresyl 2511 2Octoer17,1i8 phosphate. Some of the constants for the chemically pure tri-ortho cresyl phosphate as fumished by the manufacturers are as follows: Specific gravity at 200 C - 1.18 Boilng point at 20 mm. Hg- - 263-2650 C. Index of refraction -1.555 It is insoluble in water, and soluble in the usual lipoid solvents. P205 determination gave a value of 19.0, as against the theoretic value of 19.29 per cent. The Millon color value (11) of the cresol obtained by saponification, acidification, and steam distillation agreed with that given by a corresponding standard prepared from pure ortho cresol. The toxicity and cumulative effects of these substances were studied in rabbits upon oral administration in a solution of approximately 80 per cent alcohol to make it comparable as far as possibJe with the adulterated ginger extracts previously used. The amount of alcohol administered at one time was usually not more than 6 c. c. per kilo and never more than 8 c. c. per kilo. For comparative purposes ex- periments were made with pure phenol and with the cresols similarly administered. The results of this study are summarized in Tables 1 to 7. The subcutaneous minimum lethal dose of phenol and of the several cresols for rabbits as given by Meili, (2) Tollens, (3) Wandel, (4) and in part confirmed by ourselves is about as follows: Phenol -_----____------_--__--_----_--_____--___--500 mg. per kilo Ortho-cresol - 450 mg. per kilo Meta cresol -________--___----___ ----_____ ------500 mg. per kilo Para cresol -_____8_----__--__ 300 mg. per kilo Administered orally the phenols are somewhat less todic; thus Clarke and Brown (5) give the minimum lethal dose of phenol in rab- bits on oral administration as 0.6 gm. per kilo. When these figures are compared with the data given in Tables 1 to 7 it is at once obvious that tri-ortho cresyl phosphate stands out toxicologically apart, quantitatively as well as qualitatively, from either phenol, or the three isomeric cresols, or the phosphoric acid esters of phenol and para cresol. Thus from Table 5 it is evident that the minimum lethal dose of tri-ortho cresyl phosphate in the rabbit is 100 mg. per kilo, and as little as 50 mg. per kilo may result in definite symptoms which may occasionally prove fatal. The corre- sponding phosphoric acid ester of para cresol, on the other hand, failed to produce definite toxic effects in doses up to 700 mg. per kilo, as shown in Table 6. Furthermore, on repeated oral administration to rabbits, in daily sublethal doses, of some of the phenolic phosphoric acid esters it is shown that unlike tri-ortho cresyl phosphate they appear to be capable of detoxification at approximately the same rate as the corresponding phenols, while the action of the ortho ester is persistent and cumulative. Thus rabbits may survive as much as 1.4 Octobe IT, 19k2 25O12 to 1.5 grams per kilo of phenol, 1.4 grams per kilo of triphenyl phos- phate, 1.2 to 1.7 grams per kilo of tricresol, 1.0 to 1.5 grams per kilo of ortho cresol, and 1.0 to 1.6 grams of tn-pars cresyl phosphate, as shown in Tables 1, 2, 3, 4, and 7, respectively. Summing up the results of the experiments on rabbits it may be concluded that tni-ortho cresyl phosphate differs from the correspond- ing cresol or the other cresols or phenol or their phosphoric acid esters, first in toxicity and second in the manner of action.' The difference in toxicity is sufficiently clear from the data in the tables. The difference in manner of action may be summarized as follows: The systemic action of phenol or of the cresols is prompt. If a lethal dose is given, the symptoms appear within an hour or less, fol- lowed by coma and death in a very few hours. In case the dose is sublethal, recovery is equally prompt, so that by the following day the animal appears normal. By contrast, the systemic action of tri-ortho cresyl phosphate is slow. The first effects following the administration of a lethal dose are none other than those of the alcohol in which it is administered. After an interval of from one to several days the animals develop a strikingly characteristic group of symptoms which are unmistakable. They peculiarly combine the manifestations of mild strychnine poisoning with some of the features of phenol poisoning. There is thus a moderate degree of hyperexcitability, but never convulsions; there is a spastic incoordinated gait developing into a generalized fine tremor of the entire- musculature of the body; and- along with this there is a certain degree of emprosthotonos.