Toxicological Action and Metabolic Fate of Chlordan Ε. F. STOHLMAN and M. I. SMITH
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National Institutes of Health, Bethesda, Md.
In rabbits under light amytal anesthesia, chlordan has no direct effect on the blood pressure, but produces a type of respiration having many characteristics in common with Cheyne-Stokes type. The generalized tremors, opisthotonus, tonic and clonic convulsions, produced by chlordan were decreased or abolished and respira tion restored to normal by suitable injections of the sodium salts of amytal, phenobarbital, and pentothal. The LD of chlordan, which was about 20 mg. per kg. on intravenous administration to intact rabbits, was increased to about 60 mg. per kg. through the antidotal action of the barbiturates. An unidentified chlorine— containing degradation product with acidic properties was re covered from the urine of rabbits treated with chlordan. Ap proximately one third of its chlorine content was set free on hy drolysis at 100° C. with sodium hydroxide in either absolute alcohol or in water. 50
It was previously shown (4) that the oral L D o of chlordan i n rats was 200 to 250 mg. per kg., i n rabbits about 300 mg. per kg., and on intravenous injection i n rabbits i n Τ ween 20 about 20 mg. per kg. I t was also shown (4) that the chronic toxicity of chlordan i n both rats and rabbits was greater than that of D D T under comparable conditions. The purpose of this paper is to give some of the results obtained i n a study of the antidotal action of barbiturates i n chlordan poisoning, and of the metabolic fate of chlor dan. 5
Experimental I n this work the chlordan used was taken from the same lot employed i n previous work (4). The effects of this compound on blood pressure and respiration were studied i n rabbits under barbiturate anesthesia of light to moderate depth. F o r t y milligrams per k g . were the usual dose of barbiturate, administered intravenously. Chlordan was administered 25 to 40 minutes later intravenously as a 4 0 % solution i n Tween 20. A t this time animals having received sodium amytal were under slightly deeper anesthesia than those having received equivalent amounts of either sodium phénobarbital or sodium pentothal. Additional amounts of the barbiturates were given, when the animals developed severe general tremors and respiratory symptoms, i n order to determine their antagonistic action against chlordan. The antidotal action of the barbiturates was also investigated i n intact rabbits by the intravenous administration of both chlordan and the barbiturate. Sixty milligrams per kg. of chlordan were injected as a 4 0 % solution i n Tween 20 at a uniform rate over a period of 3 minutes. The sodium salts of the barbiturates were injected slowly as a 2.5% solution i n distilled water i n two doses. The first dose was given 2 to 5 minutes preceding 228
AGRICULTURAL CONTROL CHEMICALS Advances in Chemistry; American Chemical Society: Washington, DC, 1950.
STOHLMAN AND SMITH—TOXICOLOGICAL ACTION AND METABOLIC FATE OF CHLORDAN
229
the chlordan administration and the second, 5 to 15 minutes following i t . A l l animals were observed a minimum of 30 days.
CC-C 5
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Cft-C 6
1,2,4,5,6,7,8,8-OCTACHLORO2,3,3a,4,7,7a-HEXAHYDR0-4 7METHAN0-INDENE >
Structural Formula of Chlordan, CioHeCIs The metabolic fate of chlordan was studied i n rabbits by analysis of the relative chlorine content of chlordan added to normal rabbit's urine and of the chlorine content of the urinary excretory product. The method of analysis was similar to the one previously used (2, 4)- I n addition, hydrolysis of chlordan and of the urinary excretory productswas carried out b y adding solid sodium hydroxide to saturation to a 10-ml. solution of these substances i n hot absolute ethyl alcohol. The mixture was refluxed for 3 hours in a round-bottomed flask immersed i n boiling water and the amount of inorganic chlorine determined. Hydrolysis was similarly carried out with solutions of the respective substances i n aqueous sodium hydroxide.
Results The effects of chlordan on the blood pressure and respiration of rabbits under sodium pentothal are illustrated i n Figure 1. The injection of 60 mg. per kg. of chlordan produced a gradual drop i n blood pressure of about 20 m m . of mercury, starting during the injection and continuing for about 3 minutes after the injection (Figure I, A). I t then rapidly returned to the preinjection level. Since a similar drop i n blood pressure was produced b y the injection of an equivalent amount of the solvent, Tween 20, alone, it appears that chlordan has little if any direct effect on the blood pressure. Initially there was an increase i n respiratory rate with a slight decrease i n amplitude. This was followed b y a marked increase i n respiratory rate and amplitude with a simultaneous onset of generalized tremors. Cheyne-Stokes type of respiration followed (Figure 1,B). This consisted of alternating cycles of rapid and deep respirations with short periods of apnea, during which slight temporary elevations i n blood pressure may be seen. These cycles lasted from several minutes to 1.5 hours i n different experiments. M i l d to moderate tremors occurred more or less continuously, whereas the more severe tremors occurred i n cycles corresponding to the periods of apnea i n the Cheyne-Stokes cycle. The antagonistic effects of sodium pentothal on the blood pressure and the respiratoryeffects of chlordan are illustrated i n Figure 1, C. T h e intermittent severe tremors with concomitant Cheyne-Stokes type of respiration produced by the injection of chlordan were decreased or entirely abolished, and both the respiratory rate and depth were restored to near normal. A dose of barbiturate sufficient to diminish the tremors appreciably and to restore the respiration to normal also produced a temporary drop of 10 to. 40 m m . of mercury i n blood pressure. AGRICULTURAL CONTROL CHEMICALS Advances in Chemistry; American Chemical Society: Washington, DC, 1950.
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230
ADVANCES IN CHEMISTRY SERIES
Figure 1. Kymographic Tracing Showing Effects of Chlordan on Blood Pressure and Respiration of Rabbit under Light Pentothal Anesthesia and Antagonistic Action of Sodium Pentothal From top down: respiration, blood pressure, injection signal, and base line, time in 5 seconds; A = 60 mg./kg. chlordan, intravenously; β = 6 minutes later, Cheyne-Stokes respiration; C = 15 mg./kg. sodium pentothal 15 minutes later (respiration restored to normal and lowered blood pressure due to temporary action of pentothal); D = 20 minutes after pentothal injection T h e effects of chlordan on the blood pressure and respiration of rabbits under sodium pentothal, in general, were characteristic of those obtained under sodium amytal and so dium phénobarbital, with some variation in intensity and length of duration.
Table I.
Antidotal Action of Barbiturates in Chlordan Poisoning in Rabbits
Barbiturate Used, M g . / K g . Before Barbiturate chlordan Amytal N a Pentothal Na Phénobarbital N a Controls Controls α
20 25-30 20-30 20-30 0 0
a
After chlordan
Chlordan, Mg./Kg.«
No. D i e d / No. Used
30 15 15-50 30-40 0 0
60 40 60 60 30 20
1/13 0/3 8/14 5/14 10/10 17/23
Administered intravenously.
T h e antidotal action of the barbiturates in intact animals is shown in Table I . A l l three barbiturates in the amounts given were effective i n decreasing the severity of the intermittent general body tremors, opisthotonus, extensor rigidity, salivation, and in the prevention of convulsions. In some cases the symptoms were entirely abolished through out the critical period—the first 2 hours following chlordan administration. When deaths did occur they were generally delayed beyond the time of occurrence of death i n the nontreated groups ( 4 ) . T h e most effective antidotal action, however, was obtained with sodium amytal. T h e shorter acting sodium pentothal had considerably less antidotal effect under the conditions of these experiments. This may have been due to its relatively shorter duration of action, as a result of which the effects of chlordan were not antagonized over a sufficiently long period of time. T h e intermediate antidotal effect obtained with the long-acting phénobarbital is probably due in part to its relatively more shallow depth of anesthesia and slower action. T h e preliminary dose of the barbiturate was given in order to make the administration of the full dose of chlordan possible. Without this the severity of the tremors and convulsions developing during the injection made the complete
AGRICULTURAL CONTROL CHEMICALS Advances in Chemistry; American Chemical Society: Washington, DC, 1950.
STOHLMAN AND SMITH—TOXICOLOGICAL ACTION AND METABOLIC FATE OF CHLORDAN
Table II.
231
Fractionation of Organically Bound Chlorine Excreted in Urine of ChlordanPoisoned Rabbits Extracted, M g .
Experimental Control plus 228 mg. chlordan
With ether from acid urine, p H 3.8
From ether with dilute alkali, p H 8.6
With ether from aqueous alkali after acidification to p H 3.8
65.6 194.5 (85.3%) ·
55.0 (83.8%) 0
55.0 (83.8%) 0
Chlorine Split Off , ° ^ydrolysis at 100 C , % of Total Alcoholic Aqueous NaOH NaOH n
0
38.2 44.3
34.5 6.9
injection of the relatively large doses of chlordan difficult or impossible. As a result of the antagonistic action of these barbiturates, rabbits tolerated up to approximately three times the L D dose of chlordan. The analysis of urinary excretion of organically bound chlorine by chlordan-poisoned rabbits is shown i n Table I I . Apparently all the organic chlorine excreted i n the urine of chlordan-poisoned rabbits is extractable with ether from urines acidified to p H 4.0 or lower. Only part of it was extracted from urines acidified to p H 6.0, and relatively little was ex tracted from alkaline urines. Nearly all of the organic chlorine-containing constituents (83.8%) were extracted from ether with dilute alkali, and i n turn were again re-extracted from aqueous alkali with ether after acidification. I n contrast, chlordan added to normal rabbit urine could be extracted from either an acid or an alkaline urine with ether, to the extent of about 8 0 % of the amount added. Moreover, this was not extractable with d i lute alkali from the ether phase. About one third of the total organic chlorine i n the u r i nary excretory product was split off with either alcoholic or aqueous sodium hydroxide, whereas only about 7% of the total chlorine was removed from chlordan by aqueous so dium hydroxide under comparable conditions. These data indicate that the urinary ex cretory product probably consists of one or more acidic chlorine-containing degradation products of chlordan, which are water soluble at p H 8.6 or higher. It seems possible that the labile chlorines i n positions 5 and 6 may be split off i n the body, followed by the oxi dation of the corresponding carbons.
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Discussion The barbiturates are effective against convulsions induced experimentally from all central locations, the cerebrum, medulla, or spinal cord. They may be used clinically as well as experimentally to suppress most, if not all, varieties of convulsions of central origin (3). Since they are effective i n the prevention of the tremors, tonic and clonic convulsions, and i n the restoration of normal respiration from the Cheyne-Stokes type, produced by chlordan, it appears that these symptoms may have their origin in the central nervous system. The antidotal action of the barbiturates is probably limited to the effects of chlordan on the nervous system. They most likely have no beneficial antagonistic action against the delayed parenchymatous degenerative changes produced by chlordan (4). Therefore, they are primarily only of possible value i n acute poisoning i n which severe stimulation of the central nervous system may be the primary cause of death. The conversion i n the animal body of at least some of the water-insoluble chlordan to a water-soluble degradation product must facilitate the elimination of the poison through its excretion into the urine by the kidneys. Moreover, the degradation of chlordan as shown i n the present experiments may be a mechanism for its detoxification, as i n the case of D D T (1). Only the isolation of the degradation product, its identification, and a study of its toxicity can determine this point.
Literature Cited (1) Smith, M . I., Bauer, H . , Stohlman, E . F., and Lillie, R. D., J. Pharmacol. Exptl. Therap., 88, 359 (1946). (2) Smith, M . I., and Stohlman, E . F., U . S. Pub. Health Service, Rept. 59, 984 (1944). (3) Sollmann, Τ. Α., "Manual of Phartnacology," 7th ed., p. 681, Philadelphia, W. B. Saunders Co., 1948. (4) Stohlman, E . F., Thorp, W. T . S., and Smith, M . I., Arch. Ind. Hyg. Occupational Med., 1, 1319 (1950).
AGRICULTURAL CONTROL CHEMICALS Advances in Chemistry; American Chemical Society: Washington, DC, 1950.
