TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
36,491-501(1976)
The Dose-Dependent Pharmacokinetic Profile of 2,4,5-Trichlorophenoxy Acetic Acid Following Intravenous Administration to Rats M. W. SAUERHOFF, W. H. BRAUN, G. E. BLAU, AND P. J. GEHRINC Pharmacokinetics/Metabolism Group, Dow Lepetit, U.S.A., and Computations Research Laboratory, Dow Chemical, U,S.A., and Toxicology Research Laboratory, Health and Environmental Research, Dow Chemical, U.S.A., Midland, Michigan 48640 Received July 3,1975: accepted January, 2,1976
The Dose-DependentPharmacokineticProfile of 2,4,5-Trichlorophenoxy Acetic Acid Following Intravenous Administration to Rats. SAUERHOFF, M. W., BLAU, G. E., BRAUN, W. H., AND GEHRING, P. J. (1975). Toxicol. Appl. Pharmacol. 36, 491-501. The clearanceof 2,4$trichlorophenoxy aceticacid (2,4,5-T) from plasmaand its elimination from the body of rats were determined after single intravenous dosesof [14C]2,4,5-T. Dosedependent pharmacokinetics were found. Urinary excretion of unchanged2,4,5-T accounted for approx. 95% of the 14Cactivity excreted from the body. The results show that the distribution, and elimination of 2,4,5-T are markedly alteredwhen larger doseswere administered.The pharmacokinetic data indicate that the statistical projection of results of experimentswith largedosesof 2,4,5-T, to predict the hazardof exposure to small amounts,is not justified becausethe capability of the body to handlethe compoundhasbeenaltered. .2,4,5-Trichlorophenoxyacetic acid (2,4,5-T) is a plant growth regulator and herbicide. ‘The absorption, metabolism, and excretion of 2,4,5-T have been studied in man and experimental animals. Five human male volunteers ingested a single oral dose of 5 mg/kg without presenting detectable clinical effects (Gehring et al., 1973).The clearance of 2,4,5-T from the plasma aswell asexcretion from the body occurred by an apparent first-order rate processwith a half-life of 23 hr. Essentially all of the 2,4,5-T wasabsorbed into the body and excreted unchanged in urine. Erne (1966) demonstrated that orally administered salts of both 2,4,5-T and 2,4-dichlorophenoxyacetic acid (2,4-D) were readily absorbed in rats and eliminated primarily by the kidneys. The half-life of ,clearance from plasma of both 100 mg/kg of 2,4,5-T and 50-200 mg/kg of 2,4-D was approx 3 hr. The elimination half-life of 2,4-D from tissuesranged from 5 to 10 hr. Grunow et al. (1971) reported that 7 days after oral administration of 50mg/kg of 2,4-5-T to rats, only 69 % of the dose was recovered, all in the urine. The main constituent in urine was unchanged 2,4,5-T. Approximately 15-30 ‘A of the dose recovered in urine was found as conjugates of 2,4,5-T. Courtney (1970) found approx 90% of orally administered 2,4,5-T (SO-100 mg/kg) unchanged in the urine of rats within 72 hr. Piper et al. (1973) studied the clearance of 2,4,5-T from plasma and its elimination from the body of rats after single oral dosesof [14C]2,4,5-T. The half-life values for the clearance of 2,4,5-T from plasmaof rats given Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Bdtain
491
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ET AL
doses of 5, 50, 100, and 200 mg/kg were 4.7, 4.2, 19.4, and 25.2 hr, respectively. Halflife values for elimination from the body were 13.6, 13.1, 19.3, and 28.9 hr, respectively. These results establish dose-dependent pharmacokinetics. In interpreting the data of Piper t*t af. (1973) it is not possible to state whether dose-dependence is attributable to saturation of some excretory mechanism or to absorption. To resolve questions on absorption and subsequent eliminaton of 2,4,5-T additional pharmacokinetic studies were conducted to determine the rate of 2,4,5-T clearance from plasma, the volume of distribution, metabolism, and routes and rates of 2,4,5-T elimination from rats following a single iv dose of either 5 or 100 mg/kg of [14C]2,4,5-T. METHODS Animals. Sprague-Dawley (Spartan substrain) rats weighing 18@220 g were housed in metal metabolism cages designed for the separate collection of urine and feces. The rats were acclimated in their individual metabolism cages for 4 days prior to dosing. Food and water were available ad Zibitum to all rats throughout the acclimation and experimental periods. The cages were maintained in an air-conditioned room at a temperature of 24°C with a 12-hr light-dark cycle. Jugular cannulas were surgically implanted in all rats while anesthetized with methoxyflurane 72 hr prior to administration of the dose (Harms and Ojeda, 1974). Dosage solution. [Ring-U-14C]2,4,5-T (4.3 mCi/mmol, Mallinckrodt Chemical) was administered to rats as the sodium salt in an aqueous solution. A stoichiometric equivalent of NaOH was added and mixed with the 5- and lOO-mg/kg dosing solutions, converting the free acid of 2,4,5-T to the soluble sodium salt. The two dosing solutions were prepared equimolar with respect to sodium by adding NaCl to the 5-mg/kg dosing solution. The infrared spectrum of the sodium 2,4,5-trichlorophenoxyacetate (Na-2,4,5-T) obtained as a NUJOL mull was identical in all essential details to the spectrum of Dow Analytical Standard Na-2,4,5-T. An excessof HCl was added to an aliquot of the dosing solution and an additional infrared spectrum of the recovered free acid was obtained. This was compared to the spectrum of Dow Analytical Standard 2,4,5-T. The spectra of these materials were identical in all essential details. Thus, the addition of NaOH to 2,4,5-T did not cause chemical decomposition of the starting material. Unlabeled Dow Analytical Standard 2,4,5-T (greater than 99 %pure) and [14C]2,4,5-T were prepared to contain 1 mg of 2,4,5-T/1.5 ml for the 5-mg/kg dosing solution and 20 mg of 2,4,5-T/1.5 ml for the lOO-mg/kg dosing solution. Each solution contained approx. 5 &i/1.5 ml of [14C]2,4,5-T. Doses of 2,4,5-T were prepared to administer 5 mg/kg and 100 mg/kg of the free acid of 2,4,5-T. Dosing solutions were infused through a jugular cannula using a constant delivery syringe pump at the rate of 1 ml/min. After delivery of the dosing solution, the cannula was washed with physiological saline. The dose was determined gravimetrically by weighing the dosing syringes prior to and immediately following delivery of the dose. Five-microliter aliquots of the dose solutions were counted to quantitate the amount of 14C activity administered to each rat. The radiochemical purity of the dosing solution was determined using two thin-layer chromatography systems. Approximately 100,000 dpm were spotted on (1) alumina
PHARMACOKINETICS
OF
2,4,5-T
493
(0.25-mm thickness, Brinkman) and developed with glacial acetic acid (50): methanol (50); and (2) silica gel (0.25-mm thickness, Brinkman) and developed with chloroform (90) : methanol (5) : glacial acetic acid (5). Each plate was developed two dimensionally. The Rfl value of 2,4,5-T on both silica gel and alumina plates was 0.5. The diagonal area of each plate was scraped in l-cm sections and the scrapings counted for 14C activity in an Aquasol-water gel scintillation cocktail. The dosing solutions were shown to be 99.0 % radiochemically pure. Metabolism and balance study. Two rats of each sex received either 5 or 100 mg/kg of [14C]2,4,5-T. Urine was collected every 12 hr throughout the experimental period at both dose levels. Urine traps were immersed in Dry Ice baths. Feces were collected at 12, 36,60, and 84 hr at the 5-mg/kg dose, and every 24 hr through 168 hr at the lOOmg/kg dose. Following the last collection interval, the rats were killed by decapitation and exsanguinated. The carcasses were skinned, and the liver, kidneys, a sample of perirenal fat, and skeletal muscle were removed from each animal. Each metabolism cage was washed with water and acetone. The cage wash was subsequently counted for 14C activity. Bloodplasma study. Four rats of each sex were used to characterize the plasma concentrations of [14C]2,4,5-T. Two male and two female rats were administered either 5 or 100 mg/kg of [14C]2,4,5-T. Whole blood samples (approx. 70 ~1) were obtained by cutting the tail vein with a scalpel and collecting the blood in a heparinized capillary tube. The tubes were immediately sealed at one end and centrifuged in a hematocrit centrifuge. The tubes were broken close to the plasma red cell interface and the plasma was drained into preweighed liquid scintillation vials for subsequent 14C counting. Plasma samples were collected at 0.5, 1,2,4, 8, 12,20,28, and 36 hr after the 5-mg/kg dose, and at 0.5, 1, 2,4, 8, 12,24, 36,48, 60, and 72 hr after the lOO-mg/kg dose. Analysis of urine for 2,4,5-T metabolites. One hundred-microliter aliquots of urine were spotted on silica gel and alumina thin-layer plates and developed with chloroform (90): ethanol (5):glacial acetic acid (5) and glacial acetic acid (50):methanol (50), respectively. Standards of [14C]2,4,5-T were chromatographed with the urine samples. Plates were scanned on a Panax Thin Layer Scanner. The area on the plates containing [14C]2,4,5-T was marked, scraped, and transferred to liquid scintillation vials for subsequent 14C analysis. The remaining areas on the tic plates were scraped and handled in the same manner. The percentage of total 14C activity on each tic plate associated with [14C]2,4,5-T was calculated. Radioactivity analysis. All samples were counted in a Nuclear Chicago Mark II Liquid Scintillation System utilizing external standard ratios to determine quench correction. The validity of the quench correction curve was checked periodically using [14C]-toluene as the internal standard. Water and Aquasol were added to aliquots of urine, plasma, and cage wash, and analyzed for 14C activity. Feces, kidney, carcass, and liver samples were prepared as 33 % aqueous homogenates and oxidized in a Beckman Biological Material Oxidizer. Perirenal fat and skeletal muscle were oxidized directly. Carbon dioxide formed during the oxidation was trapped in 5 M 2-aminoethanol in 2methoxyethanol(l0 ml) and counted for 14C activity following addition of ascintillation 1 R, measured as the ratio of the distance from the origin to the top of the spot divided by the distance from the origin to the solvent front.
494
SAUERHOFF ET AL.
cocktail (10 ml). The cocktail contained 4 g of 2,5-diphenyloxazole (PPO) and 0. I g of 1: 4-di(2-(5-diphenyloxazolyl)benzene) (POPOP) per liter of 1: 1 (v/v) toluene: 2methoxyethanol. Recovery of 14C activity from spiked samples was 95 + 5 %. Gaschromatography-massspectrometry of 2,4,5-T. Urine samples (5 ml) were acidified with 1 ml of 1.ON HCl and extracted twice with 5 ml of diethyl ether. The ether extracts were methylated with diazomethane, evaporated to dryness, and redissolved in 0.5 ml of hexane. A 2-~1 aliquot of the hexane was injected into a LKB 9000 gas chromatograph-mass spectrometer (gc-ms). The gc column was glass 6 ft x 2.0 mm (i.d.), and packed with 10% OV-1 on Chromosorb W SO-100 mesh. The m/e 268, 270, and 272 peaks were monitored for sample quantitation. Recorded peaks were symmetrical; therefore peak heights and not peak areas were used for sample quantitation. RESULTS
The concentration of 2,4,5-T in the plasma of rats as determined by 14C activity is shown in Fig. 1 on a semilogarithmic plot of concentration versus time following iv doses of 5 and 100 mg/kg of [14C]2,4,5-T. Clearance of 14C activity at the 5-mg/kg dose indicates first-order clearance kinetics are operative. However, a nonlinear re-
10
20.
30
‘lo Time,
50
60
70
so
Hrr
FIG. 1. Concentration of 2,4,5-T in the plasma of rats as a function of time following a single iv dose of [%12,4,5-T at 5 mg/kg (B) and 100 mg/kg (A) as determined from the concentration of 14C activity. Data for each dose level were obtained from four rats.
PHARMACOKINETICS
OF
2,4,5-T
495
lationship is necessary to characterize the pattern for clearance with 100 mg/kg. Nonlinear parameter estimation can be used to fit the data of curve A to the equation (Wagner, 1973)
which is the Michaelis-Menten equation for a one-enzyme reaction where c is the substrate concentration: V, = 16.6 _+1.82 pg/hr/ml corresponds to the maximum rate where the enzyme is saturated and k, = 127.6 f 25.9 ,ug/g is the substrate concentration .at +V,. The pharmacokinetic parameters describing the rate of clearance of [r4C]2,4,5-T from plasma at both dose levels are presented in Table 1. The rate constant k describing TABLE 1 RATE CONSTANTS (k) AND HALF-LIFE VALUES (t+) FOR THE CLEARANCE OF [14C]2,4,5-T FROM PLASMA AND VOLUMES OF DISTRIBUTION FOLLQWING A SINGLE INTRAVENOUS DOSE OF 5 AND 100 mg/kg”
Dose (w/kg) 5 100
0.16 f O.Ol* 0.030 f 0.008* (O-36 hr) 0.13 f 0.03” (3672 hr)
4.33 f 0.27 23.1 + 4.9 (O-36 hr) 5.30 + 1.23 (36-72 hr)
190+ 8 235 + 10
DTwo male and two female rats at each dose level. * Calculated by linear regression analysis. c Maximum elimination rate constant calculated by the following equations (Wagner, 1973): Cl - cz + Km In(c1lcn) = vm(22 - td CI - ~3 + Km WI/~ = K, 0s - t,) V,,,/k, = k (maximum elimination rate constant).
the linear portion of the plasma curve at the lOO-mg/kg dose (36-72 hr) was not statistically different from the value when rats were given 5 mg/kg (O-36 hr). No sex differences were observed in the clearance of 14C activity from plasma. Projection of the blood plasma concentration curves back to the y intercept, t = 0, -allows calculation of the initial concentration of 2,4,5-T in the plasma prior to excretion. Dividing the initial plasma concentration of [14C]2,4,5-T in micrograms per milliliter into the dose in micrograms per kilogram gives the volume of distribution (V& milliliters per kilogram. V, increased with dose from 190 + 8 ml/kg to 235 f 10 ml/kg for the 5and lOO-mg/kg dose levels, respectively. Table 2 shows the excretion of r4C activity by rats following single iv doses of 5 and 100 mg/kg of [14C]2,4,5-T. Excretion is expressed as a percentage of the total dose found in urine and feces during successive time intervals. The rate constants for excretion of 14C activity from the body of rats given a single iv dose of 5 and 100 mg/kg and the corresponding half-life values (t+) are presented in Table 3. The constants were calculated by linear regression from data collected through the 84-hr collection interval. No sex differences were observed in the excretion of 14C activity from the body.
OF A SINGLE
a Values represent mesn + SD. ’ Feces collected over a 24-hr excretion interval.
96.0 f 4.55
Total
5 mdkg
DOSE OF
+ 4.42” rt 1.89 If: 2.79 + 1.35 + 0.65 + 0.05 rt 0.03
52.4 22.7 11.9 5.83 3.00 0.12 0.05
~. Urine
INTRAVENOUS
o-12 12-24 24-36 3648 48-60 60-12 72-84
Excretion interval (hr)
PERCENTAGE
TABLE
2
0.02
0.03
0.08
0.03
2.78 + 0.82
2.43 f -b 0.16 + -h 0.14 + -h 0.05 +
Feces
Total
84-96 96-120 120-144 144-168
&I2 12-24 2436 3648 48-60 60-72 72-84
Excretion interval (hr)
EXCRETED IN URINE AND SUCCESSIVE TIME INTERVALS
[“‘C]2,4,5-T FECES OF
Two FEMALE
0.23 0.10 0.20 0.17 85.8 f 3.50
k + f -t
f + f t
0.18 0.02 0.02 0.02 7.59 f 2.52
0.15 0.05 0.03 0.03
0.31 + 0.08 -1’
0.36 0.21 ++ 0.30 0.11 0.34 0.18 0.33 0.21
3.06 -h f 1 .OO
3.99 +f 0.28 0.48 2.00
DURING
-h 3.97 -t 1.63 -b
RATS
46.2 + 2.91 23.3 f 2.45 8.71 + 1.41
100 mg/kg
AND
Feces
MALE
Urine
Two
6
E% 9
E g
PHARMACOKINETICS
497
OF 2,4,5-T
TABLE 3 RATE CONSTANTS (k) AND HALF-LIFE VALUES (Q) FOR THE EXCRETIONOF [14C]2,4,5-TFROM THE BODYOF RATS FOLLOWING ASINGLEINTRAVENOUSD~SEOF~AND~OO~~/~~
(2) 5 100
0.065k 0.020 0.063f 0.020(0-84 hr)
10.7
0.010 k 0.005 (84-168 hr)
69.3
11.0
a Mean + SD for four rats. The elimination of 14C activity from the body appears to be dose-dependent. The elimination rate constants and t+ values for urinary elimination of 14C activity over a 84-hr time interval at the two dose levels are not statistically different. However, there are differences in the amount of 14C activity excreted in feces between each dosage group. Additionally, urinary elimination of 14C activity at the high dose appears to be biphasic with a slower elimination of 14C activity past the 84-hr collection interval. Eighty-four hours after administration, approx. 3.7% as compared with 0.6% of the initial dose remained in the body of rats at the lOO- and 5-mg/kg dose groups, respectively. To assess the overall recovery of 14C activity, the carcasses of rats in the excretion studies were homogenized and the 14C activity determined in aliquots of the homogenate. In addition, the cage wash was counted for 14C activity. Total recovery of 14C activity for the 5- and lOO-mg/kg dose level was 99.5 ? 5.1 % and 96.0 f 3.0 % (mean + SD). Tissue to plasma ratios were evaluated at the 5- and lOO-mg/kg dose levels 8 hr after administration of the dose. The concentration of 14C activity in plasma and selected tissues and the tissue/plasma ratios are presented in Table 4. Tissue to plasma ratios at the two dose levels were not statistically different with exception of the kidney. The results, however, indicate that the liver to plasma ratio is higher at the lOO-mg/kg dose. Kidney to plasma ratios were also evaluated at selected time intervals after the iv administration of 100 mg/kg of [14C]2,4,5-T. The concentration of 14C activity in plasma and kidney is presented in Table 5. Kidney to plasma ratios were less than 1 at the 4-hr interval. The kidney to plasma ratio increased with time following administration of the dose. At low plasma concentrations of 14C activity, the ratio was 4.95 + 0.59. The percentage of 14C activity in urine associated with [14C]2,4,5-T in two thin-layer chromatography systems is presented in Table 6. Between 96.7 and 99.3 % of 14C activity in urine appeared as [14C]2,4,5-Tat the 5-mg/kg dose level while between 93.8 and 97.1% of 14C activity appeared at the lOO-mg/kg dose level. In order to confirm the identity of the material in urine with a similar R, value to that of [14C]2,4,5-T, urine samples were analyzed by gc-ms. The amount of 2,4,5-T in each urine sample was calculated based on the specific activity of the dose and percentage of dose excreted during each collection interval. The percentage of calculated 2,4,5-T at the high dose as determined by gc-ms was 96.0 + 1.0,97.0 & 1.4, and 100.3 + 7.5 for the O-12, 12-24, and 24-36 hr collection intervals, respectively. Therefore, unchanged [14C]2,4,5-T accounted for greater than
SAUERHOFFETAL.
498
TABLE 4 CONCENTRATION OF “T ACTIVITY IN SELECTED TISSUES AND PLASMA 8 HRAFTERTHE ADMINISTRATION OF 5 AND 100 [r4C]2,4,5-T” Dose (m/k) 5
100
Tissue
Concentration (ccgk)
Tissue/Plasma ratio
Liver Kidney Brain Fat Muscle Plasma
0.94 + 0.12 9.26 _+ 2.23 -b 0.43 + 0.07 0.32 f 0.02 3.85 f 0.85
0.24 f 0.03 2.40 + 1.23
Liver Kidney Brain Fat Muscle Plasma
157+9 205 k 6.6 f 40.0 + 53.0 + 416 +
0.11 + 0.03 0.08 f 0.02 0.38 0.49 0.016 0.10 0.12
15 0.7 8.8 9.4 36
+ + f + +
0.07 0.10 0.040 0.03 0.03
’ Expressed as microgram equivalents of 2,4,5-T per gram; mean + SD of four rats. b Sample below detection limit. TABLE 5 CONCENTRATION OF 14C ACTIVITY IN KIDNEY AND PLASMA AT SELECTED TIME INTERVALS AFTER THE INTRAVENOUS ADMINISTRATION OF 100 mg/kg [r4C]2,4,5-T” Time interval 0-d 4 8 16 32 64
Plasma concentration 415 359 315 142 25
) + f + f
Kidney concentration
19 20 21 21 9
297 281 297 198 122
+ + k f *
Kidney/Plasma ratio
11 17 29 18 45
0.72 0.78 0.94 1.40 4.95
& f. f f f
0.02 0.03 0.04 0.11 0.59
a Expressed as microgram equivalents of 2,4,5-T per gram; mean ?I:SD of four rats. TABLE 6 PERCENTAGEOF URINARY 14C ACTIVITY AS [r4C]2,4,5-T” Time interval (hr) System Silica gel AL 0s Silica gel AL 03
Dose (mdkd 5 5 100 100
~ o-12 97.0 99.3 93.8 94.8
+ 1.0 of:0.6 f 5.5 + 2.5
12-24 99.3 98.7 94.7 97.1
a Mean + SD of four rats from each excretion interval.
+ + + f
24-36 1.2 2.3 3.3 2.5
99.0 96.7 95.0 96.4
f + + +
1.7 3.5 5.2 3.9
PHARMACOKINETICS
OF
2,4,5-T
93.8% of the 14C activity in urine as determined by thin-layer chromatography gc-ms at both dose levels.
499,
and
DISCUSSION
Greater than 93.8 % of the 14C activity excreted in urine was unchanged [14C]2,4,5-T. It can be inferred that this relationship also exists in plasma. Therefore, clearance of14C activity from plasma and urine represents clearance of 2,4,5-T to a good first approximation. The clearance of 2,4,5-T from plasma was clearly dose-dependent since there were marked differences in the shapes of the plasma clearance curves at the two, dose levels. It has been suggested that 2,4,5-T is cleared from the plasma by the kidney via the organic acid secretory pathway. Hook et al. (1974) have shown that kidney slices in, vitro concentrate [14C]2,4,5-T. The 2,4,5-T plasma-time profile indicates this active process may be saturated at 100 mg/kg because clearance from plasma occurs at a. maximum rate during the first 36 hr and then decreases according to a first-order process. Clearance during the first 36 hr can also be described by a first-order process. The rate constant and half-life values are shown in Table 1. The rate constant characterizing the first-order process for 36-72 hr is the same value as the rate constant valid for the entire clearance curve at the low dose level. This pattern exhibited at the high dose with decreasing substrate concentrations is characteristic of a saturable active transport process. Additional evidence in support of the concept of saturability of the excretory mechan-ism of 2,4,5-T comes from analysis of kidney tissue/plasma ratios. Eight hours after administration of 5 mg/kg the kidney/plasma ratio is 2.40 while at 100 mg/kg the ratio is 0.49. Kidney to plasma ratios at selected intervals following administration of 100 mg/kg demonstrate that as the plasma concentration of 2,4,5-T decreases, the ratio increases. The kidney cannot concentrate 2,4,5-T as effectively at the higher plasma concentrations associated with administration of 100 mg/kg. However, as plasma concentrations of 2,4,5-T decrease, the kidney can concentrate 2,4,5-T. The plasma concentration data presented in Fig. 1 reveal additional dose-dependent effects. Projection of the curve back to the y-intercept (time = 0), allows calculation of the concentration of 2,4,5-T in plasma prior to excretion. This concentration is de-pendent on the distribution of 2,4,5-T to tissues and its subsequent entry into the cells. and/or tissue spaces. The V, of 2,4,5-T is significantly larger at the high dose. If first-order processes were operative, then the Vd would not differ at these two dose levels. A comparison of liver tissue/plasma ratios 8 hr after the iv administration of 5 and 100, mg/kg of [r4C]2,4,5-T reveals that the ratios are 0.24 and 0.38, respectively. Liver tissue, therefore, is exposed to a higher concentration of 2,4,5-T than would have been predicted assuming the first-order kinetics of a 5-mg/kg dose of 2,4,5-T. While these differences are not statistically different, a trend is indicated. Examination of the excretion data (Table 2) reveals two significant features. At the lOO-mg/kg dose level, a greater fraction of the dose as 14C activity is excreted in the feces. This suggests that at high dose levels biliary excretion of 2,4,5-T and/or degradation products plays a more important role in the overall elimination of 2,4,5-T from the body.
500
SAUERHOFF
ET AL.
At the lOO-mg/kg dose level, there appears to be a second slow phase of 14C activity excreted in urine between 84 and 168 hrs. In rats given 5 mg/kg, 0.6% of the dose remained in the body after 84 hr. In those given 100 mg/kg, 3.7 ‘A of the original dose remained and was gradually eliminated. This leads to two alternative conclusions : (1) at 100 mg/kg, a larger fraction of the dose has gained entry into compartments, tissues, and cells from which it is slowly released, and/or (2) a fraction of 2,4,5-T has been metabolically converted in the body to products which are less readily excreted. This latter hypothesis is supported by data presented in Table 6. Between 96.7 and 99% of 14C activity in urine appeared as [14C]2,4,5-T at the low dose while between 93.8 and 97 % of 14C activity appeared as [14C]2,4,5-T at the high dose. While these are not significantly different, a trend is indicated. There are direct toxicological implications for the dose-dependent elimination of 2,4,5-T from plasma as well as from the body. Systemic toxicity of a drug or foreign compound is often a function of the concentration and duration of that drug in plasma. If the drug or foreign compound is eliminated at a slower rate from the plasma, and/or different metabolites are formed at a high dose, then the ability of the drug and/or metabolite to induce toxicity at the high dose will be greater. Therefore, more severe toxicological manifestations than would have been predicted will often occur at the high dose level where nonlinear kinetics are operative. However, toxic effects have not been observed with a single iv dose of 100 mg/kg of 2,4,5-T. There appear to be significant differences between the results of this study and that of Piper et al. (1973). Piper et al. administered 2,4,5-T orally, while in this study 2,4,5-T was administered by the iv route. Piper demonstrated dose-dependent elimination of 2,4,5-T from plasma. For any given oral dose, semilogarithmic plots of elimination versus time in plasma gave a linear relationship. At increasing doses, however, the elimination t, was longer which is a criterion of dose-dependentkinetics (Levy, 1968). Clearance curves did not show the classical Michaelis-Menten graphical representation of nonlinear kinetics asis shown in this study. One explanation for the data is that the study was not conducted over a sufficiently long time interval. An alternative is that oral absorption of 2,4,5-T is playing someundefined role in altering the clearance of 2,4,5-T from plasma. The pharmacokinetic data presented in this report indicate that the statistical projection of results of experiments with large dosesof 2,4,5-T to predict the hazard of exposure to small amounts is not justified becausethe capability of the body to handle the compound has been altered. REFERENCES D. K. (1970).2,4,5-T in the rat: Excretion pattern, serumlevels,placentaltransport and metabolism.In Pesticides Symposia, pp. 277-283.Halesand Assoc.,Florida. ERNE, K. (1966).Distribution and elimination of chlorinated phenoxyaceticacid in animals. COURTNEY,
Acta Vet. &and. 7, 240-256.
P. J., KRAMER, C. G., SCHWETZ, B. A., ROSE, J. Q., AND ROWE, V. K. (1973).The fate of 2,4,5-trichlorophenoxyaceticacid (2,4,5-T) following oral administration to man.
GEHRING,
Toxicol. Appl. Pharmacol. GRUNOW,
W.,
BOERNE,
26,352-361.
C., AND
BUDEZLES,
B.
(1971).Renal excretion of 2,4,5-T by rats. Fd.
Cosmet. Toxicol. 9, 667-670.
P. J., AND OJEDA, S.R. (1974).A rapid andsimpleprocedurefor thechroniccannulation of the rat jugular vein. J. Appl. Physiol. 36, 391-392.
HARMS,
PHARMACOKINETICS
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2,4,5-T
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J. B., BAILIE, M. D., JOHNSON, J. T., AND GEHRING, P. J. (1974). In vitro analysis of transport of 2,4,%richlorophenoxyacetic acid (2,4,5-T) by rat. Fd. Cosmet. Toxicol. 12, 209-218. LEVY, G. (1968). Dose dependent effects in pharmacokinetics. In Importance of Fundamental Principlesin Drug Evaluation.(D. H. Tedeschiand R. S. Tedeschi,Eds.). Raven Press,New York. PIPER, W. N., ROSE, J. Q., LENG, M. L., AND GEHRING, P. J. (1973).The fate of 2,4,Qrichlorophenoxyaceticacid (2,4,5-T) following oral administrationto rats and dogs.Toxicol. Appl. Pharmacol.26,339-351. WAGNER, J. G. (1973)Propertiesof the Michaelis-Menten equationsand its integratedform which are usefulin pharmacokinetics.J. Pharm.Biopharm.1, 103-121. HOOK,
18
AND
APPLIED
PHARMACOLOGY
36,491-501(1976)
The Dose-Dependent Pharmacokinetic Profile of 2,4,5-Trichlorophenoxy Acetic Acid Following Intravenous Administration to Rats M. W. SAUERHOFF, W. H. BRAUN, G. E. BLAU, AND P. J. GEHRINC Pharmacokinetics/Metabolism Group, Dow Lepetit, U.S.A., and Computations Research Laboratory, Dow Chemical, U,S.A., and Toxicology Research Laboratory, Health and Environmental Research, Dow Chemical, U.S.A., Midland, Michigan 48640 Received July 3,1975: accepted January, 2,1976
The Dose-DependentPharmacokineticProfile of 2,4,5-Trichlorophenoxy Acetic Acid Following Intravenous Administration to Rats. SAUERHOFF, M. W., BLAU, G. E., BRAUN, W. H., AND GEHRING, P. J. (1975). Toxicol. Appl. Pharmacol. 36, 491-501. The clearanceof 2,4$trichlorophenoxy aceticacid (2,4,5-T) from plasmaand its elimination from the body of rats were determined after single intravenous dosesof [14C]2,4,5-T. Dosedependent pharmacokinetics were found. Urinary excretion of unchanged2,4,5-T accounted for approx. 95% of the 14Cactivity excreted from the body. The results show that the distribution, and elimination of 2,4,5-T are markedly alteredwhen larger doseswere administered.The pharmacokinetic data indicate that the statistical projection of results of experimentswith largedosesof 2,4,5-T, to predict the hazardof exposure to small amounts,is not justified becausethe capability of the body to handlethe compoundhasbeenaltered. .2,4,5-Trichlorophenoxyacetic acid (2,4,5-T) is a plant growth regulator and herbicide. ‘The absorption, metabolism, and excretion of 2,4,5-T have been studied in man and experimental animals. Five human male volunteers ingested a single oral dose of 5 mg/kg without presenting detectable clinical effects (Gehring et al., 1973).The clearance of 2,4,5-T from the plasma aswell asexcretion from the body occurred by an apparent first-order rate processwith a half-life of 23 hr. Essentially all of the 2,4,5-T wasabsorbed into the body and excreted unchanged in urine. Erne (1966) demonstrated that orally administered salts of both 2,4,5-T and 2,4-dichlorophenoxyacetic acid (2,4-D) were readily absorbed in rats and eliminated primarily by the kidneys. The half-life of ,clearance from plasma of both 100 mg/kg of 2,4,5-T and 50-200 mg/kg of 2,4-D was approx 3 hr. The elimination half-life of 2,4-D from tissuesranged from 5 to 10 hr. Grunow et al. (1971) reported that 7 days after oral administration of 50mg/kg of 2,4-5-T to rats, only 69 % of the dose was recovered, all in the urine. The main constituent in urine was unchanged 2,4,5-T. Approximately 15-30 ‘A of the dose recovered in urine was found as conjugates of 2,4,5-T. Courtney (1970) found approx 90% of orally administered 2,4,5-T (SO-100 mg/kg) unchanged in the urine of rats within 72 hr. Piper et al. (1973) studied the clearance of 2,4,5-T from plasma and its elimination from the body of rats after single oral dosesof [14C]2,4,5-T. The half-life values for the clearance of 2,4,5-T from plasmaof rats given Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Bdtain
491
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ET AL
doses of 5, 50, 100, and 200 mg/kg were 4.7, 4.2, 19.4, and 25.2 hr, respectively. Halflife values for elimination from the body were 13.6, 13.1, 19.3, and 28.9 hr, respectively. These results establish dose-dependent pharmacokinetics. In interpreting the data of Piper t*t af. (1973) it is not possible to state whether dose-dependence is attributable to saturation of some excretory mechanism or to absorption. To resolve questions on absorption and subsequent eliminaton of 2,4,5-T additional pharmacokinetic studies were conducted to determine the rate of 2,4,5-T clearance from plasma, the volume of distribution, metabolism, and routes and rates of 2,4,5-T elimination from rats following a single iv dose of either 5 or 100 mg/kg of [14C]2,4,5-T. METHODS Animals. Sprague-Dawley (Spartan substrain) rats weighing 18@220 g were housed in metal metabolism cages designed for the separate collection of urine and feces. The rats were acclimated in their individual metabolism cages for 4 days prior to dosing. Food and water were available ad Zibitum to all rats throughout the acclimation and experimental periods. The cages were maintained in an air-conditioned room at a temperature of 24°C with a 12-hr light-dark cycle. Jugular cannulas were surgically implanted in all rats while anesthetized with methoxyflurane 72 hr prior to administration of the dose (Harms and Ojeda, 1974). Dosage solution. [Ring-U-14C]2,4,5-T (4.3 mCi/mmol, Mallinckrodt Chemical) was administered to rats as the sodium salt in an aqueous solution. A stoichiometric equivalent of NaOH was added and mixed with the 5- and lOO-mg/kg dosing solutions, converting the free acid of 2,4,5-T to the soluble sodium salt. The two dosing solutions were prepared equimolar with respect to sodium by adding NaCl to the 5-mg/kg dosing solution. The infrared spectrum of the sodium 2,4,5-trichlorophenoxyacetate (Na-2,4,5-T) obtained as a NUJOL mull was identical in all essential details to the spectrum of Dow Analytical Standard Na-2,4,5-T. An excessof HCl was added to an aliquot of the dosing solution and an additional infrared spectrum of the recovered free acid was obtained. This was compared to the spectrum of Dow Analytical Standard 2,4,5-T. The spectra of these materials were identical in all essential details. Thus, the addition of NaOH to 2,4,5-T did not cause chemical decomposition of the starting material. Unlabeled Dow Analytical Standard 2,4,5-T (greater than 99 %pure) and [14C]2,4,5-T were prepared to contain 1 mg of 2,4,5-T/1.5 ml for the 5-mg/kg dosing solution and 20 mg of 2,4,5-T/1.5 ml for the lOO-mg/kg dosing solution. Each solution contained approx. 5 &i/1.5 ml of [14C]2,4,5-T. Doses of 2,4,5-T were prepared to administer 5 mg/kg and 100 mg/kg of the free acid of 2,4,5-T. Dosing solutions were infused through a jugular cannula using a constant delivery syringe pump at the rate of 1 ml/min. After delivery of the dosing solution, the cannula was washed with physiological saline. The dose was determined gravimetrically by weighing the dosing syringes prior to and immediately following delivery of the dose. Five-microliter aliquots of the dose solutions were counted to quantitate the amount of 14C activity administered to each rat. The radiochemical purity of the dosing solution was determined using two thin-layer chromatography systems. Approximately 100,000 dpm were spotted on (1) alumina
PHARMACOKINETICS
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493
(0.25-mm thickness, Brinkman) and developed with glacial acetic acid (50): methanol (50); and (2) silica gel (0.25-mm thickness, Brinkman) and developed with chloroform (90) : methanol (5) : glacial acetic acid (5). Each plate was developed two dimensionally. The Rfl value of 2,4,5-T on both silica gel and alumina plates was 0.5. The diagonal area of each plate was scraped in l-cm sections and the scrapings counted for 14C activity in an Aquasol-water gel scintillation cocktail. The dosing solutions were shown to be 99.0 % radiochemically pure. Metabolism and balance study. Two rats of each sex received either 5 or 100 mg/kg of [14C]2,4,5-T. Urine was collected every 12 hr throughout the experimental period at both dose levels. Urine traps were immersed in Dry Ice baths. Feces were collected at 12, 36,60, and 84 hr at the 5-mg/kg dose, and every 24 hr through 168 hr at the lOOmg/kg dose. Following the last collection interval, the rats were killed by decapitation and exsanguinated. The carcasses were skinned, and the liver, kidneys, a sample of perirenal fat, and skeletal muscle were removed from each animal. Each metabolism cage was washed with water and acetone. The cage wash was subsequently counted for 14C activity. Bloodplasma study. Four rats of each sex were used to characterize the plasma concentrations of [14C]2,4,5-T. Two male and two female rats were administered either 5 or 100 mg/kg of [14C]2,4,5-T. Whole blood samples (approx. 70 ~1) were obtained by cutting the tail vein with a scalpel and collecting the blood in a heparinized capillary tube. The tubes were immediately sealed at one end and centrifuged in a hematocrit centrifuge. The tubes were broken close to the plasma red cell interface and the plasma was drained into preweighed liquid scintillation vials for subsequent 14C counting. Plasma samples were collected at 0.5, 1,2,4, 8, 12,20,28, and 36 hr after the 5-mg/kg dose, and at 0.5, 1, 2,4, 8, 12,24, 36,48, 60, and 72 hr after the lOO-mg/kg dose. Analysis of urine for 2,4,5-T metabolites. One hundred-microliter aliquots of urine were spotted on silica gel and alumina thin-layer plates and developed with chloroform (90): ethanol (5):glacial acetic acid (5) and glacial acetic acid (50):methanol (50), respectively. Standards of [14C]2,4,5-T were chromatographed with the urine samples. Plates were scanned on a Panax Thin Layer Scanner. The area on the plates containing [14C]2,4,5-T was marked, scraped, and transferred to liquid scintillation vials for subsequent 14C analysis. The remaining areas on the tic plates were scraped and handled in the same manner. The percentage of total 14C activity on each tic plate associated with [14C]2,4,5-T was calculated. Radioactivity analysis. All samples were counted in a Nuclear Chicago Mark II Liquid Scintillation System utilizing external standard ratios to determine quench correction. The validity of the quench correction curve was checked periodically using [14C]-toluene as the internal standard. Water and Aquasol were added to aliquots of urine, plasma, and cage wash, and analyzed for 14C activity. Feces, kidney, carcass, and liver samples were prepared as 33 % aqueous homogenates and oxidized in a Beckman Biological Material Oxidizer. Perirenal fat and skeletal muscle were oxidized directly. Carbon dioxide formed during the oxidation was trapped in 5 M 2-aminoethanol in 2methoxyethanol(l0 ml) and counted for 14C activity following addition of ascintillation 1 R, measured as the ratio of the distance from the origin to the top of the spot divided by the distance from the origin to the solvent front.
494
SAUERHOFF ET AL.
cocktail (10 ml). The cocktail contained 4 g of 2,5-diphenyloxazole (PPO) and 0. I g of 1: 4-di(2-(5-diphenyloxazolyl)benzene) (POPOP) per liter of 1: 1 (v/v) toluene: 2methoxyethanol. Recovery of 14C activity from spiked samples was 95 + 5 %. Gaschromatography-massspectrometry of 2,4,5-T. Urine samples (5 ml) were acidified with 1 ml of 1.ON HCl and extracted twice with 5 ml of diethyl ether. The ether extracts were methylated with diazomethane, evaporated to dryness, and redissolved in 0.5 ml of hexane. A 2-~1 aliquot of the hexane was injected into a LKB 9000 gas chromatograph-mass spectrometer (gc-ms). The gc column was glass 6 ft x 2.0 mm (i.d.), and packed with 10% OV-1 on Chromosorb W SO-100 mesh. The m/e 268, 270, and 272 peaks were monitored for sample quantitation. Recorded peaks were symmetrical; therefore peak heights and not peak areas were used for sample quantitation. RESULTS
The concentration of 2,4,5-T in the plasma of rats as determined by 14C activity is shown in Fig. 1 on a semilogarithmic plot of concentration versus time following iv doses of 5 and 100 mg/kg of [14C]2,4,5-T. Clearance of 14C activity at the 5-mg/kg dose indicates first-order clearance kinetics are operative. However, a nonlinear re-
10
20.
30
‘lo Time,
50
60
70
so
Hrr
FIG. 1. Concentration of 2,4,5-T in the plasma of rats as a function of time following a single iv dose of [%12,4,5-T at 5 mg/kg (B) and 100 mg/kg (A) as determined from the concentration of 14C activity. Data for each dose level were obtained from four rats.
PHARMACOKINETICS
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495
lationship is necessary to characterize the pattern for clearance with 100 mg/kg. Nonlinear parameter estimation can be used to fit the data of curve A to the equation (Wagner, 1973)
which is the Michaelis-Menten equation for a one-enzyme reaction where c is the substrate concentration: V, = 16.6 _+1.82 pg/hr/ml corresponds to the maximum rate where the enzyme is saturated and k, = 127.6 f 25.9 ,ug/g is the substrate concentration .at +V,. The pharmacokinetic parameters describing the rate of clearance of [r4C]2,4,5-T from plasma at both dose levels are presented in Table 1. The rate constant k describing TABLE 1 RATE CONSTANTS (k) AND HALF-LIFE VALUES (t+) FOR THE CLEARANCE OF [14C]2,4,5-T FROM PLASMA AND VOLUMES OF DISTRIBUTION FOLLQWING A SINGLE INTRAVENOUS DOSE OF 5 AND 100 mg/kg”
Dose (w/kg) 5 100
0.16 f O.Ol* 0.030 f 0.008* (O-36 hr) 0.13 f 0.03” (3672 hr)
4.33 f 0.27 23.1 + 4.9 (O-36 hr) 5.30 + 1.23 (36-72 hr)
190+ 8 235 + 10
DTwo male and two female rats at each dose level. * Calculated by linear regression analysis. c Maximum elimination rate constant calculated by the following equations (Wagner, 1973): Cl - cz + Km In(c1lcn) = vm(22 - td CI - ~3 + Km WI/~ = K, 0s - t,) V,,,/k, = k (maximum elimination rate constant).
the linear portion of the plasma curve at the lOO-mg/kg dose (36-72 hr) was not statistically different from the value when rats were given 5 mg/kg (O-36 hr). No sex differences were observed in the clearance of 14C activity from plasma. Projection of the blood plasma concentration curves back to the y intercept, t = 0, -allows calculation of the initial concentration of 2,4,5-T in the plasma prior to excretion. Dividing the initial plasma concentration of [14C]2,4,5-T in micrograms per milliliter into the dose in micrograms per kilogram gives the volume of distribution (V& milliliters per kilogram. V, increased with dose from 190 + 8 ml/kg to 235 f 10 ml/kg for the 5and lOO-mg/kg dose levels, respectively. Table 2 shows the excretion of r4C activity by rats following single iv doses of 5 and 100 mg/kg of [14C]2,4,5-T. Excretion is expressed as a percentage of the total dose found in urine and feces during successive time intervals. The rate constants for excretion of 14C activity from the body of rats given a single iv dose of 5 and 100 mg/kg and the corresponding half-life values (t+) are presented in Table 3. The constants were calculated by linear regression from data collected through the 84-hr collection interval. No sex differences were observed in the excretion of 14C activity from the body.
OF A SINGLE
a Values represent mesn + SD. ’ Feces collected over a 24-hr excretion interval.
96.0 f 4.55
Total
5 mdkg
DOSE OF
+ 4.42” rt 1.89 If: 2.79 + 1.35 + 0.65 + 0.05 rt 0.03
52.4 22.7 11.9 5.83 3.00 0.12 0.05
~. Urine
INTRAVENOUS
o-12 12-24 24-36 3648 48-60 60-12 72-84
Excretion interval (hr)
PERCENTAGE
TABLE
2
0.02
0.03
0.08
0.03
2.78 + 0.82
2.43 f -b 0.16 + -h 0.14 + -h 0.05 +
Feces
Total
84-96 96-120 120-144 144-168
&I2 12-24 2436 3648 48-60 60-72 72-84
Excretion interval (hr)
EXCRETED IN URINE AND SUCCESSIVE TIME INTERVALS
[“‘C]2,4,5-T FECES OF
Two FEMALE
0.23 0.10 0.20 0.17 85.8 f 3.50
k + f -t
f + f t
0.18 0.02 0.02 0.02 7.59 f 2.52
0.15 0.05 0.03 0.03
0.31 + 0.08 -1’
0.36 0.21 ++ 0.30 0.11 0.34 0.18 0.33 0.21
3.06 -h f 1 .OO
3.99 +f 0.28 0.48 2.00
DURING
-h 3.97 -t 1.63 -b
RATS
46.2 + 2.91 23.3 f 2.45 8.71 + 1.41
100 mg/kg
AND
Feces
MALE
Urine
Two
6
E% 9
E g
PHARMACOKINETICS
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OF 2,4,5-T
TABLE 3 RATE CONSTANTS (k) AND HALF-LIFE VALUES (Q) FOR THE EXCRETIONOF [14C]2,4,5-TFROM THE BODYOF RATS FOLLOWING ASINGLEINTRAVENOUSD~SEOF~AND~OO~~/~~
(2) 5 100
0.065k 0.020 0.063f 0.020(0-84 hr)
10.7
0.010 k 0.005 (84-168 hr)
69.3
11.0
a Mean + SD for four rats. The elimination of 14C activity from the body appears to be dose-dependent. The elimination rate constants and t+ values for urinary elimination of 14C activity over a 84-hr time interval at the two dose levels are not statistically different. However, there are differences in the amount of 14C activity excreted in feces between each dosage group. Additionally, urinary elimination of 14C activity at the high dose appears to be biphasic with a slower elimination of 14C activity past the 84-hr collection interval. Eighty-four hours after administration, approx. 3.7% as compared with 0.6% of the initial dose remained in the body of rats at the lOO- and 5-mg/kg dose groups, respectively. To assess the overall recovery of 14C activity, the carcasses of rats in the excretion studies were homogenized and the 14C activity determined in aliquots of the homogenate. In addition, the cage wash was counted for 14C activity. Total recovery of 14C activity for the 5- and lOO-mg/kg dose level was 99.5 ? 5.1 % and 96.0 f 3.0 % (mean + SD). Tissue to plasma ratios were evaluated at the 5- and lOO-mg/kg dose levels 8 hr after administration of the dose. The concentration of 14C activity in plasma and selected tissues and the tissue/plasma ratios are presented in Table 4. Tissue to plasma ratios at the two dose levels were not statistically different with exception of the kidney. The results, however, indicate that the liver to plasma ratio is higher at the lOO-mg/kg dose. Kidney to plasma ratios were also evaluated at selected time intervals after the iv administration of 100 mg/kg of [14C]2,4,5-T. The concentration of 14C activity in plasma and kidney is presented in Table 5. Kidney to plasma ratios were less than 1 at the 4-hr interval. The kidney to plasma ratio increased with time following administration of the dose. At low plasma concentrations of 14C activity, the ratio was 4.95 + 0.59. The percentage of 14C activity in urine associated with [14C]2,4,5-T in two thin-layer chromatography systems is presented in Table 6. Between 96.7 and 99.3 % of 14C activity in urine appeared as [14C]2,4,5-Tat the 5-mg/kg dose level while between 93.8 and 97.1% of 14C activity appeared at the lOO-mg/kg dose level. In order to confirm the identity of the material in urine with a similar R, value to that of [14C]2,4,5-T, urine samples were analyzed by gc-ms. The amount of 2,4,5-T in each urine sample was calculated based on the specific activity of the dose and percentage of dose excreted during each collection interval. The percentage of calculated 2,4,5-T at the high dose as determined by gc-ms was 96.0 + 1.0,97.0 & 1.4, and 100.3 + 7.5 for the O-12, 12-24, and 24-36 hr collection intervals, respectively. Therefore, unchanged [14C]2,4,5-T accounted for greater than
SAUERHOFFETAL.
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TABLE 4 CONCENTRATION OF “T ACTIVITY IN SELECTED TISSUES AND PLASMA 8 HRAFTERTHE ADMINISTRATION OF 5 AND 100 [r4C]2,4,5-T” Dose (m/k) 5
100
Tissue
Concentration (ccgk)
Tissue/Plasma ratio
Liver Kidney Brain Fat Muscle Plasma
0.94 + 0.12 9.26 _+ 2.23 -b 0.43 + 0.07 0.32 f 0.02 3.85 f 0.85
0.24 f 0.03 2.40 + 1.23
Liver Kidney Brain Fat Muscle Plasma
157+9 205 k 6.6 f 40.0 + 53.0 + 416 +
0.11 + 0.03 0.08 f 0.02 0.38 0.49 0.016 0.10 0.12
15 0.7 8.8 9.4 36
+ + f + +
0.07 0.10 0.040 0.03 0.03
’ Expressed as microgram equivalents of 2,4,5-T per gram; mean + SD of four rats. b Sample below detection limit. TABLE 5 CONCENTRATION OF 14C ACTIVITY IN KIDNEY AND PLASMA AT SELECTED TIME INTERVALS AFTER THE INTRAVENOUS ADMINISTRATION OF 100 mg/kg [r4C]2,4,5-T” Time interval 0-d 4 8 16 32 64
Plasma concentration 415 359 315 142 25
) + f + f
Kidney concentration
19 20 21 21 9
297 281 297 198 122
+ + k f *
Kidney/Plasma ratio
11 17 29 18 45
0.72 0.78 0.94 1.40 4.95
& f. f f f
0.02 0.03 0.04 0.11 0.59
a Expressed as microgram equivalents of 2,4,5-T per gram; mean ?I:SD of four rats. TABLE 6 PERCENTAGEOF URINARY 14C ACTIVITY AS [r4C]2,4,5-T” Time interval (hr) System Silica gel AL 0s Silica gel AL 03
Dose (mdkd 5 5 100 100
~ o-12 97.0 99.3 93.8 94.8
+ 1.0 of:0.6 f 5.5 + 2.5
12-24 99.3 98.7 94.7 97.1
a Mean + SD of four rats from each excretion interval.
+ + + f
24-36 1.2 2.3 3.3 2.5
99.0 96.7 95.0 96.4
f + + +
1.7 3.5 5.2 3.9
PHARMACOKINETICS
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2,4,5-T
93.8% of the 14C activity in urine as determined by thin-layer chromatography gc-ms at both dose levels.
499,
and
DISCUSSION
Greater than 93.8 % of the 14C activity excreted in urine was unchanged [14C]2,4,5-T. It can be inferred that this relationship also exists in plasma. Therefore, clearance of14C activity from plasma and urine represents clearance of 2,4,5-T to a good first approximation. The clearance of 2,4,5-T from plasma was clearly dose-dependent since there were marked differences in the shapes of the plasma clearance curves at the two, dose levels. It has been suggested that 2,4,5-T is cleared from the plasma by the kidney via the organic acid secretory pathway. Hook et al. (1974) have shown that kidney slices in, vitro concentrate [14C]2,4,5-T. The 2,4,5-T plasma-time profile indicates this active process may be saturated at 100 mg/kg because clearance from plasma occurs at a. maximum rate during the first 36 hr and then decreases according to a first-order process. Clearance during the first 36 hr can also be described by a first-order process. The rate constant and half-life values are shown in Table 1. The rate constant characterizing the first-order process for 36-72 hr is the same value as the rate constant valid for the entire clearance curve at the low dose level. This pattern exhibited at the high dose with decreasing substrate concentrations is characteristic of a saturable active transport process. Additional evidence in support of the concept of saturability of the excretory mechan-ism of 2,4,5-T comes from analysis of kidney tissue/plasma ratios. Eight hours after administration of 5 mg/kg the kidney/plasma ratio is 2.40 while at 100 mg/kg the ratio is 0.49. Kidney to plasma ratios at selected intervals following administration of 100 mg/kg demonstrate that as the plasma concentration of 2,4,5-T decreases, the ratio increases. The kidney cannot concentrate 2,4,5-T as effectively at the higher plasma concentrations associated with administration of 100 mg/kg. However, as plasma concentrations of 2,4,5-T decrease, the kidney can concentrate 2,4,5-T. The plasma concentration data presented in Fig. 1 reveal additional dose-dependent effects. Projection of the curve back to the y-intercept (time = 0), allows calculation of the concentration of 2,4,5-T in plasma prior to excretion. This concentration is de-pendent on the distribution of 2,4,5-T to tissues and its subsequent entry into the cells. and/or tissue spaces. The V, of 2,4,5-T is significantly larger at the high dose. If first-order processes were operative, then the Vd would not differ at these two dose levels. A comparison of liver tissue/plasma ratios 8 hr after the iv administration of 5 and 100, mg/kg of [r4C]2,4,5-T reveals that the ratios are 0.24 and 0.38, respectively. Liver tissue, therefore, is exposed to a higher concentration of 2,4,5-T than would have been predicted assuming the first-order kinetics of a 5-mg/kg dose of 2,4,5-T. While these differences are not statistically different, a trend is indicated. Examination of the excretion data (Table 2) reveals two significant features. At the lOO-mg/kg dose level, a greater fraction of the dose as 14C activity is excreted in the feces. This suggests that at high dose levels biliary excretion of 2,4,5-T and/or degradation products plays a more important role in the overall elimination of 2,4,5-T from the body.
500
SAUERHOFF
ET AL.
At the lOO-mg/kg dose level, there appears to be a second slow phase of 14C activity excreted in urine between 84 and 168 hrs. In rats given 5 mg/kg, 0.6% of the dose remained in the body after 84 hr. In those given 100 mg/kg, 3.7 ‘A of the original dose remained and was gradually eliminated. This leads to two alternative conclusions : (1) at 100 mg/kg, a larger fraction of the dose has gained entry into compartments, tissues, and cells from which it is slowly released, and/or (2) a fraction of 2,4,5-T has been metabolically converted in the body to products which are less readily excreted. This latter hypothesis is supported by data presented in Table 6. Between 96.7 and 99% of 14C activity in urine appeared as [14C]2,4,5-T at the low dose while between 93.8 and 97 % of 14C activity appeared as [14C]2,4,5-T at the high dose. While these are not significantly different, a trend is indicated. There are direct toxicological implications for the dose-dependent elimination of 2,4,5-T from plasma as well as from the body. Systemic toxicity of a drug or foreign compound is often a function of the concentration and duration of that drug in plasma. If the drug or foreign compound is eliminated at a slower rate from the plasma, and/or different metabolites are formed at a high dose, then the ability of the drug and/or metabolite to induce toxicity at the high dose will be greater. Therefore, more severe toxicological manifestations than would have been predicted will often occur at the high dose level where nonlinear kinetics are operative. However, toxic effects have not been observed with a single iv dose of 100 mg/kg of 2,4,5-T. There appear to be significant differences between the results of this study and that of Piper et al. (1973). Piper et al. administered 2,4,5-T orally, while in this study 2,4,5-T was administered by the iv route. Piper demonstrated dose-dependent elimination of 2,4,5-T from plasma. For any given oral dose, semilogarithmic plots of elimination versus time in plasma gave a linear relationship. At increasing doses, however, the elimination t, was longer which is a criterion of dose-dependentkinetics (Levy, 1968). Clearance curves did not show the classical Michaelis-Menten graphical representation of nonlinear kinetics asis shown in this study. One explanation for the data is that the study was not conducted over a sufficiently long time interval. An alternative is that oral absorption of 2,4,5-T is playing someundefined role in altering the clearance of 2,4,5-T from plasma. The pharmacokinetic data presented in this report indicate that the statistical projection of results of experiments with large dosesof 2,4,5-T to predict the hazard of exposure to small amounts is not justified becausethe capability of the body to handle the compound has been altered. REFERENCES D. K. (1970).2,4,5-T in the rat: Excretion pattern, serumlevels,placentaltransport and metabolism.In Pesticides Symposia, pp. 277-283.Halesand Assoc.,Florida. ERNE, K. (1966).Distribution and elimination of chlorinated phenoxyaceticacid in animals. COURTNEY,
Acta Vet. &and. 7, 240-256.
P. J., KRAMER, C. G., SCHWETZ, B. A., ROSE, J. Q., AND ROWE, V. K. (1973).The fate of 2,4,5-trichlorophenoxyaceticacid (2,4,5-T) following oral administration to man.
GEHRING,
Toxicol. Appl. Pharmacol. GRUNOW,
W.,
BOERNE,
26,352-361.
C., AND
BUDEZLES,
B.
(1971).Renal excretion of 2,4,5-T by rats. Fd.
Cosmet. Toxicol. 9, 667-670.
P. J., AND OJEDA, S.R. (1974).A rapid andsimpleprocedurefor thechroniccannulation of the rat jugular vein. J. Appl. Physiol. 36, 391-392.
HARMS,
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J. B., BAILIE, M. D., JOHNSON, J. T., AND GEHRING, P. J. (1974). In vitro analysis of transport of 2,4,%richlorophenoxyacetic acid (2,4,5-T) by rat. Fd. Cosmet. Toxicol. 12, 209-218. LEVY, G. (1968). Dose dependent effects in pharmacokinetics. In Importance of Fundamental Principlesin Drug Evaluation.(D. H. Tedeschiand R. S. Tedeschi,Eds.). Raven Press,New York. PIPER, W. N., ROSE, J. Q., LENG, M. L., AND GEHRING, P. J. (1973).The fate of 2,4,Qrichlorophenoxyaceticacid (2,4,5-T) following oral administrationto rats and dogs.Toxicol. Appl. Pharmacol.26,339-351. WAGNER, J. G. (1973)Propertiesof the Michaelis-Menten equationsand its integratedform which are usefulin pharmacokinetics.J. Pharm.Biopharm.1, 103-121. HOOK,
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