Dermal absorption of pesticides in mice

PESTICIDE

BIOCHEMISTRY

AND

PHYSIOLOGY

24, 119- 123 (1985)

Dermal Absorption

of Pesticides

R. E. GRISSOM,JR., C. BROWNIE, Interdepnrtmental

Toxicology

Program,

North

Carolina

State

in Mice

AND E E. GUTHRIE University,

Raleigh,

North

Carolina

27695

Received September 24. 1984: accepted November 29, 1984 The dermal penetration of 2,4-D. cyhexatin, fenvalerate, paraquat. maleic hydrazide, and captan was determined at 1, 6, and 24 hr after application to a 1.2-cm? area of the upperback of 7- to 8week-old mice (1 mgikg). The amounts of labeled pesticide were measured at the site of application and in the blood, liver, kidney, excreta. and carcass. Ninety-five percent, or greater, were recovered in all cases. These compounds were found to penetrate much slower (21% or less in 24 hr) than was shown for a group of insecticides (parathion, permethrin, carbofuran, and dieldrin. for example) in previous reports. Whereas slow penetration could be explained by low partition coefficients for most compounds, both fenvalerate and cyhexatin (high lipophilicity) were also found to have slow penetration rates. These compounds did not indicate any propensity for appreciable storage or binding in the tissues examined. The carcass contained the greatest amount of radioactivity at 24 hr. The fraction of each compound that penetrated was eliminated from the body at various rates with excretion being lowest for paraquat (50%) and highest for 2.4-D (93%) at 24 hr. d 1985 Academic Presr. Inc. INTRODUCTION

The in viva dermal absorption of a number of insecticides has been determined in target and nontarget organisms [Refs. (l3) for example]. In these studies, molecular weight, water solubility, and partition coefficients were examined in relationship to dermal penetration. Shah et al. (1) did not find a consistent relationship between physical constants and penetration although these factors are known to affect penetration (4-6). Reports of in vivo dermal penetration of pesticides is limited; therefore, the present study is directed at the dermal absorption of a diverse group of pesticides (representing herbicides, fungicides. acaricides, and insecticides) in the mouse. MATERIALS

AND METHODS

Radioactive chemicals. Captan (U-14C; sp act, 10 mCi/mmol) was obtained from Midwest Research Institute (Kansas City, MO). Cyhexatin (U-14C; sp act, 9.84 mCi/ mmol) was a gift from Dow Chemical Company, (Midland, MI). Fenvalerate (U-14C;

sp act, 19.6 mCi/mmol) was obtained from Shell Development Corporation (Houston, TX). 2,4-D (U-t4C; sp act, 1.2 mCi/mmol) and paraquat (methyl-14C; sp act, 111 mCi/ mmol) were obtained from Amersham Company (Arlington Heights, IL). Maleic hydrazide (U-14C; sp act, 5.05 mCiimmo1) was obtained from ICN (Irvine, CA). The purity of all compounds was reconfirmed with appropriate TLC systems. Experiments were conducted with compounds of 98% purity or greater. Dermal application. Procedures were similar to those previously described (1). Seven- to eight-week-old female Dublin ICR mice (27-30 g) were purchased from Dominion Laboratories (Dublin, VA).’ Upon delivery, animals were acclimated for 48 hr in the laboratory rearing room [photoperiod, 12:12; Purina rat chow (Purina, St. Louis, MO) and water ad libitum]. Twenty-four hours prior to treatment, a 3to 4-cm2 area of hair on the back and neck region was shaved with electric clippers ’ Dominion Laboratory, Dublin, VA (Conditions of laboratory described in Dominion Laboratory Brochure. Laboratory Animals. 1975). 119 0048-3575185 $3.00 CopyrIght ‘G 1985 by Academic Prear. Inc. All rights of reproduction !n any tivm recerved.

120

GRISSOM,

BROWNIE,

(type 40 blades; Oster, Milwaukee, WI), taking care to avoid skin damage. A polyethylene ring containing ventilation holes and an area of 1.2 cm2 was attached to the back with a cyanoacrylate adhesive to prevent scratching and grooming. Acetone was the vehicle for all compounds except paraquat, which was dissolved in water. and cyhexatin, which was dissolved in chloroform. Test materials (0.1 ml), containing 1 pCi of radioactivity mixed with enough nonradioactive compound to equal a total dose of 1 mg/kg body wt, were applied inside the ring. The ring was then covered with a Teflon cap. The mice were placed in mouse metabolism cages. Urine and feces were collected separately, and CO, was collected by a CO, trapping solution (R. J. Harvey Instrument Co., Hillsdale, NJ). Experiments were conducted at 23-25°C using one mouse per treatment and replicated three times for each chemical and time interval. The mice were anesthetized with ether, bled by cardiac puncture, and killed at the indicated time intervals shown in Table I. Any urine remaining in the animal was collected by gently rubbing the area around the bladder while carefully holding the animal over a collecting device. This urine was added to urine already collected in the metabolism cage. The metabolism cages were rinsed with appropriate solvents (acetone, chloroform, methanol, and/or water) and included with the urine. The skin at the site of application and specific tissues and organs (Table 2) were removed and radioactivity was determined at 1, 6, and 24 hr. The portion of the body remaining after the skin at the site of application and the organs and tissues were removed was termed carcass and was homogenized in liquid nitrogen. Whole organs or aliquots of homogenates were combusted in an oxidizer (Radiomatic Instruments and Chemical Co. Inc., Tampa, FL) equipped with a CO, trapping device containing approximately 7.4 ml of an organic amine (oxi-amin) and 12 ml of scintillation fluid (oxi-stint).

AND

GUTHRIE

Determination of radioactivity. Radioactivity was determined by a Packard TriCarb scintillation spectrometer (Packard Instrument Co., Downers Grove, IL). Quenching was corrected by internal standardization, and samples oxidized in the oxidizer were corrected to total percentage recovery for each time period. Partitiorl coefficients. Radioactive solutions containing 10 pg of pesticide were pipetted into 15ml heavy-duty centrifuge tubes. The solvent was evaporated to dryness under a gentle stream of nitrogen. Three milliliters of chloroform and 3 ml of demineralized water were added to each tube and vortex-mixed at full speed for 3 min. The tubes were then centrifgued at 25,OOOg for 30 min in a Sorvall high-speed centrifuge. One-milliliters aliquots of each layer were pipetted into test tubes. The chloroform was evaporated to dryness with nitrogen gas. Ten milliliters of Insta Gel (Packard Instrument Co., Downers Grove, IL) was added to each aliquot and then assayed. Statistical analysis. Preliminary examination of the data indicated that variation in penetration (from replicate to replicate) increased as mean penetration increased but that the coefficient of variation was relatively stable. The data were therefore transformed to the logarithmic scale in order to carry out statistical analysis. Comparisons among chemicals at a given time were performed on the log scale using the protected LSD (least significant difference) procedure at the 5% level of significance (7); that is, pairwise comparisons were made using the LSD only if an analysis of variance found a significant (P < 0.05) effect for chemicals. Results are reported in the original scale using geometric means and, where appropriate, a least significant ratio (LSR). With this representation, two geometric means are said to differ significantly if their ratio exceeds the LSR. To compare penetration for the chemicals in this study and those in a previous study (l), mean penetration after 8 hr was esti-

DERMAL

ABSORPTION

OF PESTICIDES

mated by regressing log penetration on time for each chemical in the present study. The resulting 95% confidence limits for mean penetration after 8 hr were compared with confidence limits for penetration after 8 hr for the chemicals in Table 1 of Shah et al. (1).

Recovery of radioactivity was 95% or greater in all cases. Penetration (disappearance from the site of application) is shown in Table 1, and distribution among various tissues is shown in Table 2. Both penetration and distribution are expressed as a percentage of the recovered dose. The amount of pesticide recovered from the cap was not included as part of the dose. Symptoms of toxicity were not observed at the dose levels employed in this study. The compounds examined were found to penetrate slowly in all cases. The most rapidly penetrating compound was 2,4-D at all time intervals, with approximately 7% penetration occurring in 1 hr and 21% penetra-

TABLE

Pesticide 2.4-D Fenvalerate Captan Paraquat Cyhexatin Maleic hydrazide LSRI PR > F”

between

Physical

Molecular weight 221 420 301 257 385 112

Parameters

121

tion by 24 h (Table 1). Fenvalerate, captan, paraquat, cyhexatin, and maleic hydraside were slower in penetration, with less than 10% penetration at 24 hr (Table 1). Entry was too slow to estimate T,,, values for penetration since the rate of penetration at time intervals greater than 24 hr was not known. The noninsecticidal compounds examined in this study tend to be hydrophilic (Table I), which supports this lack of penetration, but both fenvalerate and cyhexatin, lipid-soluble compounds, were also slow to penetrate. Thus, correlation between physical factors and penetration did not show a consistent pattern. The fat contained very little of the fractions of the compounds that penetrated. At 1 hr, the blood contained a maximum of 4.6% of 2,4-D, with cyhexatin also being present in the blood at a level of about 2% (Table 2); however, by 24 hr there was less than 1% of any compound in the blood. The liver contained from approximately 2% of maleic hydrazide to 10% of fenvalerate at 1 hr. The amount of compound remaining in

RESULTS

Comparison

IN MICE

I

and Percentage

H,O solubility

Partition coefficient”

620 ppm’ 2 ppb* 0.5 ppmC 5000 ppmr 1 ppb’ 6000 ppm’

6.9 x 10-s 87.3 1.67 1.69 x 10-j 5.77 9.1 x 10-4

Dermal

Penetration

in Mice

Geometric mean percentage dermal penetration” 1 hr

6 hr

24 hr

6.9“ I .9br 3.6”b 2.9b’ 0.7” 1.6’ 2.20 0.0007

8.9” 2.2’d 3.Sb 2.Q” 1.4” 1.4” I .65 0.0001

20.9” 9.1” 7.8” 6.7” 5.5” 5.4” 2.43 0.0528

u CHC1,:water. b Different superscripts in a column imply statistical differences using the protected LSD at the 5% significance level. ’ EPA Acceptable Common Names, 3rd Ed., Dec. 1975. d Shell Chemical Co., Houston, TX. c Dow Chemical Co., Midland, MI. f Least significant ratio. g P value associated with analysis of variance F test of the null hypothesis of no difference between mean penetration for chemicals.

122

GRISSOM.

BROWNIE,

AND

GUTHRIE

the liver tended to decrease with time although only 2,4-D and maleic hydrazide were present at levels less than 1% at 24 hr. The relative amounts of pesticides found in the kidneys also tended to decrease with time. The amounts in the kidneys ranged from 0.6% for maleic hydrazide to 8% for paraquat at 1 hr. By 24 hr, less than 2% of the compounds remained in the kidneys. The carcass contained the greatest percentage of the dose that penetrated at 1 hr, and ranged from 30% of 2.4-D to 84% of fenvalerate. By 24 hr, the amount of compound remaining in the carcass was reduced, with only 5.2% of the 2,4-D remaining while 40.7% of the paraquat still remained in the carcass. The amount of compound shown as excretion in Table 2 is the summation of the amount of compound detected in the CO?, urine, and feces. Excretion of all compounds was small in the CO? and feces. The majority of the pesticides were excreted in the urine. Only 1% of the fenvalerate that penetrated was excreted while 2,4-D was maximally excreted, with 58% being excreted at 1 hour. At 24 hr, excretion was increased for all compounds, with 50% of the dose being excreted for paraquat and increasing to 93% for 2,4-D (Table 2). DISCUSSION

In contrast to previous work with insecticides (l), this series of compounds (herbicides, fungicides, growth regulators, and insecticides) tended to show much slower dermal penetration. These compounds tend to have greater water solubility than the insecticides previously evaluated (1) although there are exceptions. Both fenvalerate and cyhexatin have appreciable lipid solubility but were also slow to penetrate. It is interesting to note that, while fenvalerate showed slow penetration, permethrin was among the most rapidly absorbed compounds in a previous experiment utilizing similar methodology (1). In studies with humans, 2,4-D and diquat were found to pen-

DERMAL

ABSORPTION

etrate slowly, ~1% in 24 hours (8). Whereas penetration of paraquat has been found to be slow (9), in gross accidents paraquat was found to cause mortality following dermal exposure (10). Linear regressions indicated that mean penetration for fenvalerate and cyhexatin at 8 hr would be 2.0 and 1.4%, respectively. Shah et al. (1) indicated that penetration at 8 hr ranged from 66 to 98% for the lipophilic insecticides that they evaluated. Obviously, the lipophilic compounds examined by Shah et al. (1) penetrated at a much faster rate than the compounds evaluated in the present study. Thus, a consistent correlation between physical properties of pesticides and in viva dermal penetration in the mouse remains elusive. Distribution of the fraction of the pesticides that penetrated was variable. The body contained approximately 28% of the fenvalerate, captan, and cyhexatin that penetrated, with the majority of these compounds being present in the carcass at the end of 24 hr (Table 2). The three herbicides varied widely with respect to the amount of pesticide located in the body, and ranged from 7% for 2,4-D to 50% for paraquat (Table 2). In all cases, the remaining pesticide was primarily located in the carcass. Fat did not show any tendency to accumulate any of the pesticides during the duration of the study. As noted in Table 2, the amount of compound detected in the carcass was decreased at 6 hr and further decreased at 24 hr. Metabolism may be responsible for the decrease in the body burden of these pesticides as well as being the rate-determining step. The amount of pesticide remaining in the carcass would be expected to decrease as the products of me-

OF PESTICIDES

IN MICE

123

tabolism are made available and eliminated from the body. ACKNOWLEDGMENTS This work was supported by NIEHS Grant ES00044. Paper No. 9493 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina 27695. REFERENCES 1. P. V. Shah. R. J. Monroe, and F. E. Cuthrie, Dermal penetration of pesticides in mice, To.uicol. Appl. Pharmacol. 59, 414 (1981). 2. P. V. Shah, R. J. Monroe, and F. E. Guthrie. Comparative penetration of insecticides in target and non-target species, Drug Chem. Toxicol. 6, 155 (1983). 3. J. B. Knaak, K. Yee, C. R. Ackerman, G. Zweig. D., and B. W. Wilson, Percutaneous absorption of triadimefon in the adult and young male and female rat, Toxico/. Appl. Pharmacol. 72, 406 (I 984). 4.

P. Grass0 and A. B. G. Landsdown, Methods of measuring and factors affecting percutaneous absorption, J. Sot. Cosmrr. Chems. 23, 481 (1972).

5. M. Katz and Z. I. Shaikh. Percutaneous corticosteroids absorption correlated to partition coefficient. J. Pharm. Sci. 54, 591 (1965). 6. E. J. Lien and G. L. Tong, Physiochemical properties and percutaneous absorption, J. Sot. Cosnwt.

Chern.

24, 371 (1973).

7. G. W. Snedecor and W. G. Cochran. “Statistical Methods,” Iowa State Univ. Press, Ames, Iowa. 1980. 8. R. .I. Feldman and H. I. Maibach, Percutaneous absorption of some pesticides and herbicides in man, To.yico/. Appl. Pharmacol. 28, 126 (1974). 9. R. C. Wester, H. I. Maibach, D. A. Buchs, and M. B. Aufrese, In viva percutaneous absorption of paraquat from hands. legs, and forearm of men, J. Toxicol. Environ. Health, in press. 10. D. J. Wohlfahrt, Fatal paraquat poisoning after skin absorption, Med. J. Australia 1, 512 (1982).