Dermal Exposure Related to Pesticide Use - American Chemical Society

1 Percutaneous Absorption: Interpretation of In Vitro Data and Risk Assessment C.

G.

1,2

T O B Y MATHIAS , R O B E R T S. H O W A R D I. MAIBACH

3

HINZ ,

RICHARD

3

H . GUY , and

1

1

Downloaded by COLUMBIA UNIV on March 9, 2013 | http://pubs.acs.org Publication Date: February 25, 1985 | doi: 10.1021/bk-1985-0273.ch001

2

3

Department of Dermatology, School of Medicine, University of California Medical Center, San Francisco, CA 94143 Department of Medicine, Northern California Occupational Health Center, San Francisco General Hospital, San Francisco, CA 94110 Departments of Pharmacy and Pharmaceutical Chemistry, School of Pharmacy, University of California Medical Center, San Francisco, CA 94143

Assessment of r i s k based on i n v i t r o percutaneous absorption data depends on characterization of t o t a l penetrating amounts and of k i n e t i c parameters which define the time course of absorption. Methods for determining these parameters have been reviewed, A pharmacokinetic model, which has the broad c a p a b i l i t y to predict not only the time course of absorption, but also the amounts accumulating within skin and remaining on the skin surface to be absorbed w i l l be presented. Our results suggest that when a s o l i d substance i s deposited on the skin surface from a v o l a t i l e solvent, a f r a c t i o n of the applied dose is rapidly s o l u b i l i z e d into the skin; faster absorption of this initially solubilized f r a c t i o n is f a c i l i t a t e d . Assessment of risk upon cutaneous exposure to chemical substances requires analysis of three separate aspects of toxicology: 1) the quantitative amount of exposure; 2) the quantitative degree of percutaneous absorption following exposure; and 3) measurement of a b i o l o g i c a l effect following absorption. This paper w i l l address the second of these aspects. Percutaneous absorption may be measured in vivo or i t may be determined i n v i t r o using excised skin mounted i n glass d i f f u s i o n cells. The most frequently used approaches employ radiolabelled compounds. The v a l i d i t y of i n v i t r o measurements r e l i e s on the assumption that no metabolism occurs i n skin, and that absorption into the receptor f l u i d of the diffusion c e l l s approximates absorption from dermal tissue into blood i n v i v o . These assumptions are generally unproven, and i n v i t r o measurements ultimately require confirmation i n v i v o . These limitations notwithstanding, i n v i t r o studies s t i l l provide substantially useful information, and a variety of methods have been developed. The f i r s t section of this paper w i l l focus on the general interpretation of i n v i t r o data and i t s application to r i s k assessment. 0097-£156/85/0273-0003$06.00/0 © 1985 A m e r i c a n C h e m i c a l Society

In Dermal Exposure Related to Pesticide Use; Honeycutt, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

D E R M A L E X P O S U R E R E L A T E D TO PESTICIDE USE

4


An ideal predictive description of the percutaneous absorption process should include not only the a b i l i t y to predict the time course of the penetration process through the skin and absorption into the systemic c i r c u l a t i o n , but also an a b i l i t y to predict the residual amounts remaining both on the skin surface and within the skin which are s t i l l available for absorption. In the l a t t e r half of this paper, a p o t e n t i a l l y useful pharmacokinetic model, which has this broad predictive capability, w i l l be presented, together with data suggesting that s o l i d substances deposited on the skin surface in v o l a t i l e solvents are p a r t i a l l y s o l u b i l i z e d before the solvent has evaporated; more rapid absorption of the s o l u b i l i z e d f r a c t i o n i s then l i k e l y . Interpretation of Data Percutaneous absorption studied i n v i t r o i s normally characterized either by a permeability constant or by the time course of the penetration process. Direct measurements of absorption require intermittent sampling of f l u i d contained i n the receptor half of a d i f f u s i o n c e l l . Permeability constants are frequently calculated by removing and assaying microaliquots of receptor f l u i d at various intervals during the early time course of absorption ("static" d i f f u s i o n c e l l technique), u n t i l steady state (ss) flux i s obtained; the permeability c o e f f i c i e n t s are then derived according to Fick's F i r s t Law of D i f f u s i o n : flux(ss) = Α·Ρ·Οο

(1)

where Ρ denotes the permeability c o e f f i c i e n t , Co i s the concentra tion gradient (assumed equal to the i n i t i a l concentration i n the donor half of the d i f f u s i o n c e l l ) , and A i s the surface area of skin contacted by the penetrant. If the receptor f l u i d i s not replaced, the concentration i n the receptor phase of the d i f f u s i o n c e l l w i l l eventually e q u i l i b r a t e with the donor phase concentration, at which point further net absorption w i l l cease. Thus, this technique i s unsatisfactory for measuring the complete time course of absorption. This l i m i t a t i o n may be overcome by intermittently emptying the receptor f l u i d and replacing with "fresh" solution i n order to maintain "sink" conditions ("dynamic" d i f f u s i o n c e l l technique). Techniques which u t i l i z e continuously perfused, "dynamic" d i f f u s i o n c e l l s maintain "sink" conditions, and s e r i a l samples may be collected i n automated f r a c t i o n c o l l e c t o r s . When these samples are added, the cumulative amount removed ("excreted") from the d i f f u s i o n c e l l as a function of time i s obtained. This i s not i d e n t i c a l to cumulative absorption, which i s the sum of the amount excreted (EXC) plus the residual amount (RA) s t i l l remaining i n the d i f f u s i o n c e l l . Thus, the continuous perfusion technique i s limited by the a b i l i t y of the resulting cumulative excretion curve to approximate the true cumulative percutaneous absorption curve. Two methods of approximation may be employed. Both depend on an understanding of the k i n e t i c parameters which define the d i f f u s i o n c e l l system being u t i l i z e d . Provided that there i s rapid, homogenous mixing, the volume of receptor f l u i d "cleared" (clearance, CI) of absorbed compound may be


1.

Μ ΑΤΗ IAS ET A L .

In Vitro Data and Risk Assessment

5

related to the actual volume (V) of the receptor phase of the d i f f u s i o n c e l l with a constant of elimination (Ke) by the following equation Q )


CI = Ke^V

(2)

In this situation, CI i s analogous to flow rate through the d i f f u s i o n c e l l . This relationship has been v e r i f i e d i n our laboratory by i n j e c t i n g 2 ymol of benzoic acid through excised human skin into the receptor half of a d i f f u s i o n c e l l (V-10 ml), and subsequently perfusing the c e l l at a rate of 5 ml per hour. Here, Ke predicted by Equation 2 i s 0.50 hr~ . The concentrations of collected samples were plotted semilogarithmically, and a linear f i t obtained by regression analysis (Figure 1). Measured Ke can be related to the slope (-0.22 hr"" ) of this resulting l i n e by the equation (2) 1

slope = -Ke/2.303

(3)

1

and was calculated to be 0.51 h r " , which i s indistinguishable from the prediction of Equation 2. The cumulative excretion curve generated with continuously perfused d i f f u s i o n c e l l s may approximate the true percutaneous absorption curve as the value of Ke becomes larger, provided that absorption into the d i f f u s i o n c e l l i s the rate l i m i t i n g step. Ke may be increased by increasing the flow rate, decreasing receptor phase volume, or both, as indicated by Equation 2. Figure 2 i s a series of computer generated curves, employing different values of Ke, which relate the time course of cumulative excretion to cumulative absorption, where the absorption constant (Ka) has been a r b i t r a r i l y fixed at 0.05 hr" . Assuming f i r s t order absorption into the receptor phase, with the skin as a rate l i m i t i n g membrane only, the true time course of cumulative absorption (ABS) i s described by a monoexponential function -1

ABS = Xo (l-exp(-Ka-t))

(4)

where Xo i s the t o t a l dose available for absorption. Inspection of Figure 2 reveals that the i n i t i a l r i s e of the absorption curve i s e s s e n t i a l l y linear u n t i l 10-15% of the available dose has been absorbed. The cumulative excretion curves depicted i n Figure 2 are described by the equation EXC - Xo[l-Ke«exp(-Ka»t)/(Ke-Ka) - Ka-exp(-Ke.t)/(Ka-Ke)]

(5)

The p r o f i l e s demonstrate that when Ke i s only s l i g h t l y larger than Ka, a substantial lag occurs before peak flux (rate of r i s e of the cumulative excretion curve) i s obtained. As Ke increases, the term exp(-Ke-t) approaches 0 more quickly, and Equation 5 reduces to Equation 4. We may effect a reduction of Equation 5 to Equation 4 by assuming that this exponential approaches 0 when i t i s 95% complete, i . e . ,




6




MATHIAS ET A L .


8


(6)

exp(-Ke»t) = .05 Solving Equation 6 for t, we obtain

(7)


t » 3/Ke

Once this value of t has been exceeded, the reduction to Equation 4 has been effected and the remainder of the cumulative excretion curve i s dependent on the absorption process only. Thus, as the value of Ke increases, the excretion curve approximates the absorption curve more closely. If a r e l i a b l e estimate of Ρ i s to be obtained from a cumulative excretion curve generated by a continuously perfused, "dynamic" d i f f u s i o n c e l l , data points must be selected after the value of t defined by Equation 7 has been surpassed (when the shape of the excretion curve becomes dependent on absorption only), but before 10-15% of the t o t a l available dose has been absorbed ( i . e . , during the "steady state" period of absorption). Inspection of Figure 2 demonstrates that data points selected before or after these boundary conditions may lead to determinations of Ρ which are f a l s e l y low. Excretion data may also be transformed to approximate the true cumulative absorption (3). This method involves p l o t t i n g excretion rates (determined from the collected samples) against time, and calculating the area under the resulting curve (AUC). These excretion rates are assumed to approximate the instantaneous rates at the midpoint of the sample c o l l e c t i o n intervals (4), and must be appropriately plotted along the time axis. AUC thus approximates t o t a l cumulative excretion to the midpoint ( i n time) of the l a s t collected sample. Figure 3 depicts t y p i c a l excretion rate data for benzoic acid (4.8 μπιοί deposited i n acetone over 3.14 cm of excised human skin, Ke-0.5 h r " ) . Using an analogous midpoint approximation, the concentration of the l a s t collected sample i s assumed to approximate the concentration of the residual receptor phase at the midpoint of the sample c o l l e c t i o n i n t e r v a l . The residual amount (RA) l e f t i n the d i f f u s i o n c e l l i s determined by multiplying the approximated concentration by the receptor volume. An approximated cumulative absorption curve may thus be generated from excretion rate data by adding RA to AUC for each collected sample, and Ρ subsequently determined i n the usual fashion. Figure 4 compares the cumulative absorption curve for benzoic acid (4.8 umol deposited i n acetone over 3.14 cm of excised human skin) approximated by this technique, compared to the true absorption curve obtained by manually emptying a d i f f u s i o n c e l l at hourly i n t e r v a l s , then r e f i l l i n g with "fresh" solution to maintain sink conditions. The results are e s s e n t i a l l y i d e n t i c a l . The time course of the penetration process may be reasonably predicted from Ρ (derived from any of the methods discussed above) according to Equation 1, assuming that Co never changes s i g n i f i c a n t l y over the period of observation ( " i n f i n i t e " dose). This s i t u a t i o n may be obtained when the skin i s exposed for a short period of time or to a large volume per unit surface area, such as immersion i n a tank of solution. When the concentration i n contact with the skin i s l i k e l y to change over the period of exposure, 2

1


Μ ΑΤΗ IAS ET AL.



1000ί

ο ·

&

lOOr

10r

10

_J

20

I

30

40

50

TIME (HOURS)

Figure 3·

Excretion rate data for benzoic acid, u t i l i z i n g midpoint approximations.



10

( " f i n i t e dose", e.g., small volumes of solution saturating clothing), Ρ alone i s i n s u f f i c i e n t to describe the penetration time course. Here, the k i n e t i c s are more appropriately characterized by Ka, the f i r s t order absorption rate constant. A p o t e n t i a l l y useful mathematical relationship between Ρ and Ka may be derived. CI i s a constant which relates flux out of a compartment to the concentration of the cleared solution. CI may be used to describe the i n i t i a l steady state f l u x from an externally applied solution through skin according to the relationship (1)


flux(ss) = Cl-Co

(8)

As defined i n Equation 2, CI i s also related to the volume of the cleared solution by a constant, which i s analogous to Ka i n t h i s s i t u a t i o n . Substituting Equation 2 into Equation 8, the new equation becomes: flux(ss) = Ka.V-Co Setting Equation 9 equal to Equation 1 and relationship

(9) solving for Ka,

the

Ka = Α·Ρ/ν

(10)

i s obtained. Thus, the complete time course of the penetration process may be characterized by transforming Ρ into Ka and u t i l i z i n g Equation 4. It should be emphasized that this relationship i s applicable only when the substance contacting the skin i s i n solution, and an i n i t i a l value of Co i s known. When a s o l i d substance i s deposited on the skin from a v o l a t i l e solvent, no i n i t i a l Co (or V) can be determined and Ρ cannot be obtained.

Pharmacokinetic Model An i d e a l pharmacokinetic model of the percutaneous absorption process should be capable of describing not only the time course of penetration through skin and into blood (or receptor f l u i d i n a d i f f u s i o n c e l l ) , but also the time course of disappearance from the skin surface and accumulation (reservoir effect) of penetrant within the skin membrane. Neither Fick's F i r s t Law of Diffusion nor a simple k i n e t i c model considering skin as a rate l i m i t i n g membrane only i s s a t i s f a c t o r y , since neither can account for an accumulation of penetrant within skin. To resolve this dilemma, we have analyzed the i n v i t r o time course of absorption of radiolabeled benzoic acid (a rapid penetrant) and paraquat (a poor penetrant) through hairless mouse skin using a l i n e a r three compartment k i n e t i c model (Figure 5). The three compartments correspond to the skin surface (where the i n i t i a l dose i s deposited), the skin i t s e l f (considered as a separate compartment), and the receptor f l u i d i n the d i f f u s i o n c e l l . The i n i t i a l amount deposited on the skin surface i s symbolized by Χ10, and K12 and K23 are f i r s t order rate constants. Both compounds were studied at equivalent doses of 1 umol per cm , deposited i n approximately 16 u l per cm of an appropriate


1.

M A T H I A S ET A L .


3000 γ


δ

11

*****

2000

1000 \

10

20

30

40

50

Time (hours)

Figure 4.

Cumulative absorption of benzoic acid, comparing the curve approximated by the AUC + RA technique (closed c i r c l e s ) to the true absorption curve (open circles)·

SKIN SURFACE

Figure 5.

SKIN COMPRIMENT

DIFFUSION CELL

Linear 3 compartment pharmacokinetic model, considering the skin as a separate compartment. See text for explanation of symbols.



12


v o l a t i l e solvent (acetone i n the case of benzoic acid, methanol i n the case of paraquat). Evaporation of solvent from the skin surface was allowed to occur at ambient conditions and was usually complete within 3-4 minutes. Continuously perfused d i f f u s i o n c e l l s (receptor volume = 10 ml) were u t i l i z e d (3). The rate of perfusion was 10 ml per hour, and samples were collected every 30-60 minutes. Cumulative absorption curves were approximated using the AUC + RA method from excretion rate data as described above. The t o t a l dose available for absorption was considered to be the sum of a l l the amounts recovered from skin surface washings, tissue digestion, and cumulative absorption through skin (see below). The mathematical solution of the pharmacokinetic model depicted by Figure 5 i s described by Equation 5, where K12 and K23 are f i r s t order rate constants analogous to Ka and Ke, respectively. This solution was applied to the data and "best f i t " parameters estimated by i t e r a t i v e computational methods. The " f i t " of the data to the k i n e t i c model was analyzed by least squares nonlinear regression analysis (5). The s u i t a b i l i t y of the model i n describing the percutaneous penetration process was established by the following experiments. 1) The input function (amount remaining on the skin surface for absorption over time) was determined by rinsing the skin surface with d i s t i l l e d water (3 consecutive rinses of 10-15 seconds each) at various time periods after the i n i t i a l application: 1/2 hr, 1 hr, 2 hr, 4 hr, 8 hr, 24 hr, and 48 hr. Analysis of the recovered amounts by i t e r a t i v e computational estimation allowed "best f i t " parameters describing this process to be obtained. 2) The output function characterizing the movement of penetrant from the skin compartment into the receptor f l u i d of the d i f f u s i o n c e l l was determined as follows. T h i r t y minutes after i n i t i a l application, the skin surface was rinsed as described above to remove any surface residual. The d i f f u s i o n c e l l was emptied, rinsed and r e f i l l e d with fresh solution. A l l radiolabel recovered over the next 12 hours was assumed to have originated from the " f i l l e d " skin compartment, rather than the skin surface. "Best f i t " parameters were obtained by computational methods as before. Having determined the k i n e t i c parameters of the simulation by the input and output function experiments, the appropriateness of the k i n e t i c model was confirmed by measuring the t o t a l amounts which had both penetrated through and accumulated within the skin at various time i n t e r v a l s . These data were obtained as part of the same experiments to determine the input function. Following skin washing to remove surface residual, the amount remaining within the skin was determined by subsequent digestion i n a tissue s o l u b i l i z e r (Soluene 350, Packard Instruments, Downers Grove, I L ) . The r e s u l t i n g data were compared to values predicted by the pharmacokinetic model, using the parameters determined by the input and output function experiments. Results of the input function experiments for both benzoic acid and paraquat indicate that the residual amounts recovered from the skin surface over time are best described by a monoexponential function (Figure 6), with a f r a c t i o n of the applied dose having



M A T H IAS ET A L .


I00

r

Time (hr)

Figure 6.

Amounts of benzoic acid (closed c i r c l e s ) and paraquat (open c i r c l e s ) on the skin surface remaining to be absorbed, as a function of time.



14

DERMAL EXPOSURE RELATED TO PESTICIDE USE

disappeared completely from the surface at an rapid rate within the f i r s t half hour. Each data point represents the average of 4 separate samples of hairless mouse skin. This suggests that the v o l a t i l e solvent vehicle may influence the percutaneous absorption process, either s o l u b i l i z i n g part of the applied dose i n the s u p e r f i c i a l skin surface l i p i d layer, before i t has evaporated (making i t available for faster absorption), or possibly by carrying a f r a c t i o n of the applied dose into the skin compartment as a portion of the solvent i t s e l f penetrates the skin. The k i n e t i c model depicted i n Figure 5 may adequately account for this rapid disappearance from the skin surface i f i t i s assumed that a f r a c t i o n of the applied dose (X20) immediately p a r t i t i o n s into the skin compartment at time zero (r f o r benzoic acid=0.95, r for paraquat-0.97). Although the amount of paraquat (a poor penetrant), which may rapidly s o l u b i l i z e into the skin compartment i s rather small (3%), the corresponding f r a c t i o n for benzoic acid appears to be quite large (77%). The slower phase of disappearance from the skin surface depicted i n Figure 6 may then r e f l e c t dissolution of the residual applied substance (X10) from a dried s o l i d phase on the skin surface as a l i m i t i n g step. Average K12 values for this slow phase of absorption were 0.25 hr" f o r benzoic acid and 2x10 hr for paraquat. Results of the output function experiments for benzoic acid are summarized i n Table I.

Table I.

Skin Sample #

Output F u n c t i o n Parameter

F-X20(%)

Estimates f o r Benzoic

m/hr"

1

(SE)

1 2 3

100 100 100

.68 (.007) .62 (.005) .28 (.002)

AVE"

TOO

.53

Acid

R

2

.83 .93 .91

(.21)

These indicate that the entire amount remaining to be absorbed from the skin compartment after surface washing may be reasonably described by a monoexponential function, with an average K23 value of 0.53 hr . On the other hand, a monoexponential function does not adequately account for the entire dose of paraquat remaining to be absorbed, and suggests that a substantial amount of paraquat may bind to skin and be unavailable for absorption. The amount of absorbed paraquat which may be reasonably described by a monoexponential output function (F«X20) i s only about 10% (assuming i r r e v e r s i b l e binding), with an average K23 value of 0.82 hr (Table II). The "best f i t " parameter estimates obtained from the input and output function experiments for both benzoic acid and paraquat are summarized i n Table I I I .


1.

In Vitro Data

MATHIAS ET AL.

Table I I .

15

Assessment

Output F u n c t i o n Parameter E s t i m a t e s

Skin Sample #


and Risk

K23/hr

F»X20(%) (SE)

1

f o r Paraquat

1 2 3

12.5 (.43) 10.8 (.28) 7.8 (.17)

0.59 (.009) 0.67 (.008) 1.20 (.19)

AVE

10.4 (2.4)

.82 (.33)

Table I I I .

R

(SE)

2

.73 .79 .49

Summary o f "Best F i t " Parameter Estimates

-1

A

PeneX10(%)(SE) trant

K12/hr (SE)

X20(%)

F.X20(%)(SE)

Benzoic 33.2(2.9) acid

0.23(.005)

76.8

100

0.53(.21)

3.1

10.4(2.4)

0.82(.33)

Paraquat

96.9(0.5)

3

4

2xl0" (2xl0" )

K23/hr (SE)

Using these estimates, the k i n e t i c model depicted i n Figure 5 was applied to data describing the time course of penetration through skin (Figure 7) and accumulation within skin (Figure 8). Again, a l l data points represent average values obtained from 4 separate samples of hairless mouse skin. The s o l i d lines represent values predicted by the model. For benzoic acid, the " f i t " of the k i n e t i c model i s i n r e l a t i v e l y good agreement with the observed data, without any consideration given for potential skin binding (r f o r t o t a l penetration=0.94, r for skin accumulation^.83). In the case of paraquat, the binding observed i n the output function experiments was assumed to be i r r e v e r s i b l e , and to occur only with X20. Although the early time courses are predicted reasonably well, the late time behavior i s not ( r for t o t a l penetration=0.56, r f o r skin accumulâtion=0.89)· Further studies are i n progress to consider p o t e n t i a l l y reversible binding f o r paraquat, which could account for these observations. 2



DERMAL EXPOSURE RELATED TO PESTICIDE USE

0

4

8

16

24 Time

Figure 7·

32

40

48

(hr)

Amounts of benzoic acid (closed c i r c l e s ) and paraquat (open c i r c l e s ) penetrating completely through skin as a function of time.

I00r-

m

Figure 8.

Amounts of benzoic acid (closed c i r c l e s ) and paraquat (open c i r c l e s ) accumulating within the skin as a function of time.


1.

M A T H I A S ET A L .

In Vitro Data and Risk

Assessment

17


Summary The r i s k of an adverse systemic reaction following skin contact with a potentially toxic chemical depends on the speed and t o t a l amount of absorption through the s k i n . Several techniques have been devised to measure these parameters across isolated skin mounted i n diffusion c e l l s . The mathematical and k i n e t i c relationships which define the operation of these various systems have been reviewed. A novel k i n e t i c description of the penetration process has been developed and applied to skin absorption measurements for benzoic acid and paraquat. The results suggest that when these substances are deposited on the skin surface in a v o l a t i l e solvent, a portion of the applied dose i s rapidly s o l u b i l i z e d into the skin and available for fast absorption, while the remainder is absorbed more slowly.

Acknowledgment s This work was supported, in part, by NIOSH grant OH-01830 (to RHG). We thank Andrea Mazel for preparing the manuscript.

Literature Cited 1. Rowland, M.; Tozer, T.N. "Clinical Pharmacokinetics"; Lea and Febiger: Philadelphia, 1980; p. 84. 2. Gibaldi, M.; Perrier, D. "Pharmacokinetics"; Marcel Dekker: New York, 1975; pp. 2-6. 3. Mathias, C.G.T. Clin. Research 1983, 31, 586A. 4. Gibaldi, M.; Perrier, D. "Pharmacokinetics"; Marcel Dekker: New York, 1975; pp. 301-305. 5. Peck, C.; Barrett, B.B. J. Pharmacokin. Biopharm., 1979, 5, 537. RECEIVED November 27, 1984


Dermal Exposure Related to Pesticide Use - American Chemical Society

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