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PA R T I

Concepts

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CHAPTER

1 2 3 4 5 6 7 8 9 0 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 RONALD C WESTER AND HOWARD I MAIBACH

Skin Permeability

Contents
1.1 1.2 1.3 1.4 1.5 Introduction Method analyses: Atrazine Method analyses: Borates Limitations Discussion

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SKIN PERMEABILITY

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1.1 INTRODUCTION
The rate determining step for human risk assessment is bioavailability, that amount of chemical in the environment which gets into the human body. If the exposure includes skin, then skin permeability becomes a rate determining step. Various methods are available to assess skin permeability. These include in vivo, in vitro and computer model methods. Cost/benet would favor the in vitro system (this is assumed) and certainly the computer calculated permeability is cost friendly (not to mention manpower friendly). The down side is that errors can cost money and human suffering. This chapter gives examples of the different methodologies, showing when they work and where validation points out method shortcomings.

1.2 METHOD ANALYSES: ATRAZINE

Table 1.1 gives the in vivo human percutaneous absorption of [14C] atrazine. Two dose levels, 6.7 and 79 g/cm2, were applied to the ventral forearm of volunteers (from whom consent had been obtained) and total urinary and fecal radioactivity determined. A previous in vivo intravenous study in the rhesus monkey showed that all of the iv dose was excreted within 7 days, and this was the case with the human volunteers with topical dose application. Total percent dose absorbed was 5.6 3.0 and total dose accountability (absorbed plus washes) was 101.2 3.4 percent for the 6.7 g/cm2 dose. Similar results were obtained TABLE 1.1: Atrazine human in vivo percutaneous absorption
Dose Aa (n = 4) Dose Excretron: 6.7 g/cm2 5.0 0.6 5.6 5.6 101.2 0.0156 17.5 Dose Ba (n = 6) 79 g/cm2 1.1 0.1 1.2 1.2 92.3 0.0379 24.5

Urinary (%) Fecal (%) Total (%) Dose absorbed (%) Total dose accountability (%) Flux (g/cm2/hr) Half-life (hr) 14C

2.9 0.3 3.0 3.0 3.4 0.0084 5.4

0.9 0.1 1.0 10 2.8 0.0332 9.0

Dose applied to ventral forearm, covered with non-occlusive raised patch for 24 hours, then dose side washed with soap and water.
a

Mean SD

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DERMATOTOXICOLOGY

for the higher dose. This is considered the gold standard for skin permeability. Denitive percent dose absorbed and ux are obtained and all of the applied dose is accounted for. The in vivo urine samples were further validated. Split urine aliquots were analyzed by accelerator mass spectrometry (Gilman et al., 1998). Data from these two methods (scintillation counting and accelerator MS) have a correlation coefcient of 0.998 for a linear plot of the entire sample set. Urinary metabolites were also determined using HPLCaccelerator mass spectrometry (Buchholz et al., 1999). Table 1.2 gives atrazine in vitro percutaneous absorption through human skin (Ademola et al., 1993). The human skin was used under conditions which ensure skin viability (Wester et al., 1998a) and atrazine metabolites were determined. In this in vitro study receptor uid accumulation and skin content (at end of study) were determined for skin permeability. A basic question with in vitro methodology is: does one use only receptor uid content or both receptor uid and skin content to determine skin permeability. Without knowledge of in vivo human absorption (Table 4.1) which is the proper choice? Table 1.3 summarizes atrazine flux in humans using the in vivo data (0.0156 g/cm /hr) and in vitro data (0.0081 g/cm /hr for receptor uid only and 0.038 g/cm2/hr using combined receptor fluid and skin content). For comparison purposes the flux was calculated using Guy and Potts (1992) as 0.044 g/cm2/hr. All three ux calculations are relatively in agreement. Atrazine is a friendly chemical for these types of analysis because the molecular weight (215.69), water solubility (34.7 mg/L) and Log P (octanolwater) of 2.61 are amendable to all systems. However, there are exceptions to the rule.
2 2

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TABLE 1.2: Atrazine in vitro percutaneous absorption human skin

Distribution Receptor uid Skin Surface wash Total recovery
Dose is 4.6 g/cm2 Each value: mean SEM (n = 14) for 20 hours Source: Ademola et al. (1993)

29 30 31 32 33 34 35 36 37 38

Percent Dose Absorbed 3.5 12.8 66.8 83.0 0.3 1.2 6.9 7.3

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TABLE 1.3: Atrazine Flux In Humans

Method In vivo human In vitro Receptor uid Receptor uid and skin Calculatedb
a Based upon 6.7 g/cm2 dose Source: Guy and Potts (1992)

Flux (g/cm2/hr) 0.016 0.008 0.038 0.044

1.3 METHOD ANALYSES: BORATES

Boron is an ubiquitous element in rocks; soil and water. A small amount of boron is essential to life. Borates come in contact with human skin in many ways (mining, detergent, fertilizer, wood treatment, organic insecticide). Table 1.4 gives the in vivo percutaneous absorption in human volunteers (from whom informed consent was obtained) for the borates 5 percent boric acid, 5 percent borax and 10 percent disodium octaborate tetrahydrate (DOT). These dose concentration are near water solubility limitation (Wester et al.,

TABLE 1.4: In vivo absorption, ux and permeability content for 10boron as 5% boric acid, 5% borax, and 10% disodium octaborate tetrahydrate (dot) in normal human volunteers
Dose 5% boric acid No treatment SLS treatmenta 5% borax No treatment SLS treatmenta 10% DOT No treatment SLS treatmenta
a

Dose 10B (g)

Percentage Flux of dose (g/cm2/h)

Permeability constant (Kp) (cm/h) 1.9 2.0 1.8 1.5 1.0 0.9 107 107 107 107 107 107

14,200 14,200 9270 9220 34,700 34,800

0.226 0.239 0.210 0.185 0.122 0.107

0.00912 0.00966 0.00855 0.00746 0.00975 0.00878

SLS = sodium lauryl sulfate Dose was spread over 900 cm2 area of the back

Source: Wester et al., 1998b

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DERMATOTOXICOLOGY

TABLE 1.5: In vitro percutaneous absorption of boron administrated as boric acid, borax, and disodium octaborate tetrahydrate (DOT) in normal human skin
Dosing solution Percentage of dose absorbed geometric mean (95% CI) Flux (g/cm2/h) Kp (cm/h)

1 2 3 4 5 6 7
1.75 (0.1817) 0.70 (0.0726.81) 0.28 (0.0292.72) 1.20 (0.01211.7) 0.41 (0.0423.99) 0.19 (0.0181.81) 0.07 14.58 0.58 0.25 8.5 7.9 1.4 2.9 1.2 5.0 106 104 104 104

Boric acid (w/v) 5% at 2l/cm2 5% at 1000l/cm2 0.5 at 1000l/cm2 0.05% at 1000l/cm2 Borax 5% at 1000l/cm2 DOT 10% at 1000l/cm2
Source: Wester et al. (1998b)

8 9 0 11 12 13 14 15 16

1.7 104 0.8 104

1998b): the in vivo permeability contants (Kp) range from 1 2 10 for these
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borates. The human skin in vitro percutaneous absorption is in Table 1.5. Comparison of Tables 1.4 and 1.5 yield some interesting data relative to in vivo and in vitro methodology. The in vitro permeability coefcient (Kp) for the 5 percent boric acid, 5 percent borax and 10 percent DOT range from 0.8 2.9 10-4. This is a 1000-fold increase over in vivo Kps. The in vivo studies were done with a dose of 2l/cm2 (any more would run off the skin). The in vitro doses were at 1000 l/cm . However one in vitro 5 percent boric acid was dosed at 2 l/cm2. Interestingly, the 5 percent boxic acid Kp at 1000 l/cm was 2.9 10 while the 5 percent boric acid Kp at 2 l/cm
2 -4 2 2

was 1.4 10-6, a 200-fold difference. The amount of vehicle (water) was the determining factor in boric acid in vitro human percutaneous absorption. The relationship between ux and permeability coefcient (ux is concentration dependent while Kp is independent) was true for this in vitro study (Figure 1.1).

1.4 LIMITATIONS
Regulatory agencies have developed an afnity for a calculated permeability coefcient (Kp) for risk assessment. Permeability coefcients are easiest determined from the time course of chemical diffusion from a vehicle across the skin

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FLUX AND PERMEABILITY COEFFICIENT

20

10

FLUX (g/sq cm/hr) PERMEABILITY COEFFICIENT (cm /hr E-4)

0 5 0.5 PERCENT BORIC ACID DOSE

Figure 1.1: Boric acid dose response at 1000 l/cm2.

0.05

barrier into a receptor uid. Table 1.6 compares in vitro diffusion receptor uid absorption with in vivo percutaneous absorption. Receptor uid accumulation for the higher logP chemicals (Table 1.7) is negligible. This is due to basic chemistrythe compounds are not soluble in the water based receptor uid. Based on these receptor uid accumulations these chemicals are not absorbed skin. Risk assessment would contain an extreme false negative component. That point where the diffusion system and receptor uid accumulation gives a true Kp or manufactures a false Kp has not been determined. Regulatory agents should have some in vivo validation before blindly accepting an in vitro Kp. Also, computer models based on in vitro data have the same risk.

1.5 DISCUSSION
Human skin was developed during evolutionary history, basically designed as a physical barrier to the environment and to contain our water based body chemistry. The industrial revolution introduced a new wave of chemicals for the skin to deal with. Considering skins barrier properties, a lot of protection is provided. However, chemicals do permeate the skin barrier and human health

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DERMATOTOXICOLOGY

TABLE 1.6: In vitro receptor uid versus in vivo percutaneous absorption

Percent dose In vitro Receptor uid 0.08 0.04 0.09 0.01 0.07 0.04 0.6 0.01 0.3 0.04 0.1 0.04 0.02 0.9 0.03 0.4 0.03 0.07 0.06 0.02 0.01 0.06 0.06 0.06 0.05 0.09 0.00

1 2 3 4 5 6
9.4 0.5 22.0 3.4 2.8 1.8 5.8 6.4 8.5 8.3 8.3 1.0 3.6 8.3 8.5 2.7 2.1 4.7 1.2 1.9

Compound DDT Benzo[a]pyrene Chlordane Pentachlorophenol PCBs (1242)

Vehicle Acetone Soil Acetone Soil Acetone Soil Acetone Soil Acetone TCB Mineral oil Soil Acetone TCB Mineral oil Soil Acetone Soil Water Soil Water Soil Water Soil

In vivo 18.9 3.3 51.0 13.2 6.0 4.2 29.2 24.4 21.4 18.0 20.8 14.1 14.6 20.8 20.4 13.8 2.6 15.9 2.0 3.2

7 8 9 0 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

0.6 0.05

PCBs (1254)

0.07 0.05 0.01 1.1 0.5 0.2 0.02 0.01 0.01

2,4-D Arsenic Cadmium Mercury

TABLE 1.7: Octanol / water partition coefcients of compounds

Compounds DDT Benzo[a]pyrene Chlordane Pentachlorophenol 2,4-D PCBs Aroclor 1242 Aroclor 1254 LogP 6.91 5.97 5.58 5.12 2.81 mixture (high logP) (high logP)

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requires knowledge and regulation to maintain safety. Skin permeability can best be determined in vivo in human volunteers (gold standard). in vitro diffusion methodology and predictive models can aid in predicting skin permeability, but they do have limitation. If these limitations are not vigorously dened and validated, the consequences can be severe. False positive errors can be nancially costly and false negative errors can be deadly.

REFERENCES
ADEMOLA, J.I., SEDIK, L.E., WESTER, R.C. and MAIBACH, H.I. (1993) In vitro percutaneous absorption and metabolism in man of 2-chloro-4-ethylamino6-isopropylamine-5-triazine (Atrazine). Arch. Toxicol. 67, 8591. BUCHHOLZ, B.A., FULTZ, E., HAACK, K.W., VOGEL, S., GILMAN, S.D., GEE, S.J., HAMMOCK, B.D., HUI, X., WESTER, R.C., MAIBACH, H.I. (1999) APLCaccelerator MS measurement of atrazine metabolites in human urine after dermal exposure. Anal. Chem. 71, 35193525. GILMAN, S.D., GEE, S.J., HAMMOCK, B.D., VOGEL, J.S., HAACK, K.W., BUCHHOLZ, B.A., FREEMAN, S.P.H.T., WESTER, R.C., HUI, X., MAIBACH, H.I. (1998) Analytical performance of accelerator mass spectrometry and liquid scintillation counting for detection of
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C-labeled atrazine metabolites in

human urine. Anal. Chem. 70, 34633469. GUY, R.H. and POTTS, R.O. (1992) Structure-permeability relationship in percutaneous penetration. J. Pharm. Sci. 81, 603604. WESTER, R.C., CHRISTOPHER, J., HARTWAY, T., POBLETE, N., MAIBACH, H.I. and FORSELL, J., (1998a) Human cadaver skin viability for in vitro percutaneous absorption: storage and detrimental effects of heat-separation and freezing. Pharm. Res. 15, 8284. WESTER, R.C., HUI, X., HAACK, K.W., POBLETE, N., MAIBACH, H.I., BELL, K., SCHELL, M.J., NORTINGTON, D.J., STRONG, P. and CULVER, B.D. (1998b) In vivo percutaneous absorption of boric acid, borax, and disodium octaborate tetrahydrate in humans compared to in vitro absorption in human skin from innite and nite doses. Toxicol. Sci. 45, 4251.

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