PB85-17':694
REVIEW OF DERMAL ABSORPTION
U.S Environmental Protection Agency
Washington, DC
Oct 84
U.S. DEPARTMENT OF COMMERCE
National Technical Information Service
NITS
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DISCLAIMER
This report has been reviewed in accordance with U.S. Environmental
Protection Agency policy and approved for publication. Mention of trade names
or commercial products does not constitute endorsement or recommendation or
use.
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CONTENTS
Foreword vii
Abstract viil
1. Introduction 1
2. Conclusions 2
3. Recommendations 4
4. Skin as a Barrier to Absorption 7
4.1 Structure of the Skin
4.2 Factors Affecting Absorption
5. Techniques for Measuring Absorption 15
5.1 In Vivo Human Studies
5.2 In Vivo Animal Studies
5.2 Tn Yftro
6. Theoretical Treatment of Dermal Absorption 38
6.1 Pick's Law Applied to Dermal Absorption
6.2 Non-Steady State Diffusion
6.3 Calculation of kp and Km
6.4 Prediction of Permeability for Some Alcohols and Steroids
7. Dermal Absorption in Exposure Assessments 58
7.1 Current Agency Practice
7.2 Examples of Dermal Intake Calculations
7.2.1 Dermal Exposure from Swimming
7.2.2 Pesticide Application
7.2.3 Treated Field Reentry
7.2.4 Hypothetical Dermal Intake of PCBs
8. Analysis/Future Research Needs 69
9. References 74
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LIST OF TABLES
Page
1. Relationship Between Dose and Efficiency of Absorption ....... 10
2 a. Ratio Comparing Absorption of Various Sites to Forearm ..... 12
b. Penetration Indices for Five Anatomic Sites ........... 12
c. Body Surface Areas for Five Anatomic Sites ........... 12
3. Percutaneous Absorption of Steroids in Man ............. 16
4. Percutaneous Absorption of Some Organic Compounds in Man ...... 19
5. Percutaneous Absorption of Some Pesticides and Herbicides in Man. . 22
6. In Vivo Percutaneous Absorption by Rat, Rabbit, Pig, and Man. ... 24
7. In Vivo Percutaneous Absorption of Several Pesticides by Rabbit,
TTg, Squirrel Monkey, and Man ................... 26
8. Geometric Means of Percentage C Recovered in Various Fractions
at 5, 15, and 60 Min Post Application ................ 27
9. Comparison Between Physical Parameters and Geometric Means of
Rate of Penetrations ........................ 28
10. Comparison of Human In Vivo and In Vitro Absorption ......... 32
11. Comparison of Human In Vivo and In Vitro Absorption Using
Refined Techniques ......................... 34
12 a. Percutaneous Absorption of Acetyl salicylic Acid in Rats ..... 36
b. In Vivo vs. In Vitro Percutaneous Values with Those of Other
"STudPTe? ............................. 36
c. Comparison of Permeation Values with Those of Other Studies . . .36
13. Membrane Permeability and Partition Coefficients ..... ..... 45
14. Permeability Constants and Partition Coefficients .......... 47
15. Steroid Fluxes ................. . ......... 49
16. In Vitro Permeability of Aliphatic Alcohols Through Human
"Epidermis .............................. 54
17. a. In Vitro Permeability of Steroids Through Human Epidermis ... .55
b. Trii Vitro Permeability of Steroids Through Human Epidermis ... .56
IV
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LIST OF TABLES (Continued)
Page
18. a. Relative Contribution to Total Dermal Exposure of Body Areas
to Pesticides as Studied by the California Department of Food
and Agriculture 63
b. Relative Contributions to Total Dermal Exposure of Body Areas
by Job Activity 63
19. Worker Reentry Exposure 65
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LIST OF FIGURES
Page
1. Skin Cross Section 7
2. Schematic Representation of a Diffusion Cell with Top Open to
the Ambient Environment. 30
3. Diagram Showing the Concentration Profile Across Stratum Corneum
During Steady-State Absorption 39
4. The Pattern of Changing Absorption Rate for Small Amounts of
Penetrant Per Unit Area of Skin 42
5. The Extrapolation to Zero Absorption of the Steady-State,
Linear Region of the Graph of Total Amount Absorbed Versus
Time Yields the Lag Time 48
6. Dependence of the Permeability Constant on the Stratum Corneum/
Water Partition Coefficient 53
VI
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FOREWORD
The Exposure Assessment Group of EPA's Office of Research and Development
has three main functions: 1) to conduct exposure assessments; 2) to review
assessments and related documents; and 3) to cievelop guidelines for Agency
exposure assessments. These exposure assessments are critical to the evaluation
of the public health risks that are presented by toxic substances. The activities
under each of these three functions are supported by and respond to the needs of
the various EPA program offices. In relation to the third function, the Exposure
Assessment Group conducts projects for the purpose of developing or refining
techniques used in exposure assessments.
The current project was initiated to provide a review of human dermal
absorption since this is a mechanism by which toxicants can enter the body.
To properly conduct an exposure assessment, all routes of exposure should be
determined. Besides reviewing current dermal absorption studies, future
research needs are addressed which could further elucidate the methods needed
for dermal exposure calculations. The Exposure Assessment Group eventually
hopes to provide similar reviews for all routes of exposure.
James W. Falco, Ph.D., P.E.
Director
Exposure Assessment Group
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ABSTRACT
Dermal absorption is one of the three main routes of human exposure. This
report is a review of the current dermal absorption literature with an emphasis
on applications for exposure assessments. The structure of the skin is described
in the first technical chapter as well as factors such as occlusion or abrasion
which affect absorption rates. The next chapter presents comprehensive tables
of in vivo human dermal studies, in vivo animal studies, and in vitro results
using either excised animal or human skin. Theoretical treatments of dermal
absorption are discussed next, including kinetic expressions for various dermal
exposure situations and ways to predict absorption using partition coefficients.
Following this theoretical review some calculations of typical dermal exposure
scenarios are presented such as chemicals in swimming pools or reentry into
pesticide treated fields. Lastly, an analysis of some data gaps in dermal
absorption knowledge is coupled with suggestions for future research.
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1. INTRODUCTION
Three main routes of human exposure to toxic compounds have been identified,
namely inhalation, ingestion, and dermal penetration. It. is recognized that
for certain scenarios, such as farm worker reentry following pesticide application
or occupational handling of pure liquids or solids, exposure via the dermal
route is the most critical exposure pathway. In the past, when the Agency was
faced with estimating the dermal absorption of a compound without the support
of scientific studies, a value of 10 percent absorption was assumed. Recently,
the Office of Pesticide Program's Scientific Advisory Panel stated that if no
literature was available on a compound to show otherwise, then a value of 100
percent of the penetrant on the" skin should be used to represent the dermal
absorption. This value of 100 percent is a conservative approach and leads to
the overestimation of actual dermal absorption for most compounds. The intent
of this document is to review the current state of knowledge of dermal exposure
focusing on ways to assign more realistic values for absorption (i.e., <100
percent).
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2. CONCLUSIONS
The review of the literature on dermal absorption presented in this report
has brought out a number of factors which help to explain how dermal absorption
of a penetrant occurs. It has been shown that the thin outermost layer of
skin, the stratum corneum, is the rate limiting membrane for diffusion. For
most penetrants, absorption through the general skin surface is the preferred
route over "holes" in the skin caused by hair follicles and sweat ducts. The
site of dermal exposure is important too as studies have shown up to a ten-fold
difference in absorption rate depending on where on the body a penetrant is
applied. There are a number of other factors which can affect the amount of
penetrant absorption such as condition of the skin, occlusion or covering of
the applied dose, frequency of application, metabolism of a penetrant by the
skin, and the solvent or formulation used to deliver a penetrant to the skin.
In vivo studies in man have been done for approximately 50 chemicals,
mostly pesticides and steroids. Studies have shown up to a 5-fold variation
in skin absorption between subjects. Toxicity concerns prohibit further testing
in man, thus in vivo studies are done on animals. There is a large variation
in penetrant absorption in the animal species tested with the rhesus monkey
and miniature swine appearing to be closest to man. In vitro studies using
either excised human or animals skin give qualitative agreement with in vivo
results and show the potential for quantitative use.
Attempts have been ^ade tc predict dermal absorption from partition
coefficients, particularly with octanol/water or olive oil/water. It has been
found, however, that a compound has to be both lipid soluble and water soluble
to be a good penetrant. The solubility of the penetrant within the stratum
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corneum relative to its solubility in the vehicle (i.e., the membrane partition
coefficient, Km) is a better indicator of permeability than octanol/water or
olive oil/water partition coefficients. Values for K^ have been used to
predict the permeability of the linear primary alcohol series with good success.
The absorption rate is just one factor needed to calculate dermal intake
for an exposure assessment. In addition, one needs to know the duration of
the exposure per each event, the frequency of events expressed in number of
exposures per some unit time, the ambient concentration of the penetrant as a
function of time plus the medium or vehicle in which it is applied to the skin,
and the area of exposed skin. The uncertainty contained in these factors is
large and may necessitate the use of approximations.
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3. RECOMMENDATIONS
One stated goal of this review is to work towards assigning a more realistic
value for the percent absorption of an untested penetrant rather than the
current conservative approach of using 100 percent. Since so few compounds
have been tested in vivo in man and toxicity concerns prevent the testing of
many compounds, it is necessary to study penetrants by in vivo animal tests, _i£
vitro animal or human tissue tests, or by calculating potential absorption from
partition coefficients or other physical parameters. In order to work towards
the goal of being able to assign a value of less than 100 percent for the
absorption rate of an untested penetrant, it is recommended that:
- A data base be established of Km and diffusivity values for a number of
chemicals representing the various chemical classes.
- Quantitate the factors affecting absorption such as vehicle, site of
application, condition of the skin, occlusion, frequency of application,
and metabolism. In particular, the effect of the carrier solvent
(vehicle) on the rate of absorption should be determined by measuring
the rate for a pure penetrant and then dissolving the penetrant in a
number of solvents (such as those commonly found in industry).
- Develop kinetic expressions to represent the various dermal exposure
situations that occur such as finite amount of a fast penetrant, finite
amount of a slow penetrant, excess of a penetrant where the diffusion
through the stratum corneum is not rate limiting, buildup of penetrant
in blood such that the concentration (C0) is fQ, etc. Also, it is
necessary to develop procedures for identifying which kinetic expression
is appropriate for use with a given penetrant.
- Determine the most suitable animals species (probably the rhesus monkey
or miniature swine, or rat because of ease of handling and economy) and
do all animal in vivo work only on th.is species. The relationship
between in vivo in man versus in vivo in animals should be established
by comprehensive testing of penetrants that have already been studied
in man.
- For in vitro work, decide from what part of the body tissue should be
taken (forearm, back, stomach). Investigate problem of low water
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solubility penetrants by using solvent/water or straight solvent for
the receptor cell (the bottom part of the diffusion cell apparatus).
Work on making this method more quantitative so that in vitro results
can substitute for in vivo results. Also, the relative permeability of
all body tissue that could potentially be exposed should be measured
and correlated to the body tissue area selected for the in vitro studies.
- Establish the relationship between dermal absorption and the various
partition coefficients, particularly Km and Koctanol» and see if tne
methods used to calculate partition coefficients such as structure-
activity, number and kind of functional groups, etc., can be used to
calculate dermal absorption directly.
- Determine the feasibility of grouping penetrants into a numerical system
like 100-10-1-.!% absorption depending on in vivo or in vitro studies,
or physical parameters like Km or other partition coeTficients.
It may be possible to develop a model which includes all possible parameters
affecting dermal intake based on the results of the recommended studies. One
could store in the model such factors as: skin area for total body and for
each body region depending on age, sex, and body weight; thickness of skin for
different body regions; differences in percent or rate of absorption (based on
some unity scale) for various body sites; kinetic expressions to determine the
absorption for a range of scenarios; representative K^ and diffusion constants
for chemical classes and/or functional groups, etc. Then one could enter data
specific for a particular exposure, such as penetrant partition coefficients,
volatility, concentration, and number and kind of functional groups, vehicle
(including any binding to soil, or other substrates), contact time, skin area
plus where on the body, etc., and have the model calculate the dermal intake.
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4. SKIN AS A BARRIER TO ABSORPTION
4.1 STRUCTURE OF SKIN
Skin is one of the largest organs of the body comprising approximately 10
percent of the normal body weight. The skin consists of two different layers
with the thinner outer layer known as epidermis and the thicker, inner layer
as dermis (Fig. 1). Although skin thickness varies with location in humans,
the epidermis is approximately 0.1 mm and the dermis 2-4 mm thick (Rongone,
1983). The outermost layer of the epidermis is called the stratum corneum and
is from 10 to 50 urn thick. In this document we are mainly concerned about the
stratum corneum as it has been shown that the stratum corneum is at least
three, and frequently as much as five, orders of magnitude less permeable to
most substances as the dermis (Michaels et al., 1975). Also, the permeability
of the entire epidermis is indistinguishable from that of the stratum corneum
alone. Thus, the skin can be thought of as a three layer laminate of stratum
corneum, remainder of the epidermis, and dermis with permeation occurring by
diffusion through the three layers in series.
The stratum corneum is a heterogeneous structure containing about 40%
water, about 40% protein (primarily keratin), and about 15 to 20% lipids (mainly
triglycerides, free fatty acids, cholesterol, and phospholipids) (Michaels et
al., 1975 and Anderson and Cassidy, 1973). The stratum corneum can absorb up
to six times its own weight in water, and in its fully hydrated state, its
permeability to water and other low molecular weight penetrants is increased
(Scheuplein, 1967). The lipid components of the stratum corneum may be the
chief reason for its uniquely low permeability as when this tissue is extracted
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Epidermis
Living
dividing
cells
Blood
vessel
Subcutaneous
.tissue
Figure 1. Skin Cross Section
(By Permission from the New York Times)
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with a fat solvent and then rehydrated, the water permeabil ity of the stratum
corneum and its permeability to larger molecules is greatly enhanced (Scheuplein,
1967).
A recent study by Eli as, et al. (1981) related dermal absorption to stratum
corneum structure and lipid composition. The extreme sensitivity of the
permeability barrior to damage by lipid solvents suggest that lipids are
important determinants of skin penetration. In addition to lipids, several
other stratum corneum structural parameters, including thickness, number of
cell layers, and geometric organization could determine stratum corneum
permeability. The authors correlated the in vitro penetration of water and
salicylic acid across leg and abdominal stratum corneum with both the lipid
composition and the structure of the same samples. One finding from this study
is that there is an apparent noncorrelation of penetration of both substances
with either stratum corneum thickness or number of cell layers. The results
suggest that dermal absorption of both substances correlate with the lipid
content by weight of the sample. Also, the study suggests that relatively
small inherent variations in lipid concentration may explain observed differences
in permeation across different topographic regions. The authors predict that
regions of high permeability such as palms and soles would have a low lipid
weight percent, whereas those of low permeability such as facial or perineal
stratum corneum, would have a relatively high lipid weight percent.
The role of skin appendages such as hair follicles and sweat ducts on skin
permeability has been studied extensively. One would think that these "holes"
in the skin would facilitate the passage of a penetrant; however, their total
area is relatively small and, for most penetrants, absorption through the
general skin surface is the preferred route (Dugard, 1983). In the case of some
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molecules that penetrate the bulk of the stratum corneum slowly, such as
electrolytes and polar molecules with three or more labile polar groups, the
route through follicles and ducts may predominate (Scheuplein, 1980).
4.2 FACTORS AFFECTING ABSORPTION
A general definition of percutaneous absorption can be given as the
penetration of substances from the outside into the skin and through the skin
into the blood stream. Schaefer and Schalla (1980) have broken this process into
individual parts as: (1) penetration is considered to be the process of entrance
into 1 layer; (2) permeation is the migration through 1 or several skin layers;
(3) resorption is the uptake by the cutaneous microcirculation; and (4) absorption
is the sum of all these processes.
There are a number of parameters which can affect the amount of penetrant
absorption. The concentration of applied dose and surface area are the two
most important factors, with the greatest potential for absorption occurring
when a high concentration of a penetrant is spread over a large surface area of
the body (Wester and Noonan, 1980a). The relationship between the concentration
of applied dose and the efficiency of absorption is not necessarily a linear
one as studies have shown the efficiency to decrease with increasing dose (Table
1). The site of application was the forearm for both man and rhesus monkey in
Table 1; note the similarity in percent absorbed. The absolute dose absorbed
increases, of course, as the dose applied is increased.
The solvent or formulation used to deliver a penetrant to the skin (vehicle)
can have a decided impact on the efficiency of absorption. The ability of a
compound to penetrate the skin and exert its effect is dependent on two
consecutive physical events. The compound must first Diffuse out of the vehicle
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TABLE 1. RELATIONSHIP BETWEEN DOSE AND EFFICIENCY OF ABSORPTION
Penetrant
Hydrocortlsone
Benzole Add
Testosterone
Dose (ug/cm2)
4
40
4
40
2000
4
400
Percent
Rhesus
2.9
2.1
59.2
33.6
17.4
18.4
2.2
of Dose Absorbed
Man
1.9
0.6
42.6
25.7
14.4
13.2
2.8
from Wester and Noonan 1980a
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to the skin surface, and then must penetrate the skin in route to the site of
action (Ostreng et al., 1971). If the membrane diffusion constant for the
penetrant and the thickness and solvent properties of the membrane are unchanged
by the nature of the external vehicle, then the rate of absorption is proportional
to the chemical potential in the vehicle. When the chemical potential and
penetrant concentration are linearly related, Pick's law of diffusion (see
Chap. 6) is obeyed for the vehicle (Dugard, 1983). Some solvents, such as
DMSO, actually dissolve lipids, destroying the barrier function and carrying
the penetrant along with it into the body. The effect of a solvent on the rate
of absorption of a penetrant should be studied further.
The site of application of a penetrant can also be an important fact in
the efficiency of absorption. Maibach and Feldmann (1971) have measured the
absorption of several compounds at various sites on the body. Their results
show a wide range of values with the palm allowing approximately the same
penetration as the forearm, the abdomen and dorsum of the hand having twice the
penetration of the forearm, the scalp, angle of the jaw, postauriacular area,
and forehead having four-fold greater penetration, and the scrotum allowing
almost total absorption (Table 2a).
Guy and Maibach (1984) divided the body into five regions for the purpose
of calculating correlation factors for use with forearm penetration data:
genitals, arms, legs, trunk, and head. Table 2b gives "penetration indices" or
the ratio of skin penetration for one of the five anatomic sites divided by
skin penetration for the forearm. These penetration indices are derived from
hydrocortisone skin penetration data and from absorption results using the
pesticides malathion and parathion. The authors show the relative proportion
of body area and actual skin area for the five anatomic sites in Table 2c.
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TABLE 2a. RATIO COMPARING ABSORPTION OF VARIOUS SITES TO FOREARM
Site
Parathion
From Malbach and Feldmann, 1971.
Mai athi on
Hydrocortlsone
Forearm
Palm
Foot, ball
Abdomen
Hand dorsum
Scalp
Jaw angle
Forehead
Scrotum
1.0
1.3
1.6
2.1
2.4
3.7
3.9
4.2
11.8
1.0
0.9
1.0
1.4
1.8
...
...
3.4
...
1.0
0.8
—
...
...
3.5
13.0
6.0
42.0
TABLE 2b. PENETRATION INDICES FOR FIVE ANATOMIC SITES
Site
Penetration Index based
on hydrocortlsone
Penetration Index based
on pesticide data
Genitals
Arms
Legs
Trunk
Head
40
1
0.5
2.5
5
12
1
1
3
4
From Guy and Malbach, 1984 (see original article for the number of footnotes
describing how these Indices were calculated)
TABLE 2c. BODY SURFACE AREAS FOR FIVE ANATOMIC SITES
Adult8 Ch1ldb Neonatec
Body area (X) Area (cm2) Body area (S) Area (cm2) Body area (*) Area (cm2)
Genitals
Arms
Legs
Trunk
Head
1
18
36
36
9
190
3420
6840
6840
1710
1
19
34
33
13
75
1425
2550
2475
975
1
19
30
31
19
19
365
576
595
365
Total
19000
7500
1920
Fran Guy and Malbach, 1984
aAdult: weight =
bChild: weight =
cNeonate: we1ght
70 kg; height - 1.83m
19 kg; height = 1.10m
= 3 kg; height - 0.49m
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The condition of the skin, such as loss of barrier function of the stratum
corneum through disease or damage, will also affect absorption. Absorption can
be virtually 100% if all barrier function is removed. Occulusion or covering
of the applied dose as with bandaging or putting on clothing after a dermal
application will increase absorption. Occlusion changes the hydration and
temperature of the skin and also prevents the accidental wiping off or evapora-
tion of an applied dose (Wester and Noonan, 1980a).
The frequency of dermal applications is also a factor which could affect
absorption. In one study the absorption of a single application of a high
concentration of penetrant was greater than when the equivalent concentration
was applied in equally divided doses (Wester et al., 1977).
In another study the effect of repeated applications of a penetrant was
tested. The absorption on the 8th day of application of the same penetrant
dose was found to be significantly higher than on the first day. The authors
suggest that the initial applications of penetrant altered the barrier function
of the stratum corneum, resulting in increased absorption for subsequent
applications (Wester et al., 1980b).
The skin contains many of the same enzymes as the liver, thus penetrant
metabolism by the skin could affect absorption. The metabolizing potential of
skin has been estimated to be about 2% of the liver with most of the enzyme
activity localized in the epidermal layer (Pannatier et al., 1978). The slower
a penetrant is absorbed through the skin the greater the opportunity for some
metabolism to occur. In vitro penetrant absorption studies using excised human
skin will not show the results of any potential metabolism and, thus, may not
reflect the actual in vivo absorption for some penetrants. The metabolism
potential of skin should be studied further.
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CHAPTER 5 - TECHNIQUES FOR MEASURING ABSORPTION
5.1 IN VIVO
Percutaneous absorption In vivo is usually determined by measuring radio-
activity in excreta following the topical application of a labeled compound.
The penetrant under study is labeled with carbon 14 or tritium and following
application, the total amount of radioactivity excreted in urine or urine plus
feces is determined. The radioactivity in the excreta can be a mixture of the
parent compound and any metabolites. The amount of radioactivity retained in
the body or excreted through expiration or sweat can be determined by measuring
the amount of radioactivity excreted following an intravenous (i.v.) injection;
the fraction determined by i.v. dose is then used to correct the amount of
radioactivity found after topical administration (Wester and Maibach, 1983).
Another in vivo method of measuring absorption is the detection of plasma
radioactivity following topical administration. This method may be difficult
to apply because penetrant concentrations in blood after topical administration
are often very low. Determining the loss of material from the surface as it
penetrates the skin is another way to measure in vivo absorption. It is assumed
that the difference between applied dose and residual dose is the amount of
penetrant absorbed. The difficulties inherent in skin recovery, volatility of
penetrant, and errors associated with using the difference between amount
applied and amount left make this method less quantitative. Biological or
pharmacological responses, such as vasodialation, have also been used to estimate
absorption for a limited number of compounds (Stoughton et al., 1960)
The main source of human in vivo data is the work of Feldmann and Maibach
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(1969 and 1970). These authors studied the percutaneous absorption of some
steroids, pesticides, and organic compounds using radiolabeled (Cl4) doses
administered intravenously and topically to human volunteers. The intravenous
dose was used to correct for the amount of radiolabeled penetrant that is
percutaneously absorbed but not recovered in urine samples (i.e., excreted in
feces or retained in the body). The topical dose of 4 ug/cm2 was dissolved
in acetone and applied to a 13 cm^ circular area of the ventral forearm.
All urine was collected for five days divided into suitable time periods so
that either the absorption rate (%/hr) or total absorption (% of dose) could
be calculated. The skin sites were not protected and not washed for 24 hours.
Tables 3-5 are a compilation of the 49 penetrants studied.
Table 3 shows the percutaneous penetration of some steroids in man. For
the intravenous dose, one microcurie (14C) of the steroid was dissolved in 5 to
20 ml of saline (or saline plus ethanol) and injected. The half life, which is
an indicator of the relative speed of elimination of the injected dose, was
obtained from plotting the amount of l^C excreted divided by the time period
expressed as the percent of dose administered versus the collection t.ime. The
values for percent recovery are given in the first part of Table 3 with
testosterone showing almost complete elimination from the body and fluocinolone
yielding only one-third of the injected dose. Cortisone, corticosterone, 17-OH
desoxycorticosterone, desoxycorticosterone, and 17-OH progesterone are assumed
to be excreted similarly as hydrocortisone. Likewise, the value for testosterone
was used for the acetate and propionate, plus dehydrocortisone and androstenedione,
The second part of Table 3 shows absorption after topical administration. The
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TABLE 3. PERCUTANEOUS ABSORPTION OF STEROIDS IN MAN
I.V. Administration —
Steroid.- 8 Recovered Half Life (hrs)
Hydrocortlsone
Hydrocortlsone acetate
Estradlol
Testosterone
Progesterone
Fluoclnolone acetonlde
Dexamethasone
65.4
68.9
51.6
92.1
68.7
37.0
47.4
5
5
8
4
4
5.5
4
Topical Administration*.-
Total
Steroid Absorption Rate (t/hr) Absorption No.
Time (hrs) 7
0-12 12-24 24-48 48-72 72-96 96-120 % of dose S.O.
Subjects
Hydrocortlsone .005 .023 .019 .018 .016 .010 1.87 1.59 15
Hydrocortlsone
acetate .020 .069 .032 .024 .015 .008 2.65 1.80 6
Cortisone .015 0037 .039 .036 .032 .024 3.38 1.64 7
Cortlcosterone .013 .065 .139 .070 .050 .039 8.78 5.35 6
17-OH DOC .041 .101 .084 .076 .062 .055 8.41 4.28 5
Desoxycortlcos-
terone .197 .313 .143 .069 .035 .020 12.55 8.53 6
17-OH Proges-
terone .042 .120 .213 .211 .078 .031 14.76 11.35 7
Continued on following page
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Table 3 Continued -
Topical Administration*—
Steroid
Progesterone
Hud not one
acetonlde
Dexamethasone
Estradlol
Testosterone
Testosterone
acetate
Testosterone
proplonate
Dehydroeplan-
drosterone
Androstenedlone
Tirtie (hrs)
0-12
.208
.002
.005
.008
.147
.103 '
.061
.265
.183
Absorption
12-24 24.48
.264
.011
.003
.056
.364
.133
.096
.446
.334
.135
.012
.004
.099
.156
.048
.035
.249
.155
Rate (X/hr)
48-72
.045
.016
.003
.101
.066
.015
.015
*
.091
.076
72-96
.024
.008
.002
.107
.036
.007
.009
.046
.043
96-120
.011
.005
.002
.103
.018
.004
.005
.028
.028
Total
Absorption
% of dose
10.81
1.34
.40
10.62
13.24
4.62
3.44
18.45
13.47
S.T).
5.78
1.05
.23
4.86
3.04
2.28
1.03
7.71
5.56
No.
Subjects
6
9
3
3
17
6
9
6
11
From Feldmann and Haibach. 1969
•Corrected for l.v. recovery
-17-
-------
average amount of absorption for this series of steroids is roughly 10 percent
with a fifty-fold difference between the least and the most absorbed. However,
inspection of the standard deviation column (S.D.) shows that there is a great
deal of uncertainty in this methodology with penetran-;s like hydrocortisone
having a standard deviation of ^ 85 percent. The authors point out a physical
relationship between the number of hydroxyl groups on the steroids and their
total absorption; corticosterone and 17-OH desoxycorticosterone each have two
while desoxycorticosterone and 17-OH progesterone each have one. For each pair,
the absorptions are reasonably close. Also, the series of steroids shows a
stepwise increase in absorption for each hydroxyl removed (Feldmann and Maibach,
1969).
Table 4 presegts further in vivo percutaneous absorption studies by Feldmann
and Maibach (1970) on some organic compounds. The values for percent recovery
shown in the first part of the table indicate that several of the compounds are
poorly absorbed and excreted (i.e., the correction factor for hexachlorophene
is greater than 20 which means that the percent recovery for this compound in
urine after topical administration has to be multiplied by this factor). Three
compounds were deemed too toxic to test intravenously in man and thus were
administered to guinea pigs. Nicotinic acid, nicotinamide, hippuric acid and
phenol were assumed to be excreted similarly to salicylic acid, while thiourea
was assumed to behave like urea. The second part of Table 4 gives the absorption
after topical administration. Three compounds, caffeine, dinitrochlorobenzene,
and benzoic acid, had total absorptions of about 50 percent after being corrected
for incomplete absorption following i.v. administration. Three other compounds,
nicotinic acid, hippuric acid, and thiourea displayed total absorptions of less
-18-
-------
TABLE 4. PERCUTANEOUS ABSORPTION OF SOME ORGANIC COMPOUNDS IN MAN
I.V. Administration—
Compound
w-
Caffeine
Chloramphenlcol
Colchlclne
Dlethyltoluamlde
D1n1trochlorobenzene
Hexachlorophene
Nitrobenzene
Potassium thlocyanate
Salicylic add
Urea
Butter Yellow*
Malathlon*
Methyl chol anthrene*
X Recovered
59.4
67.4
27.9
52.3
64.0
4.4^
60.5
10.2
89.8
71.7 .
58.6
76.0
18.2
Half Life (hrs)
6 r
6
4
4
4
48
20
12
4
8
6
4
14
•Recovery In urine after l.v. administration In guinea pigs.
-19-
-------
Table 4 continued—
Topical Administration*—
Compound
Absorption Rate
ITjme (nrsj
1 0-12
Acetyl salicylic
•eld
Benzole add
Butter yellow
Caffeine
Chloramphenlcol
Colchlclne
Dlnltrochloro-
benzene
Dlethyltoluamlde
Hexachlorophene
Hlppurlc acid
Malathlon
Methyl chol anthrene
Nicotfnlc add
Nlcotlnamlde
Nitrobenzene
Pa raaminoben zoic-
acid
Phenol
Potassium
thiocyanate
Salicylic add
Thlourea
Urea
.141
3.036
.215
.559
.007
.036
3.450
.773
.029
.005
.313
.062
.000
.019
.022
.159
.254.
.051
.116
.046
.008
12-24
.438
.340
.685
1.384
.019
.038
.565
.331
.031
.003
.170
.329
.002
.168
.022
.648
.091
.060
.535
.035
.021
24.48
.334
.055
.289
.855
.021
.033
.134
.084
.020
.001
.044
.258
.001
.177
.013
.444
.010
.078
.356
.010
.051
(Wose/hr)
48-72
.147
.000
.083
.109
.022
.040
.045
.036
.028
.001
.017
.127
.001
.088
.013
.196
.601
.097
.156
.008
.073
72-96
.076
.000
.054
.032
.015
.025
.018
.016
.034
.001
.011
.064
.002
.052
.011
.058
.000
.100
.080
.007
.075
96-120
.060 .
.000
.022
.014
.012
.004
.009
.012
.030
.001
.006
.045
.007
.031
.006
.044
.000
.093
.033
.007
.034
Total
Absorption
S of dose
21.81
42.62
21.57
47.56
2.04
3.69
53.14
16.71
3.10
.21
7.84
16.81
.34
11.08
1.53
28.37
. 4.40
10.15
22.78
.88
5.99
S.O
No.
Subjects
r
3.11 S
16.45 <
4.88 4
20.99 12
2.46 6
2.50 6
12.41
4
5.10 4
1.09 7
.09 7
2.71
5.16
7
3
.09 3
6.17 7
.84 6
2.43 13
2.43
3
6.60 6
13.25 17
.22 3
1.91
4
From Feldmann and Maibach, 1970
•Corrected for l.vf recovery.
-20-
-------
than 1 percent of the applied dose. The range for total absorption for these
compounds is greater than 250-fold, much larger than the series of steroids in
Table 3. However, as with the steroids, the standard deviations are very high
for the compounds, indicating a large degree of uncertainty, with chloramphenicol
showing a standard deviation of greater than 100 percent. The authors point
out two examples of closely related compounds showing great differences in
penetration: benzoic acid was absorbed 200 times more than its glycine conjugate,
hippuric acid; nicotinic acid showed minimal penetration while 10 percent of
its amide, nicotinamide, was absorbed. The authors also generally found a
good correlation between the maximum penetration rate for each compound and
its total absorption.
Table 5 presents similar in vivo absorption studies by Feldmann and Maibach
(1974) on some pesticides and herbicides. The authors used the same experimental
method as discussed previously to study 5 organophosphates, 3 chlorinated
hydrocarbons, 2 carbamates, and 2 herbicides. The total excretion from the
i.v. dose varied over a wide range with dieldrin, aldrin, and carbaryl at 3-7
percent and malathion, baygon, and 2,4-D being over 80 percent. For the topical
administration, diquat was the only compound with only slight penetration;
carbaryl, on the other hand, was nearly completely absorbed after multiplying
the topical results by the large correction factor obtained from the i.v.
administration. The authors discuss the rather large standard deviations of
1/3 to 1/2 of the rcson value found in their studies. They claim that the
difference is due to actual differences between people rather than experimental
error as repeat studies on the same subject gave similar results. The authors
assume a normal distribution and find that 1 person in 10 will absorb twice
-21-
-------
TABLE S. PERCUTANEOUS PENETRATION OF SOME PESTICIDES AND HERBICIDES IN HAN
I.V. Administration—
i
ro
Absorption rate (X dose/hr)(t1me period In hr)
Total Absorption
Compound
Azodrln
Ethlon
Gut hi on
Ma lath Ion
Parathlon
Baygon
Carbaryl
Aldrln
Dleldrln
Llndane
2.4-D
Dlquat
Time (hrs)
^ 0-4 4-8
1.816
0.832
1.513
12.949
0.035
10.361
0.459
0.224
0.038
0.688
3.001
9.328
2.721
1.041
1.204
5.571
1.321
7.290
0.394
0.091
0.067
0.611
4.063
1.544
8-12
1.701
1.892
1.590
2.420
2.508
1.478
0.211
0.113
0.074
0.552
5.312
1.825
12-24
1.000
0.791
1.041
0.368
1.124
0.192
0.102
0.040
0.046
0.244
1.728
0.292
24-48
0.679
0.316
0.813
0.052
0.469
0.064
0.037
0.023
0.046
0.232
0.737
0.127
48-72
0.341
0.123
0.458
0.017
0.135
0.053
0.021
0.013
0.013
0.132
0.275
0.059
72-96
0.173
0.071
0.257
0.008
0.069
0.047
0.011
0.011
0.015
0.125
0.153
0.054
96-120
0.088
0.065
0.127
0.004
0.037
0.043
0.008
0.008
0.008
0.102
0.097
0.045
% Dose
67.7
38.4
69.5
90.2
45.8
83.8
7.4
3.6
3.3
24.6
100.0
61.2
Half-life
SO flir)
5.3
3.6
6.9
9.7
5.3
7.2
2.2
0.9
1.0
6.1
2.5
16.0
20
14
30
3
8
8
9
6
28
26
13
4
Topical Admlnlstratlon--
Azodrln
Ethlon
Guthlon
Halathlon
Parathlon
Baygon
Carbaryl
Aldrln
Dleldrln
Llndane
2.4-D
Dlquat
0.057
0.004
0.044
0.089
0.007
0.351
0.005
0.086
0.143
0.064
0.009
0.005
0.048
0.005
0.202
0.408
0.116
0.705
1.212
0.074
0.137
0.135
0.012
0.002
0.092
0.026
0.294
0.396
0.243
1.204
3.027
0.078
0.135
0.245
0.020
0.005
0.121
0.036
0.276
0.149
0.202
0.543 -
1.944
0.079
0.093
0.113
0.029
0.003
0.183
0.044
0.207
0.029
0.110
0.093
0.853
0.066
0.066
0.088
0.068
0.003
0.147
0.041
0.125
0.008
0.062
0.023
0.277
0.053
0.060
0.066
0.082
0.003
0.113
0.015
0.059
0.006
0.036
0.020
0.154
0.060
0.043
0.051
0.043
0.002
0.073
0.011
0.040
0.005
0.029
0.028
0.105
0.061
0.034
0.048
0.027
0.001
14.7
3.3
15.9
8.2
9.7
19.6
73.9
7.8
7.7
9.3
5.8
0.3
7.1
1.1
7.9
2.7
5.9
• " 5.2
21.0
2.9
3.2
3.7
2.4
0.1
From Feldmann and Malbach, 1974
•Corrected for l.v. recovery. There were 6 subjects for each compound for both l.v. and topical administration.
-------
the mean value while 1 in 20 will absorb 3 times this amount. They have also
found that experimental subjects differ by a factor of 5 in the amount of
percutaneous absorption. The authors also comment that the experimental subjects
did not sweat extensively such as field worker might when exposed to these
pesticides; the effect.of sweating should be studied. Also, Larry Hall (personal
communication) points out that the skin penetration tables in Feldmann and
Maibach's studies are accurate only if the rate of elimination is much greater than
the rate of skin absorption.
5.2 IN VIVO ANIMAL STUDIES
The toxicity of many penetrants prohibits in vivo human studies, thus
researchers have had to use animal models to obtain absorption data. Unfortunately
there are a number of problems associated with the extrapolation of animal data
to humans. Animal species variation, different sites of application, shaved
skin vs. unshaved, occluding or restraining devices, and skin metabolism (or
lack thereof) are some of the factors which need to be considered when using
animal studies to predict human absorption.
Bartek et al. (1972) compared percutaneous absorption in rats, rabbits, minia-
ture swine, and man; Table 6 shows the results of their study. Radiolabeled
compounds were applied to the shaved skin of the back and protected by a non-
occluding device. In general, the penetration through the skin of the pigs
and man was similar and much slower than it was through rat and rabbit skin.
For the compounds studied, haloprogin was completely absorbed by the rat and
rabbit, while only 11 percent was absorbed by man. About half of the caffeine
applied was absorbed by all of the animals plus man. The results of this
study show that absorption in the rabbit and rat would not be predictive of that
in man while the miniature swine appears closest to man.
-23-
-------
TABLE 6. IN VIVO PERCUTANEOUS ABSORPTION BY RAT. RABBIT, PIG. AND MAN
Penetrant
Haloprogln
N-Acetylcycte1ne
Testosterone
Cortisone
Caffeine
Butter Yellow
Percent
Rat
95.8
3.50
47.4
24.7
53.1
48.2
dose absorbed*
Rabbit
113.0
1.98
69.6
30.3
69.2
100.0
Pig
19.7
6.00
29.4
4.06
32.4
41.9
-Nan
11.0
r
2.43
13.2"
3.38"
47.6"
21.6"
From Bartek, laBudde, and Malbach. 1972.
•Corrected for recovery following l.v. administration
"Human data taken from Feldmann and Malbach, 1969 and 1970.
-24-
-------
Bartek and LaBudde (1975) also studied the absorption of 4 pesticides in
the rabbit, pig and squirrel monkey using the same techniques as their previous
study. The results were compared to man where the site of application was the
forearm (Table 7). The in vivo absorption of pesticides in the rabbit was
greater than in man, while absorption in the pig and squirrel monkey was closer
to that in man.
Shah^^l_. (1981) investigated the dermal penetration of some insecticides
in mice. Acetone solutions of the radiolabeled test penetrants were applied
to shaved backs of female mice. Mice were placed in metabolism cages equipped
with C02 trapping devices and urine collection jars. Urine, blood, specific
tissues, and organs were sampled at various time intervals. The remaining
portion of the body was termed carcass. Table 8 shows the percent of radio-
labeled penetrant recovered in various fractions at 5, 15, and 60 minutes
following topical administration. Recovery of radioactivity was 90% or more
in all cases. The authors also tried to relate physical properties such as
molecular weight, solubility, and partition coefficients to dermal absorption.
Partition coefficients for chloroform-water, olive oil-water, and benzene-water
were determined using radiolabeled compounds and a high-speed centrifuge.
Table 9 presents a comparison between physical parameters and rates of penetra-
tion. The authors did not draw any conclusions from their study other than
that there is a lack of correlation among partition systems themselves. One
interesting aspect of this study is that an aqueous wettable powder formulation
of carbaryl penetrated more rapidly than did the acetone formulation.
Researchers have attempted to bridge in vivo animal and human dermal
absorption studies by transplanting human skin to the athymic (nude) mouse.
-25-
-------
TABLE 7. IN VIVO PERCUTANEOUS ABSORPTION OF SEVERAL PESTICIDES BY RABBIT, PIG,
SQUIRREL MONKEY, AND HAN
Percent Dose absorbed*
Pesticide
DDT
Llndane
Parathlon
Malathion
Rabbit
46.3
51,2
97.5
64.6
Pig
43.4
37.6
14.5
15.5
Monkey
1.5
16.0
30.3
19.3
Man**
10.4
9.3
9.7
8.2
From Bartek and LaBudde, 1975
•Corrected for recovery following 1,v. administration (except the monkey data).
**Human data from Feldmann and Malbach, 1974
-26-
-------
TABLE 8. GEOMETRIC MEANS OF PERCENTAGE 14C RECOVERED IN VARIOUS FRACTIONS AT 5, 15.
AND 60 MIN POST APPLICATION
Toxl cant
Carbamates
Carbaryl
Methomyl
Carbofuran
Organophosphates
Parathlon
Malathlon
Chlorpyrlfos (methyl)
Chlorpyrlfos
Botanical type
Nicotine
Permethrln
Chlorinated hydrocarbons
DDT
Hexachloroblphenylb
4-Chloroblphenyl
Chlordecone
Dleldrln
Blood
5 15
mln mln
0.1 0.3
0.1 1.8
<0.1 1.7
<0.1 0.2
0.3 0.9
0.3 0.7
<0.1 0.4
0.6 2.1
<0.1 1.0
<0.1 <0.1
-------
TABLE 9. COMPARISON BETWEEN PHYSICAL PARAMETERS AND GEOMETRIC MEANS OF RATE OF PENETRATIONS
ro
oo
Partition Coefficients
Molecular ^2°
Toxicant Weight Solubility
Carbamates
Carbaryl
Met homy 1
Carbofuran
Organophosphates
203
162
221
Parathlon 292
Malathlon 330
Chlorpyrlfos (methyl )318
Chlorpyrlfos 350
Botanical type
Nicotine
Permethrln
162
390
40 ppm
58.000 ppm
700 ppm
24 ppm
145 ppm
4.7 ppm
2 ppm
Mlsdble
0.07 ppm
CHC13
Water
277
15
32
433
112
92
374
2
269
Olive Oil-
Water
46
0.1
5
1738
56
245
1044
0.02
360
Benzene- TO. 8
Water (mln)
139
40
659
91
116
480
0.4
80
12.8+4.1
13.3+2.8
7.7+1.5
66.0+21.9
129.7+47.7
51.6+6.5
20.6T5.9
18.2+2.4
5.9+1.3
of
1
mln
22.2
18.8
28.5
4.4
5.5
8.3
15.7
5.2
36.2
Geometric
percentage
5
mln
31.5
24.7
32.6
9.8
13.4
14.3
28.8
27.9
40.9
15
mln
56.0
55.5
71.7
8.3
22.7
32.2
64.4
59.5
63.1
means
penetration
60 480 2880
mln mln mln
71.7 88.5
84.5 88.3
76.1 94.7
31.9 85.4
24.6 66.7
54.4 78.2
69.0 73.9
71.5 90.7
79.7 88.1
98.9
99.6
97.8
Chlorinated hydrocarbons
DOT
Hexachl orobl pheny 1 a
4-Ch1orob1pheny1
Chlordecone
Dleldrfn
355
361
188
490
384
1.2 ppb
0.953 ppb
<0.6 ppm
0.4 ppm
0.18 ppm
532
2465
684
190
1775
887
482
185
282
170
1398
144
105.4+25.6
43.8+7.0
16.8T2.3
41.3T8.8
71.7+17.3
3.9
17.3
6.5
18.8
1.2
12.3
28.7
13.5
34.4
3.1
21.7
44.7
53.5
42.5
26.1
34.1 71.1
55.3 66.8
84.5 97.5
54.0 65.9
33.7 82.6
91.3
94.0
From Shah, et al. 1981 (and references therein)
-------
Krueger and Shelby (1981) used human skin obtained from skin grafts or organ
donors to graft to nude mice. The authors found that the human skin grafts
undergo a proliferative response when 10 ng of the tumor promoter 12-0-
tetradecanoyl phorbol 13-acetate is applied while nude mice do not respond to
this dose. The authors state that their studies show that the unit function of
human skin after transplant is similar to that prior to transplant.
5.3 IN VITRO
In vitro studies have also been used extensively to estimate absorption.
For these studies, a piece of excised skin is attached to a diffusion apparatus
which usually consists of a top chamber to hold the applied dose of the penetrant
plus any solvent, an 0-ring to hold the skin in place, and a temperature con-
trolled bottom chamber containing saline or other solvents plus a sampling
port to withdraw fractions for analysis (Fig. 2). Human forearm skin is difficult
to obtain, thus it is common practice to use abdominal skin collected at autopsy.
For most studies, the stratum corneum is heat separated from the epidermis and
dermis, then studied by itself.
Franz (1975) studied the in vitro permeability of 12 organic compounds which
had been previously studied in vivo in man. Special emphasis was given to
duplicate the in vivo conditions, such as amount of dose applied, in order to
show how accurately in vitro absorption studies can reflect the living state.
Each piece of skin was mounted in a diffusion cell (diffusion area of either
1 cm^ or 2.5 cm^) with the epidermal side of the skin exposed to ambient air
while the dermal side was bathed in a saline solution containing an antibacterial/
antifungal plus buffers. The temperature was maintained at 37°C by a water
-29-
-------
Donor
Skin
"0" Ring
Temperature
Jacket
Sampling Port
Outflow
Inflow
Stirring Bar
Figure 2. Schematic representation of a diffusion cell with top
open to the ambient environment.(Franz, 1975)
-30-
-------
jacket which surrounded the chamber. A small amount of the radiolabeled penetrant
in the same dose range as the in vivo studies was dissolved in acetone and
spread across the entire exposed surface with the acetone evaporating in less
than one minute. At selected intervals after the addition of penetrant to the
epidermis the dermal bathing solution was removed in its entirety, gelled and
analyzed in a liquid scintillation spectometer. Either the total absorption
was determined by one sample taken at 24-hr intervals or the kinetics of the
absorption process were determined by taking frequent samples throughout the
day.
Table 10 shows the total absorption of 12 organic chemicals that have been
studied both in vivo and in vitro. Highly water-insoluble compounds were not
selected for in vitro study since their permeability might be limited due to
insolubility in the dermal bathing (saline) solution. In viewing the data from
Table 10 only two compounds, chloramphenicol and benzoic acid appear to be in
quantitative agreement. The in vivo studies are from Feldmann and Maibach and,
as discussed previously, are from 5-day urine collection of an applied dose to
the forearm. The in vitro studies, on the other hand, use human adominal
tissue, last for two days, and have a much smaller receptor volume when compared
to the entire body (i.e., you might see a build-up of penetrant concentration
in the receptor cell that you would not see when the dose is applied in vivo).
Franz (1978) further investigated the apparent differences between the jji
vivo and in vitro approaches. Since in only two of the twelve compounds studied
was there no radiolabeled compound in the urine on day 5, the author thought
that this collection period might be inadequate and lead to underestimation of
-31-
-------
TABLE 10. COMPARISON OF HUMAN IN VIVO AND W VITRO ABSORPTION
Total Absorption (expressed as percent of applied dose)
Compound
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
Hlppurlc add
Nlcotlnlc acid
Thlourea
Chloramphenicol
Phenol
Urea
Nlcotlnamlde
Acetyl salicylic add
Salicylic acid
Benzole add
Caffeine
D1 n1 1 rochl orobenzene
In V1vo«
0.2 + 0.1 [7]
0.3 + 0.1 [3]
0.9 l 0.2 [3]
2.0 + 2.5 [6] .
4.4 + 2.4 [3]
6.0 +_ 1.9 [4]
11.1 +6.2 [7]
21.8 +_ 3.1 [3]
22.8 + 13.2 [17]
42.6 +_ 16.5 [6]
47.6 ^ 21.0 [12]
53.1 + 12.4 [4]
In V1trob
1.2 (0.8. 2.7) [15]
3.3 (0.7, 8.3) [19]
3.4 (2.4. 5.5) [52]
2.9 (1.0. 5.7) [12]
10.9 (7.7, 26) [7]
11.1 (5.2. 29) [22]
28.8 (16, 65) [21]
40.5 (17, 49) [14]
12.0 (2.3, 23) [10]
44.9 (29, 53) [18]
9.0 (5.5, 20) [17]
27.5 (19. 33) [18]
From Franz, 1975 (In vivo data from Feldroann and Malbach. 1970)
'Mean + standard deviation. The figure 1n brackets Is the number of subjects
studied*.
"Median with 95$ confidence Interval given 1n parentheses.
-32-
-------
the actual amount absorbed. Also, there are differences in the permeability
of skin from different sites of the body. Franz restudied four compounds
(hippuric acid, nicotinic acid, thiourea, and caffeine) for which there was
poor agreement between the in vivo and in vitro results. This time adominal
skin was used for both the in vivo and in vitro tests plus urine was collected
in the in vivo tests until background levels of radioactivity were reached.
Also, to minimize the differences that might be caused by desquamation of the
epidermis in vivo, the surface of the skin was washed with acetone and water
24 hours after application of the test compounds in both sets of experiments.
Table 11 shows the comparison between human in vivo and in vitro absorption,
using Franz's refined techniques, for the four compounds originally showing
poor agreement. Hippuric acid, thiourea, and caffeine gave excellent quantitative
agreement, but nicotinic acid displayed the lack of agreement found in the
previous study (see Table 10). Franz found that less than 15 percent of an
intravenously administered dose of ^C-nicotinic acid was excreted in the urine;
Feldmann and Maibach (1970) did not directly measure the excretion of nicotinic
acid following an i.v. dose, but assumed on the basis of similar chemical
structures that it would behave like salicylic acid, a compound in which 90
percent of the i.v. dose is excreted. Thus, when the value of 0.32 percent
absorption observed in vivo in humans is corrected for incomplete urinary
excretion, the value of 2.1 percent is in better agreement with the value
observed in vitro. Franz (1978) states that based on the data obtained from the
compounds studied to date, it appears that in vitro studies accurately portray
the phenomenon of absorption as it occurs in living man.
-33-
-------
TABLE 11. COMPARISON OF HUMAN IN VIVO AND IN VITRO ABSORPTION
USING REFINED TECHNIQUES
Total Absorption (expressed as percent of applied dose)
Compound
1. N1cot1n1c Acid
2. Hlppurlc Add
3. TMourea
4. Caffeine
In V1voa
0^32 +. 0.10 [3]
1«0 + 0.4 [6]
3.7 + 1.3 [4]
22.1 +. 15.8 [4]
Tb
21
3
21
7
In V1troa
2.3 _+ 0.9 [4]
1.25^0.5 [4]
4.6 + 2.3 [5]
24.1 +; 7.8 [4]
From Franz, 1978
aHean ± SO. The figure 1n brackets Is the number of subjects studied.
bNumber of days urine was collected.
-34-
-------
Bronaugh et al. (1982) compared in vivo and in vitro absorption through
female rat skin using benzoic acid, acetylsalicyclic acid, urea, and caffeine.
For the in vivo studies, petrolatum was used as a nonvolatile vehicle because
of its ability to adhere to the skin; in this manner the concentration was
known and, therefore, permeability constants could be calculated. Full thick-
ness, lightly shaved skin (with the subcutaneous fat removed) was used in a
standard diffusion cell for the in vitro experiments. Since the permeability
n
constant (kp) is defined as the steady-state rate of absorption (amount/cm /hr)
o
divided by the concentration of solute applied to the skin (amount/cm ), a kp
value (cm/hr) is obtained by the diffusion cell procedure. The determination
of a kp value from in vivo data is based on the measurement of absorption rate.
This rate is estimated by considering the absorbed compound to accumulate in
both the body of the animal and the excreted waste. The kp was calculated from
the rate of body accumulation plus the rate of excretion divided by the
concentration of solute in vehicle. The quantitative agreement between the i_n_
vivo and in vitro work of Bronaugh et al. (1982) appears good as shown in
Tables 12 a, b, and c.
-35-
-------
TABLE 12a. PERCUTANEOUS ABSORPTION OF ACETYLSALICYLIC ACID IN RATS
Absorption (X of applied dose)3
Days In V1vo In Vitro
1
2
3
4
5
Total
8.5 i 1.6
7.9 +. 2.0
4.0 + 0.9
2.8\+ 0.5
1.9 + 0.5
24.8 + 4.4
8.8 +_ 1.2
8.5+^ 1.2
4.6+0.5
4.3^0.4
2.9 + 0.1
29.0 _+ 0.1
From Bronaugn et al. 1982
aResults are expressed as the Y+_ SE of four or five determinations.
TABLE 12b. Iti VIVO VS. IN VITRO PERCUTANEOUS ABSORPTION THROUGH RAT SKIN8
Rate (ng/hr/cm^) Permeability constant
Test Compound
Caffeine
Acetyl salicylic add
Body
16.3
0
Urine
27.8
11.4
In V1vo In Vitro
2.1 x 10-4(7) 3.1 x 10-4(6)
5.2 x 10'5(7) 6.5 x 10-5(5)
From Bronaugh et al. 1982
'Compounds In a petrolatum vehicle were applied to a 2.0-cm^ area of skin on the
living animals and 1n diffusion cells. Results are the means of the number of
determinations In parenthesis.
TABLE 12c. COMPARISON OF PERMEATION VALUES WITH THOSE OF OTHER STUDIES
In V1vo In Vitro
Test Compound
Benzole add
Acetyl salicylic acid
Urea
Rat
(Petrolatum)
37.1
24.8
8.1
Human0
(Acetone)
42.6
21.8
6.0
Rat
(Petrolatum)
49.1
29.0
7.2
Human"
(Acetone)
44.9
40.5
11.1
From Bronaugh et al. 1982
aValues from Feldmann and Mai bach (1970)
bValues from Franz (1975)
-36-
-------
CHAPTER 6 - THEORETICAL TREATMENT OF DERMAL ABSORPTION
6.1 PICK'S LAW APPLIED TO DERMAL ABSORPTION
For those unfamiliar with Pick's Laws of Diffusion, it may be helpful
to reference a basic physical chemistry text such as Moore, 1962. Scheuplein
and Blank (1971) developed an integrated form of Pick's law to express diffusion
through the skin barrier. The flow across the membrane is called the flux,
Js, and the expression for the steady-state flux of solute across an inert
membrane is given by:
Js = D(d - C?)
O 1
where Js = steady-state flux of solute (moles cm"'1 hr"1)
D = Average membrane diffusion coefficient for solute
o 1
(cnrsec"1) (sometimes interchanged with Dm to represent
diffusion coefficient through skin membrane)
C = Concentration of solute
6 = Membrane thickness (cm)
For skin, the stratum corneum is not an inert structure but one with an affinity
for the applied solute; thus, the concentrations at the surfaces of the membrane
are hot usually equal to the concentrations in the external solutions. The
correlation between external and surface concentrations can be stated in
terms of the sol vent -membrane distribution coefficient, Km. The intergrated
form of Pick's law then becomes Js = KmDAC<;
6
and kp = Km D
6
where ACS = Concentration difference of solute across membrane
(moles cm~3)
-37-
-------
k. = Permeability constant for solute (cm hr~)
Js = kp ACS
In the application of the expanded form of Pick's law, i.e., Js = kp&Cs =
, only the skin membrane thickness, diffusivity, and the partition
6
coefficient for the solute-solvent membrane (Km) are considered. This equation
describes reasonably well the permeability of the skin to non-electrolytes, but
this simplified expression applies only to steady-state permeability. The time
it takes for the permeability to reach a steady-state is called the lag time
(T) for diffusion. For an simple, ideal membrane, the lag time is related to
the diffusion constant by T = «2 (Dugard, 1983). Another factor of interest is
6Dm
the amount of penetrant remaining dissolved within the stratum corneum at the
end of a short period of contact. The penetrant that is within the stratum
corneum cannot be quickly washed away and eventually will enter the body (unless
volatile or lost through desquamation), leading to the term "reservoir effect."
A diagram showing the concentration profile across stratum corneum during steady-
state absorption, assuming uniform properties across the membrane, is shown in
Figure 3.
A corollary of Pick's law is that the chemical potential of a penetrant is
maximal in a saturated solution and therefore a maximum absorption rate occurs
from a saturated solution. The chemical potential of a particular chemical is
the same in all saturated solutions (i.e., neat liquid or solid), regardless of
solvent. This means that there is a single maximum absorption rate definable
for any penetrant. The absorption rate for an ideal system is proportional to
the degree of saturation, and, thus, all half-saturated solutions of a penetrant
have equal chemical potential and all give half the maximum possible absorption
-38-
-------
Penetrant
Concentration
i
to
vo
I
c=o
Outer
Solution
Membrane
Inner
Solution
Figure 3. Diagram showing the concentration profile across
stratum corneum during steady-state absorption
(From Dugard, 1983)
-------
rate (Dugard, 1983 and ref. therein).
Knowledge of any physical equilibrium condition may be used to relate the
chemical potential in one vehicle or physical state to that in another.
Predictive treatments based on solubilities or on equilibria are largely untested
and depend on several systems behaving close to the ideal, thus any predictions
should be regarded as qualitative. Reasons for the breakdown of solubility-
derived predictions include solvent damage to stratum corneum, deviations from
ideality near penetrant saturation in vehicles, variations in stratum corneum
hydration in contact with different solvents, and entry of vehicle components
into the stratum corneum to alter its solvent properties (Dugard, 1983).
6.2 NON-STEADY STATE DIFFUSION '
The diffusion of the penetrant across the stratum corneum is usually the
rate determining step in the dermal absorption process; however, there are
instances where this is not the case. Higuchi (1962) has considered the
condition where the penetrant is entirely dissolved in the vehicle and its
diffusion in this medium is slow. Assuming that any penetrant reaching the
stratum corneum is immediately absorbed and that not more than 30% of the
original amount of penetrant is absorbed, then the total amount of penetrant
(Qt) released from the vehicle by time t is approximately
Qt = 2c AL
where Dv is the diffusion constant of the penetrant and Cv is the concentration
of the penetrant in the vehicle.
A second situation where diffusion across the stratum corneum is not rate
limiting occurs when the vehicle contains suspended penetrant whose dissolution
is rate limiting. Higuchi (1960) described the release and absorption of the
-40-
-------
penetrant when it is in small particles evenly dispersed in the vehicle as
Qt = (2 Av * Sv> [. A* c t ic 11/2
1 + Z [AV-5V)/5VJ
where Av is the total amount of penetrant, dissolved and suspended, in the
vehicle per unit volume, and Sv is its solubility in the vehicle. The rate
of absorption at a given time t is obtained by differentiating Q^ (Dugard
1983, and references therein) to yield
W = 1/2 ki^jsj1/2
If Av is much greater than Sv, these equations reduce to
Qt = (2AvDvSyt)1/2 and dQ ,/Av D Sy\1/2
Another type of non-steady state absorption can occur when a small amount
of penetrant is applied. "Small" means that the source of the penetrant is
significantly reduced in quantity by the absorption process. Figure 4 shows
the manner in which the absorption rate changes with time for a fast penetrant,
moderate penetrant, and a very slow penetrant, when depletion of the external
source of penetrant occurs (Dugard, 1983).
Looking at Figure 4 we see that for curves A and B a specific maximum
rate is achieved. The time taken to reach the maximum rate, Tmax, is obtained
from the relationship Tmax = 62-h2 (Scheuplein and Ross, 1974). The thickness
^
of the penetrant layer, h, is usually small in comparison with
-------
ro
i
o
o
vt
.Q
03
CC
Time
Figure 4. The pattern of changing absorption rate for small
amounts of penetrant per unit area of skin. Curve A,
moderately fast penetrant; curve B, slower penetrant;
curve C, very slow penetrant. Curves A and B show the
effect of the depletion of penetrant source. (Dugard, 1983).
-------
Ross (1974)'also state that the peak rate of absorption is directly proportional
to the amount of penetrant applied per unit area, which they designate as the
specific dose A.
For very slow penetrants such as cortisone, the authors found the rate of
absorption to be constant for a relatively long period of time (such as curve C
in Figure 4). This rate is approximately proportional to the specific dose,
thus the flux can be expressed as:
J = k^A (slow penetrant, 6>h)
where k^ is a transfer coefficient. Instead of permeability constant (kp)
defined in terms of flux and concentration, the transfer coefficient (k^)
is defined in terms of flux and specific dose (A). Unlike the permeability
constant, as A increases k^ gradually decreases because the source of penetrant
comes to contain excess material where additions cause no further increase in
absorption rate (Dugard, 1983).
6.3 CALCULATION OF kp and Km
Scheuplein (1965) calculated kp values for the homologous series of normal
primary alcohols Cj - CQ by measuring the in vitro permeability of the alcohols
through human abdominal skin in a diffusion cell. The sol vent-membrane distribution
coefficients Km, or partition coefficients, were computed from the loss in
concentration of the original solution after equilibrium with the tissue, i.e.:
Km = moles alcohol absorbed per unit mass of dry tissue
moles alcohol in solution per unit mass of water
Penetration rates (Js) were obtained from the rate of increase of concentration
on the receptor (bottom half of the diffusion cell). Permeability constants kp
were computed directly from the linear portion of the accumulation curve or by
graphical procedures.
-43-
-------
Table.13 gives a summary of the membrane permeability and partition
coefficients for the normal primary alcohol series. The molecular volume
increases by a factor of 4 within the series but its effect on the rate of
diffusion is less than a factor of 1.6. The Table also shows that the permea-
bility constant increases as the molecular weight increases instead of
decreasing; this is a result of the increasing alcohol-membrane solubility as
a consequence of the decreasing polar character of the alcohols.
It has been known for some time that the permeability of nonelectrolytes
through membranes increases as the membrane solubility of the penetrating
molecule increases. More precisely it is the solubility of the penetrating
molecule within the membrane relative to the solubility in the solvent (i.e.,
the membrane partition coefficient, Km) which directly influences the permeability.
The actual partition coefficients of lipophilic membranes are usually estimated
by approximating their lipophilic solubility with olive oil-water partition
coefficients. Aqueous membrane partition coefficients Km have been measured
for the alcohols and are compared with the olive-oil water values K0 in Table
13. It is apparent that there are large differences between the two partition
coefficients. The olive oil-water coefficient is a poor approximation to Km
except in a narrow range near a value of K0 = Km = 10.0. The deviation near
the origin stems from the fact that Km must be greater than 0.6, the lowest
conceivable weight fraction of water in the tissue, while K0 values for water and
the very polar alcohols are in the order of 10"^ owing to their low solubility in
olive oil. From the deviation at higher K values it is clear that stratum
corneum is a less potent absorbent for strongly nonpolar molecules than is
olive oil (Scheuplein, 1965).
-44-
-------
TABLE 13. MEMBRANE PERMEABILITY AND PARTITION COEFFICIENTS*
Water
Methanol
Ethanol
Propanol
Butanol
Pentanol
Hexanol
Heptanol
Octanol
If
Km
3.3
1.7
1.7
0.7
1.0
1.2
1.3
1.07
1.04
k V1/3
-£
8.65
5.85
6.64
2.95
4.51
5.72
6.50
5.55
5.60
«n
0.3
0.6
0.6
2.0
2.5
5.0
10.0
30.0
50.0
kp
1.0
1.0
1.0
1.4
2.5
6.0
13.0
32.0
52.0
"
18.02
32.04
46.07
60.09
74.12
88.15
102.2
116.2
130.2
V
18.02
40.05
59.3
74.9
91.5
107.9
124.8
141.3
157.4
"o
0.000
0.008
0.03
0.17
0.5
5.0
11.5
62.0
220.0
* from Scheupleln, 1965
kp « Permeability constant
Km « Sol vent-membrane distribution coefficient
K0 - olive oil-partition coefficient
V • Molecular volume
M - Molecular weight
-45-
-------
Scheuplein et al. (1969) also studied a series of steroids to see how
permeable these larger molecules, some with polyfunctional character, are
through skin. Since steroid molecules have considerably larger molecular
volumes than the linear primary alcohols and usually have several polar groups,
lower diffusion rates are expected. The diffusion constants for the linear
primary alcohols are approximately the same with D = 10~9 cm^ sec"1, thus the
diffusion constants for the steroids are expected to be lower. Similar
s
experimental procedures (as for the linear alcohol series) were used to obtain
permeability constants for 14 common steroids, except that partition coefficients
with amyl caproate and hexadecane were also measured.
The data in Table 14 were computed from the steady state portions of flux
vs. time curves; lag times (T) were extrapolated from the steady state portions
of these "penetrations curves" (see Fig. 5). The concentration gradients used
in the experiments were extremely small as the aqueous donor solutions were
not saturated with steroid. The observed fluxes listed in column 2, Table
15, therefore do not represent maximum obtainable fluxes from water solutions
of the steroids. Since Pick's law is obeyed at the very dilute concentrations
characteristic of saturated aqueous solutions of steroids, maximum obtainable
fluxes may be computed. These are listed in column 3 of Table 15 (Scheuplein
et al. 1969).
Scheuplein et al. (1969) chose the steroids for this study to include as
wide a range in polarity as possible within a certain range of molecular weight.
Only a 25% difference in molecular weight exists between the lowest steroid
(estrone, MW = 270.3) and the highest (hydrocortisone, MW = 360.4). However,
there is approximately a 1000-fold difference in permeability between these two
compounds. The difference in kp must lie in the respective values for Km and
-46-
-------
TABLE 14. PERMEABILITY CONSTANTS AND PARTITION COEFFICIENTS FOR SOME STEROIDS
Steroid
Progesterone
Pregnenolone
Hydroxypregnenol one
Hydroxyprogesterone
Cortexone
Testosterone
Cortexolone
Cortlcosterone
Cortisone
Hydrocortlsone
Aldosterone
Estrone •
Estradfol
Estrlol
iCs
2.0
5.1
2.9
10.0
2.4
10
10
1.7
0.97
1.8
0.74
2.5
2.5
7.0
kp
1500
1500
600
600
450
400
75
60
10
3
3
3600
300
40
D
160
220
155
166
135
195
36.1
39.2
13.1
4.8
4.9
870
72.4
19.3
*m
104
50
43
40
37
23
23
17
8.5
7
6.8
46
46
23
*ac
56
52
49
46
30
16
11.2
6.8
1.52
1.30
80
66
1.64
Khex
17.0
4.2
1.6
2.5
3.0
2.6
0.1
0.024
0.28
0.009
3.0
0.63
0.23
From Scheupleln et al. (1969)
AC « Initial donor concentration 1n moles/cc x 10"9
kn « Permeability constant 1n cm hr"1 x 10"6 (e.g. estrone kn • 3600 cm hr'1 x
P 10-6j P
D » Diffusion constant 1n crn^ see"* x lO"1^
Km = Stratum corneum/water partition coefficient
Kac * A"1*1 caproate/water partition coefficient
Knex > Hexadecane/water partition coefficient
\
-47-
-------
-------
TABLE 15. STEROID FLUXES
The second and third columns are the observed (J.exp) and the maximum
obtainable fluxes (J.max) with aqueous steroid solutions. In the last column
are calculated values from 1n vivo penetration measurements where aqueous
solutions were not used.
Steroid
Progesterone
Pregnenolone
Hydroxypregnanol one
Hy d roxyp rogeste rone
Cortexone
Testosterone
Cortexolone
Corflcosterone
Cortisone
Hydrocorflsone
Aldosterone
Estrone
Estradlol
Estrlol
Js (exp)
30
51.3
17
60
10.9
40
7.5
1.04
0.097
0.055
0.022
J« (max) J« (In vivo
430
810
555
171
1350
349 150
[433]
287
65
23 7.0 - 20.0
[75]
173
13.2
58.4
From Scheuplein et al. (1969), and references therein.
Js (exp) » Flux observed experimentally moles cm^hr"1 x 10"13.
Js (max) • Predicted flux for saturated aqueous steroid solutions.
Js (in vivo) = Values obtained from the literature.
[ ] » Indicates estimates of solubility used for calculation.
-49-
-------
Dm. As shown in Table 14 the membrane-water partition coefficient Km changes
only 15-fold through the group of steroids in comparison to the approximately
1000-fold change in kn. Thus, it is apparent that the principal determining
factor which decreases the permeability of the steroids is the decrease in the
diffusion constant Dm. Since the molecular volume of the steroids is about 3-4
times that of an average small molecule such as pentanol, we expect a decrease
in Dm from this factor alone arising from the increased degree of chemical
interaction between the larger steroid molecule and the lipid-protein-H20 matrix
within the stratum corneum membrane. Introducing additional polar groups into
the molecule lowers the diffusion constant still further. Because of this
change in Dm within the series of steroids one cannot expect a proportionality
between permeability constant and partition coefficient. Reference to Table 14
shows that there is no systematic relationship between k« and Km values.
Partition coefficients for the steroids between an ester, amyl caproate,
and water and between an alkane, hexadecane, and water were also measured.
Comparison of these data shows that the stratum corneum is much more similar in
its solvating properties to partially polar amyl caproate than to non-polar
hexadecane. Although there doesn't appear to be any direct proportionality
between Km and kp within this group of steroids, there does exist the possibility
of an useful correlation between the solubility of a steroid in a particular
solvent and its permeability. From the data in Table 14, Scheuplein et al.
(1969) showed that one may expect a steroid with an amyl caproate-water
distribution coefficient from 20-50 to have a permeability from 400-1000 x 10~6
cm hr'l. The rest of the steroids may be broadly grouped as:
-50-
-------
kp x IP"6 cm hr"1
1-2 1-10
2-10 10-70
10 - 20 70 - 400
20 - 50 400 - 1000
A similar tabulation could be made for Khex and for Km but the latter values
cannot be obtained as accurately and hexadecane does not appear to serve well
as an approximation for the solvating property of hydrated stratum corneum.
6.4 PREDICTION OF PERMEABILITY FOR SOME ALCOHOLS AND STEROIDS
Lien and Tong (1973) have tried to correlate percutaneous absorption data
for different types of drugs or organic compounds with a number of physiochemical
constants by using a computerized multiple regression analysis program. By
comparing the equations of different sets of data the authors sought to obtain
useful quantitative guidelines for predicting percutaneous absorption.
The authors took the absorption data and most of the chemical constants
from the literature while octanol-water partition coefficients were either
experimentally determined or calculated. The equation used in the computerized
o
regression analysis program is log BR = - kj (log P)£ + ^2 log P + kj
(electronic term) + k4 (steric) + k$ where BR is the biological response
expressed as the molar concentration (C) of the drug absorbed, 1/C for a standard
response (such as erthema or vasoconstriction), or the permeability constant
(kp). The coefficients kj through ks are obtained using the method of
least squares. For some cases the solubility in water appeared to be important;
therefore a log S term was also included in the analysis. The addition of
other steric or electronic terms, such as molar refraction, Taft's polar sub-
-51-
-------
stitute constant, and molecular weight, significantly improved the correlation
in some cases.
For the alcohols and steroids, the variation in permeability constants is
primarily due to the difference in lipophilic character. By comparing an
equation derived from the permeability experiment on aliphatic alcohols through
human epidermis with an equation from similar experiments on steroids, i.e.,
(alcohols): log kp = 0.934 log Km - 2.891 (n = 8, r = 0.986, s = 0.121)
(steroids): log kp = 2.626 log Km -7.537 (n = 14, r = 0.931, s = 0.377)
we see that the permeability of steroids through the epidermis is much more
dependent on the partition coefficient into stratum corneum (Km) as compared
with the alcohols. The lower intercept of the equation for steroids as compared
to the equation for alcohols shows the stronger hydrophobic interactions between
the steroids and the epidermis than between the alcohols and the epidermis (see
Fig. 6). Tables 16 and 17 display the good correlations found for both alcohols
and steroids indicating that this approach enables a chemist to predict the
relative degree of penetrant absorption through skin from the physicochemical
constants of a series of compounds plus the absorption data of a few parent
molecules (Lien and Tong, 1973).
-52-
-------
Figure 6. Dependence of the permeability constant (log kp) on
the stratum corneum/water partition coefficient (log Km)
Equation A, log kp«0,934 log Km-2.891 is derived from
the data of a alcohols absorbed through human epidermis;
Equation B, log kp»2.626 log Km-7.537 is derived from
the data of alcohols absorbed through human epidermis
(From Lien and Tong, 1973).
-53-
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TABLE 16. IN VITRO PERMEABILITY OF ALIPHATIC ALCOHOLS THROUGH HUMAN EPIDERMIS*
Compound
Hater
Methanol
Ethanol
n-Propanol
n-Butanol
n-Pentanol
n-Hexanol
n-Heptanol
n-Octanol
•From Lien and
Log K0
• • •
-2.10
-1.52
-0.77
-0.30
0.70
1.06
1.79
2.34
long 1973
Log PO
• • •
-0.66a
-0.168
0.34b
0.84b
1.34b
1.84°
2.34b
2.84b
Log Km
-0.52
-0.22
-0.22
0.30
0.40
0.70
1.00
1.48
1.70
Obsd.
-3.00
-3.00
-3.00
-2.85
-2.60
-2.22
-1.89
-1.49
-1.28
Calcd.
• • •
-3.24
-2.97
-2.70
-2.43
-2.16
-1,88
-1.61
-1.34
D1ff.
• • •
0.24
-0.03
-0.15
-0.17
-0.06
-0.01
0.12
0.06
•Experimental determined value from Leo et al., 1971.
Calculated value
P0 « Octanol/water partition coefficient.
K0 « Olive oil/water partition coefficient from Scheupleln (1965)
KID • Stratum corneum/water partition coefficient from Scheupleln (1965)
kp » Permeability constant: observed from Scheupleln (1965), calculated from
log kp - 0.544 log P - 2.884 n - 8. r - .979. s - 0.150
-54-
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TABLE 17a. IN VITRO PERMEABILITY OF STEROIDS THROUGH HUMAN EPIDERMIS*
Steroid Log Knex Log Kac Log Km Log Pe
Progesterone
Pregnenolone
Hydroxypregnenolone
Hydroxyprogesterone
Cortexone
Testosterone
Cortexolone
Cortlcosterone
Cortisone
Hydrocortlsone
Aldosterone
Estrone
Estradlol
Estrlol
1.23
0.62
0.20
0.40
0.48
0.42
-1.00
-1.62
-0.55
-2.05
. . .
0.48
-0.20
-0.64
1.75
1.72
1.69
1.66
1.48
1.20
1.05
0.83
0.18
0.11
. . .
1.90
1.82
0.21
2.02
1.70
1.63
1.60
1.59
1.36
1.36
1.23
0.93
0.85
0.83
1.66
1.66
1.36
2.78
(2.82)
(2.24)
(2.17)
1.72
1.94
(1.11)
0.66
0.15
0.20
•FromLlen and Tong, 1973
Knex « Hexadecane/water partition coefficient from Scheuplein et al., 1969
Kac » amyl caproate/water partition coefficient from Scheuplein et al., 1969
KID » stratum corneum/water partition coefficient from Scheuplein et al., 1969
Pe * ether/water partition coefficient: experimental values without parentheses;
values 1n parentheses were calculated from Flynn, 1971.
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TABLE 17b. IN VITRO PERMEABILITY OF STEROIDS THROUGH HUMAN EPIDERMIS*
Steroid Log K (cm/hr) Log D (cn^/sec x IP"13)
Obsd.° Ca1td.b D1ff. "flbsd.3 Calcd.*- D1ff.
Progesterone
Pregnenolone
Hydroxypregnenol one
Hydroxyprogesterone
Cortexone
Testosterone
Cortexol one
Cortlcosterone
Cortisone
Hydrocortlsone
A1 dosterone
Estrone
Estradlol
Estrlol
-2.82
-2.82
-3.22
-3.22
-3.35
-3.40
-4.12
-4.22
-5.00
-5.52
-5.52
-2.44
-3.52
-4.40
-2.23
-3.07
-3.26
-3.34
-3.36
-3.97
-3.97
-4.31
-5.10
-5.31
-5.36
-3.18
-3.18
-3.97
-0.59
0.25
0.04
0.12
0.01
0.57
-0.15
0.09
0.10
-0.21
-0.16
0.74
-0.34
-0.43
2.20
2.34
2.19
2.22
2.13
2.29
1.56
1.59
1.12
0.68
2.47
2.49
2.18
2.14
1.91
2.02
1.59
1.35
1.08
1.11
-0.27
-0.15
0.01
0.08
0.22
0.27
-0.03
0.24
0.04
-0.43
8 From Scheupleln et al. 1969
b Calculated from log kp - 2.626 Kra - 7.537 (n - 14, r - 0.931. s - 0.377)
c Calculated from log D (cm2/sec x 10'13) - - 0.221 (log PJ2 + 1.170 log Pe + 0.734
(n « 10, r - 0.961, s • 0.180)
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CHAPTER 7 - DERMAL ABSORPTION IN EXPOSURE ASSESSMENTS
7.1 CURRENT AGENCY PRACTICE
As stated in the introduction of this report the current Agency policy is
to use a value of 100 percent to represent the dermal absorption of a penetrant
unless a lower value can be supported by scientific studies. For most compounds
this is a large overestimation of the actual dermal absorption rate or percent
fraction of penetration. In Table 4 there are three compounds, hippuric acid,
nicotinic acid, and thiourea, that have a total dermal absorption of less than
1 percent of the applied dose after 5 days; the predicted absorbed dose of
these three compounds would be two orders of magnitude high if their dermal
absorption was assumed to be 100 percent. On the other hand, compounds like
carbaryl, caffeine, dinitrochlorobenzene, and DMSO are much more thorough
penetrants and their absorption could be fairly well approximated by using 100
percent. From Table 5 it appears that the majority of the pesticides are
close to 10 percent for their penetration value.
«
There are many other parameters that go into calculating dermal exposure and
intake besides the dermal absorption factor. In addition, one needs to know
the area of exposed skin, the concentration of the penetrant, the duration of
the exposure per each event, and the frequency of events expressed in number of
exposures per some unit time (usually per year). The Exposure Evaluation
Division of OTS in conjuncion with Versar, Inc., developed a general scheme
for calculating the dermal intake of humans (Freed, et al. 1983). One scenario
is that of a thin film of penetrant on the skin. For this finite mass situa-
tion the exposure is calculated by multiplying (concentration) x (skin area) x
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(frequency) times the thickness of the film layer. The dermal intake is then
calculated by multiplying the exposure times the dermal absorption factor
expressed as a percent or as %/hr times the duration. Another scenario is that
of an excess amount of penetrant on the skin. In this case, the thickness of
the penetrant layer is not calculated; steady-state kinetics are assumed and
the dermal intake is calculated by multiplying (skin area) x (concentration) x
(frequency) x (duration) x. (permeability constant, kp).
Freed et al. (1983) give a sample calculation in their document showing
how the parameters above are derived for the case of ambient human exposure to
penetrants in water from swimming. Information on the frequency and duration
of outdoor swimming was found in a report by the Bureau of Outdoor Recreation
which was based on a survey of 11,000 people. For this group, 34 percent swam
in rivers, lakes, and oceans, the average frequency of swimming was 7 days per
year, and the average duration was 2.6 hours per day yielding a periodicity of
18.2 hours/year. The availability for dermal exposure in this example was
assumed to equal the total amount of human skin surface area which is 17,000
cm2 for the average adult and 7,700 cm2 for the average child (1-10 yrs). The
ambient aqueous concentration of a penetrant can be determined by using
monitoring data and/or by modeling. Multiplying (skin area) x (cone.) x
(frequency) x (duration) times the absorption rate for the specific penetrant
will give the dermal intake.
In another study, Brown et al. (1984) looked at the role of skin absorption
as a route of exposure for volatile organic compounds (VOCs) in drinking water.
They estimated absorption levels for an adult taking a 15-minute bath and
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drinking two liters of water, an infant bathed 15 minutes and fed one liter of
water, and a 48-pound child swimming one hour and drinking a liter of water.
Concentrations of each VOC were varied from 0.005 mg/1 of water to 0.5 mg/1.
The authors found that for the swimmer, a minimum of 83 percent and possibly 91
percent of the chemicals entering the body came through the skin. An interesting
result is that the highest doses resulted from water with the smallest concentration
of the penetrant. Scheuplein and Blank (1973) have also shown the permeation
rates are actually increased with dilute solutions as compared to pure liquids.
They illustrate this with the example of hexanol: liquid hexanol (8.2 M) is
approximately 150 times more concentrated than saturated aqueous hexanol (0.055
M), yet the permeation rate of aqueous hexanol, far from being 150 times less
than the pure liquid, is almost twice as great. This apparently anomalous
behavior is attributed to the compaction and dehydration of the stratum corneum
when in contact with pure liquids making it less porous, as well as to the
distribution factors of changing gradients and partition coefficients understood
in Pick's Law.
7.2 EXAMPLES OF DERMAL INTAKE CALCULATIONS
7.2.1 Dermal Exposure from Swimming
There are several specific penetrant examples of how to calculate dermal
absorption of a penetrant in swimming water. Scow et al. (1979) prepared a
document for the Office of Water Planning and Standards in which the exposure
levels for heptachlor and chlordane were calculated. The water flux (J) through
the skin is taken to be between 0.2 to 0.5 mg/cm2-hr while the flux of the
solute (penetrant) is estimated by multiplying the water flux by the weight
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fraction of penetrant in the water. At a concentration of 1 ppb, the flux of
penetrant would be 0.2 x 10'9 to 0.5 x 10'9 mg/cm2-hr. Using a representative
body surface area of 1.8 x 10* cm2, if one swam for 4 hours in any given day in
water containing 1 ppb chlordane, then from 0.013 to 0.036 ug chlordane might
be adsorbed.
Beech (1980) calculated the amount of chloroform absorbed over a 3 hour
period by a 6 year-old boy swimming in water containing 500 ug chloroform per
/
liter. The average 6 year-old is assumed to weigh 21.9 kg and have a surface
area of 0.88 x 10* cm2. The permeability constant (kp) for chloroform in
aqueous solution was assigned the value of 125 x 10~3 cm/hour. The flux through
the skin per hour is: 125 x 10~^ cm/hour x 500 ug/liter x 1 liter/1000 cnr* x
1000 mg/1 ug = 62.5 x 10'6 mg/cm2 hour.
The total dermal intake is then: 3 hr x 62.5 x 10'6 mg/cm2 hr x 0.88 x 104 cm2
= 1.65 mg chloroform.
7.2.2 Pesticide Application
Another area of potentially significant dermal exposure is that of pesticide
application and worker reentry to areas where crops have been sprayed. Maddy
et al. (1983) developed exposure monitoring techniques designed to investigate
the factors influencing pesticide exposure to workers during the application
process. The authors conducted dermal exposure monitoring of workers involved
in the application of parathion, meinphos, nitrofen, DEF/Folex, and chloroben-
zilate. Exposures of mixers/loaders, ground applicators, mixer/loader/ground
applicators (workers performing all three activities during a single application),
aerial applicators, and flaggers were determined in a total of 102 individual
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exposure situations. California regulations require use of clean outer clothing
to reduce the potential dermal exposure of workers to pesticides by decreasing
the area of bare skin available for contact with the chemicals. Rubber or
some other type of waterproof gloves were worn by all workers except the aerial
applicators and flaggers.
Hand exposure was determined by rinsing the hands in a predetermined
solvent containing either water, soap and water, ethanol, or a combination of
the three. Hands were rinsed prior to and upon completion of the applications.
Exposures to the hands, face, and neck were estimated by placing small patches
on the upper collar of the coveralls in the front and back, or, in some cases,
placing patches directly on the face. Values were extrapolated to the entire
surface areas of these body parts. Potential exposure to skin protected by
coveralls was also measured with patches.
Table 18 summarizes the average percentage of total dermal exposure found
on various regions by individual chemical and job activity. The results show
that estimated exposure to protected body areas represented only 23.3 percent
of the total dermal exposure. Hand exposure exceeded exposure to all other
areas; workers who wore waterproof gloves still experienced hand exposure
representing 40.9 percent of their total dermal exposure. This result indicates
why the hands are not considered to be a protected area even though waterproof
gloves were usually worn. The author's possible explanations for the relative
ineffectiveness of the gloves include (1) contamination of the inside material
of the gloves, (2) removal of gloves during mechanical adjustments to the
application equipment, and (3) the handling of the outside of contaminated
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gloves while putting them on or taking them off.
Maddy et al. (1983) conclude that more attention should be given to the
monitoring of the hands, head, face, and neck, and less attention to monitoring
protected areas. They also suggest that since hand exposure was responsible
for more than 42 percent of the total dermal exposure, estimates of total
dermal exposure could be derived by multiplying the hand exposure by a factor
of 2.5.
7.2.3 Treated Field Reentry
A second example of dermal intake resulting from pesticide application is
the worker reentry study of Popendorf et al. (1979). Five peach orchards were
harvested each for three days at decreasing post-application intervals. Both
aerosol and dermal exposure estimates were made for the organophosphate pesticide
Zolone plus its metabolite Zoloxon. Low levels of Guthion (also an organophosphate)
were present in some of the orchards from prior applications. Aerosol samples
were collected at 1.7 1pm for one to two hours during the workday from near the
breathing zone of each worker. Respiratory doses were calculated from the
airborne concentrations and estimated respiratory volumes of 24 m^ during each
three day work sequence. Dermal doses to pickers were estimated using 4x4
inch gauze patches taped to the skin. A knit nylon glove backed by a pad was
used to monitor hand exposure. At the end of each week, the patches from each
location were pooled for extraction and analysis. The resulting value was
adjusted for exposure time and patch area at each location and then multiplied
by its proportionate body surface area; these were summed to estimate the whole
body dermal dose of each compound for each week (see Table 19). Approximately
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TABLE 18A. RELATIVE CONTRIBUTIONS TO TOTAL DERMAL EXPOSURE OF BODY AREAS
TO PESTICIDES AS STUDIED BY THE CALIFORNIA DEPARTMENT OF FOOD AND AGRICULTURE
Chemical
Parathi on
Mevinphos
TDK
DEF/Folex
Chi orobenz. late
Total
Average Percentage
Number of of Total Dermal
Exposures Exposure Found
Monitored on Hands
3
22
24
32
21
102
23.4
48.0
49.2
46.8
27.1
42.9
Average Percentage
of Total Dermal
Exposure Found on
Head. Face and Neck
67.4
34.6
22.7
23.7
59.4
33.8
Average Percentage
of Total Dermal
Exposure Found on
Hands and Head,
Face and Neck
90.8
82.6
71.9
70.5
86.5
76.*7
Average Percentage
of Total Dermal
Exposure Found on
Protected Area
9.2
17.4
28.1
29.5
13.5
23.3
TABLE 188. RELATIVE CONTRIBUTIONS TO TOTAL DERMAL EXPOSURE OF BODY AREAS BY JOB ACTIVITY
Job Activity
Mixer/Loader.
Ground App'. icator
Mixer/Loader
Aerial Applicator
Ground Appi Icator
Fl agger
Total
Number of
Exposures
Monitored
4
36
18
25
19
102
Average Percentage
of Total Dermal
Exposure Found
on Hands
18.1
50.7
54.6
30.4
38.7
42.9
Average Percentage
of Total Dermal
Exposure Found on
Head, Face and Neck
57.5
22.0
27.4
47.9
38.6
33.8
Average Percentage
of Total Dermal
Exposure Found on
Hands and Head,
Face and Neck
75.6
72.7
82.0
78.3
77.3
76.7
Average Percentage
of Total Dermal
Exposure Found on
Protected Areas
24.4
27.3
18.0
21.7
22.7
23.3
From Maddy et al. 1983
-------
98-99% of the total doses thus calculated were attributed to the dermal rather
than respiratory route. These calculations do not account for possible variations
in the rate of chemical absorption at different skin locations.
7.2.4 Hypothetical Dermal Intake of PCBs
Versar, Inc. (1983a) in conjunction with the Exposure Evaluation Division
of OTS has estimated maximum probable dermal intake to PCBs for several
scenarios. The first example deals with dermal exposures in the occupational
environment. Annual dermal intake of PCBs resulting from use of a specific
chemical or product (assuming that no protective clothing or equipment is worn)
can be estimated from
Amount PCBs PCBs available Frequency of
absorbed = for absorption x exposure x Absorption
(mg/yr) (mg/event) (events/yr) (%)
where, for liquids, PCBs available for absorption =TxLxCxS
- T (liquid film thickness) is assumed to be 0.0018 cm. This is the
average of the measured film thicknesses of five solutions on the skin
after immersion of hands into the solution followed by a partial wipe
with a rag: mineral oil, cooking oil, bath oil, 50% bath oil/50% water,
and water (Versar, 1983b)
- L (density of liquid) is assumed to be 1.6 x ID-* mg/cm^.
- C is the PCB concentration in the liquid (kg/kg)
- S (skin area exposed) is assumed to be the entire surface area of both
hands which is taken to be 870 cm2.
and, for dusts, PCBs available for absorption = M x C x S
- M is the maximum mass of a dust that can adhere to one cm2 of skin
which is 2.77 mg/cm2 (Versar, 1982)
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TABLE 19. WORKER REENTRY EXPOSURE
INTEGRATED DERMAL DOSE TO EACH LOCATION, ALL VALUES IN mg
Zolone
Week
Hands
Forearms
Upper arms
Head
Neck
Shoulders
Chest
Back
Hips
Th1 ghs
Calves
Feet
Total without hands
Overall
Hands
Forearms
Upper arms
Head
Neck
Shoulders
Chest
Back
H1ps
Thighs
Cal ves
Feet
Total without hands
Overall
95.0
9.8
4.3
7.3
1.6
0.4
1.0
0.6
0.2
0.7
1.4
0.2
27.3
122.3
Gut Mont
20.8
2.82
0.57
1.38
0.30
0.06
0.24
0.06
0.04
0.14
< 0.02
5.62
26.4
116.1
17.6
14.5
11.6
2.4
1.7
1.8
1.7
0.2
1.0
0.3
0.1
52.9
169.0
Zoloxon
2.17
0.28
0.17
0.17
0.03
0.04
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
0.69
2.86
84.2
12.2
13.4
23.0
2.4
1.4
1.1
2.0
0.6
2.3
0.5
0.1
58.9
143.1
Zoloxon
1.82
0.29
0.35
0.68
0.05
0.04
0.03
0.05
0.02
0.06
1.56
3.38
158.0
12.3
13.1
24.1
2.6
. 0.7
2.5
3.8
1.3
5.1
7.9
0.9
74.3
232.1
Zoloxon
1.39
0.08
0.11
0.22
0.04
0.01
0.03
0.10
0.02
0.07
0.01
0.01
0.74
2.13
tA total dose of 2.6 mg Zoloxon (2.5 to the hands) in week 1 Is not listed.
From Popendorf et al. 1979-
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- C is the PCB concentration in dust (kg/kg)
- Sis the skin area exposed per event (cm^/event)
A sample calculation for the exposure scenario of loading/unloading a
liquid containing 50 mg/kg PCB with a frequency of 96 times/yr (with absorption
assumed to be 100%) is:
Amount PCBs = TxLxCxSx96 events/yr. x 100%
absorbed (mg/yr)
= 0.0018cm x 1.6 x 103 mg/cm3 x 50 mg/kg x 870cmz x 96
events/yr
= 12 mg/yr
The second example is the calculation of the maximum probable individual
dermal intake to PCBs resulting from routine use of general household cleaners
containing incidental PCBs. The dermal intake calculations apply to typical use
of a solid household detergent designed to be mixed with water. The following
assumptions were made:
- 96 gm of detergent is used per cleaning job and detergent is mixed with
1 gallon (3.785 1) of water.
- Detergent contains 25 percent by weight of the PCB - contaminated
chemical.
- A 0.0024cm thick film of the cleaning solution remains on the hand and
part of the forearm, covering a skin surface area of 500 cm2, after
each immersion. This is the measured film thickness for a solution of
50% water/50% bath oil retained on the skin after immersion of hands
into the solution followed by a partial wipe with a rag (Versar, 1983b).
Therefore, 0.0024 cm x 500 cm2 =1.2 cm3 (ml) of solution remain on the
skin.
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- Detergent is used once a week for a total annual frequency of 52 times
a year.
- One hand is immersed 20 times in the bucket during weekly cleaning
sessions and no gloves are worn.
- All PCBs in the solution remaining on the skin after each immersion are
available for dermal absorption.
The PCBs available for absorption (mg/event) =AxBxCxD
A = PCB in constituents (mg/kg)
B = Aqueous dilution of detergent; quantity detergent per volume of water
(kg/ml)
C = Weight fraction of PCB - contaminated constituent
D = Volume of solution on skin per immersion (mg/event)
Frequency = 20 immersions per week x 52 weeks per year = 1,040 events/yr
Absorption = 100 percent
A sample calculation for PCB concentration of 50 mg/kg is :
Amount PCBs =AxBxCxDx Frequency x Absorption
absorbed
= 50 mg x .096 kg x .25 x 1.2 ml_ x 1,040 events
kg 3.785 x 103 ml event yr
= .4 mg/yr
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CHAPTER 8 - ANALYSIS/FUTURE RESEARCH NEEDS
The most direct method of obtaining percutaneous absorption data for a
penetrant is to conduct a human in vivo study. We have seen, however, that
there are many problems with this direct approach such as toxicity concerns,
having to monitor urine output for 21 days for slow penetrants, and the 5-fold
variation possible between subjects. Kligman (1983), in a recent review of
percutaneous absorption, states in comparing in vivo to in vitro results that
in vitro data are more credible on technical grounds alone as the methods are
more precise, involve less experimental error, and the variables are under much
greater control. From the limited number of in vitro results presented in this
review, it appears that the in vitro studies are more reproducible than j_n_
vivo studies. Because of the problems associated with in vivo studies, it is
'suggested that research be focused on compiling absorption data with in vitro
studies using 1 type of skin which then can be compared to the permeability of
skin from all other parts of the body (i.e., adominal skin could be tested and
ratios such as found in Table 2 could be used to determine absorption at other
sites like the forearm or palm). The in vitro results could then represent
actual penetration in vivo; however, it may be prudent to include a "safety
factor" of 5 (or other number derived from more extensive studies) to account
for the variation between subjects as noted in the in vivo studies.
The previous chapters have shown the importance of the stratum corneum/water
partition coefficient, Km. Unfortunately, there is only a limited data set
of measured Km values, thus its utility as a predictor of dermal absorption
is largely unknown. Furthermore, the accuracy of measured Km values is questioned
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by Scheuplein et al. (1969) who state that partition coefficients like Kamyi
caproate (^ac) or ^hexane can &e obtained more accurately than Km.
It is suggested that a program to measure Km for a large number of
chemicals and covering a variety of functional groups be established. In this
manner we could determine how well in vitro Km measurements represent the
actual in vivo partition between the penetrant and skin. Also, we would like
to know if Km can be calculated from other partition expressions like Kac
°r Kolive oil* For example, a regression analysis was done for the
b
linear alcohol series giving Km = aK0iive 01-] with r .= .97, a = 3.748,
and b = .448 showing a good correlation for this series. However, a similar
regression analysis for the steroids (the only other chemicals with measured
Km values) had a poorer fit with r = .78, a = 8.742, and b = .4288, possibly
due to inaccuracies in the measurement of Km.
The second parameter of importance needed to calculate permeability
is the diffusivity (D). We have see that for the linear primary alcohols
D is relatively constant (D = 10"^ cm^ sec"*), while for the series of
steriods, D is not constant, varying by approximately 200-fold. This
variation can be explained in part by changes in molecular volume and polar
functional groups. As with Km, it is suggested that D values be tabulated
for a number of chemicals covering a variety of functional groups so that
the dynamic range of D can be determined. It may be possible to approximate
D for an unknown chemical by comparison to chemicals with measured D values.
The compilation of Km and D values for a number of representative
chemicals will facilitate the estimation of kp for unknown chemicals. In
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order to promote consistency in the compilation of these values, it would
be prudent to decide what tissue (man or animal) to use in diffusion
cells and partition measurements of Km plus from what body site. If
.animals are to be used either for in vivo or in vitro studies, it would
be most helpful for comparative purposes to decide what animal species is
closest to man and use only the species chose. The rhesus monkey and
miniature swine are closest in absorption properties to man, but the rat
may be the species of choice due to economy and ease of handling. Also,
it is suggested that clearcut procedures be developed to measure Km
and D values for penetrants of low water solubility. In vitro diffusion
cell measurements are currently restricted to penetrants that are at
least partially water solubile, although some effort is underway to use
sol vent/water mixtures for the receptor bath.
Values for Km and D are required to calculate the permeability when
there is an excess of penetrant and steady-state kinetics are followed.
However, there are other scenarios, such as when a finite amount of
penetrant (i.e., thickness of penetrant layer is smaller than the thickness
of the stratum corneum) is applied and the penetrant is fast; Fickian
diffusion kinetics may not be followed for this case. It would be useful
to establish expressions for kinetic flux for all possible scenarios such
as: finite amount of a fast penetrant, finite amount of a slow penetrant,
excess of a penetrant where the diffusion through the stratum corneum is
not rate limiting, buildup of penetrant in blood so that C0 7* 0, etc. In
this manner, once the appropriate scenario is determined from the exposure
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situation, the proper kinetic expression can be used to determine the flux.
The use of partition systems to predict dermal absorption has been
discussed in the previous chapters. Partition coefficients themselves
have been predicted from structure-activity relationships, number and
kind of functional groups, and by other techniques (Sato and Nakajima 1979,
Hansch et al. 1972 and 1973, Fujita et al. 1964, Leo and Hansch 1971a and
1971b, and Katz and Shaikh 1965). Thus, it may be possible to use the
methods developed to predict partition coefficients to calculate dermal
absorption once the relationship between a partition coefficient and
permeability is established.
We have seen in the previous chapters that there are two approaches
used to represent the amount of penetrant diffusing through the stratum
corneum. The first and most common method is to use the total percent
adsorption calculated or estimated (such as 100% absorption); this method
does not take into account the contact time or the particular kinetics
that may apply depending on the speed of the penetrant, its thickness on
the skin, or if it is bound to the vehicle in some manner. The second
method is to use an absorption rate which then has to be coupled with a
contact time. It would be valuable to review the advantages/disadvantages
of these approaches, particularly in terms of their relative uncertainty.
Since the conservative approach when no data is available for a pene-
trant is to use 100 percent absorption, it may be possible to group
penetrants into a numerical system such as 100% - 10% - 1% - .1% absorption
depending on physical parameters such as Km and D, on partition coefficients,
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or in vitro tests. This grouping of penetrants into a numerical system
based on physical parameters is similar in intent to the grouping of
steroids by their Kac value as is done by Scheuplein et al. (1969)
(see Chapter 6). The use of a numerical system is an "order of magnitude"
approach which may be justified when the uncertainty of all the factors
leading to dermal intake are taken into consideration.
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9. REFERENCES
Anderson, S.L., and Cassidy, J.M. 1973. Variations in physical dimensions and
chemical composition of human stratum corneum. J. Invest. Dermatol. 61:
30-32.
Bartek, M.J., LaBudde, J.A., and Mai bach, H.I. 1972. Skin permeability
in vivo; comparison in rat, rabbit, pig, and man. J. Invest. Dermatol.
58:114-123.
Bartek, M.J., and LaBudde, J.A. 1975. Percutaneous Absorption In Vitro. In:
H.I. Maibach (ed), Animal Models in Dermatology. ChurchhilT~Livingstone,
New York, p. 103.
Beech, J.A. 1980. Estimated worst case trihalomethane body burden of a child
using a swimming pool. Medical Hypotheses. 6:303-307.
Bronaugh, R.L., Stewart, R.F., Congdon, E.R., and Giles, A.L. 1982. Methods
for in vitro percutaneous absorption studies. I. Comparison with in vivo
results"Tox. Applied Pharmacol. 62:474-480.
Brown, H.S., Bishop, D.R., and Rowan, C.A. 1984. The role of skin absorption
as a route of exposure for volatile organic compounds in drinking water.
Am. J. Public Health 74:479-484.
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