3 In Vitro Methods for the Percutaneous Absorption of Pesticides ROBERT L. BRONAUGH Downloaded by COLUMBIA UNIV on March 9, 2013 | http://pubs.acs.org Publication Date: February 25, 1985 | doi: 10.1021/bk-1985-0273.ch003
Division of Toxicology, Food and Drug Administration, Washington, DC 20204 The primary barrier to the entry of chemicals through skin is the upper layer of the epidermis, the stratum corneum. The passive diffusion through this nonliving tissue forms the rationale for using in vitro diffusion cell systems to measure skin absorption. This procedure has been validated in our laboratory and by others by comparison with in vivo results. Compounds that lack water solubility can present difficulties in the standard in vitro method using an aqueous receptor solution beneath the skin. This fluid must be modified so that it is sufficiently lipophilic to allow partitioning of the test compound from skin into the diffusion cell receptor. We have used a nonionic surfactant, oleth 20, to increase the absorption of this type of compound without apparent damage to the skin. The permeation (percent of applied dose) of the hydrophobic pesticide DDT through human skin was 1.1% with saline in the receptor and4.5%using 6% oleth 20. These results with a model compound illustrate the need to consider the solubility properties of a compound in a percutaneous absorption study. The results of a number of comparative studies (1-4) indicate that the percutaneous absorption of chemicals can be measured accurately by i n v i t r o techniques. When discrepancies have been found between i n vivo and i n v i t r o values, they are often the result of experimental differences. Errors i n the measurement of percutaneous absorption are possible with i n vivo techniques because of the d i f f i c u l t i e s i n observing absorption through skin by measuring amounts of compound excreted i n the urine and feces. As with any technique, i n v i t r o absorption measurements are meaningful only when done with sound methodology and an awareness of the potential problems and l i m i t a t i o n s . Probably the greatest problem i s in the measurement of absorption of compounds that are insoluble i n water. These compounds w i l l not l i k e l y p a r t i t i o n T h i s chapter not subject to U . S . copyright. P u b l i s h e d 1985, A m e r i c a n C h e m i c a l Society
In Dermal Exposure Related to Pesticide Use; Honeycutt, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.
34
DERMAL EXPOSURE RELATED TO PESTICIDE USE
freely into the aqueous d i f f u s i o n c e l l receptor f l u i d routinely used. A number of pesticides are subject to t h i s p i t f a l l because of t h e i r poor water s o l u b i l i t y . Methods for i n v i t r o absorption measurements w i l l be presented, including techniques for studying hydrophobic compounds. The absorption of two pesticides with limited water s o l u b i l i t y (DDT and parathion) has been measured through excised human skin.
Downloaded by COLUMBIA UNIV on March 9, 2013 | http://pubs.acs.org Publication Date: February 25, 1985 | doi: 10.1021/bk-1985-0273.ch003
Diffusion C e l l Methodology In v i t r o percutaneous absorption experiments are sometimes conducted with the p r i n c i p l e s of d i f f u s i o n rigorously i n mind. An aqueous solution of the penetrant i s applied on one side of the skin and i t s d i f f u s i o n followed into i d e n t i c a l aqueous f l u i d on the other side of the membrane. S t i r r i n g devices are used on both sides of the membrane (2-chambered c e l l technique) (5). Currently, skin absorption i s frequently measured after application of the test substance i n a small amount of vehicle to the surface of the skin. Permeation i s followed by removal of aliquots from the s t i r r e d solution i n the receptor below the skin (1-chamber s t a t i c c e l l ) (jL, 6). This procedure more closely simulates the i n vivo situation because the skin i s exposed to ambient conditions and i s not excessively hydrated as i n the 2-chamber procedure. A further refinement i n absorption measurements i s the use of a perfusion f l u i d below the skin to take up penetrating substances (7). Sampling i s f a c i l i t a t e d by c o l l e c t i o n of the effluent i n v i a l s i n a f r a c t i o n c o l l e c t o r . The flow-through d i f f u s i o n c e l l i s a 1-chambered c e l l with many advantages. Automatic sampling allows a savings of labor i n addition to around-the-clock determination of the absorption p r o f i l e . Sink conditions are e a s i l y maintained even in absorption studies where large amounts of nonradiolabeled material are applied. A more physiological assessment of percutaneous absorption might be obtained for compounds of limited water s o l u b i l i t y since the c e l l s may serve to mimic the effect of blood flow through the skin by taking up and carrying away the absorbed compounds. The volume of the receptor i s c r i t i c a l . To completely remove the material that has penetrated into the receptor i n a given time, the volume of f l u i d pumped into the c e l l must be many times the volume of the receptor. This requires a small receptor volume so that the volume of effluent from the c e l l i s manageable. The skin permeability of a hydrophobic fragrance material (cinnamyl anthranilate) was examined using 1-chambered s t a t i c d i f f u s i o n c e l l techniques (8). In v i t r o percutaneous absorption measurements with rat skin using a normal saline receptor solution resulted i n values that were much lower than the corresponding i n vivo data for the compound. This difference i s shown i n the time course for the absorption of cinnamyl anthranilate (Figure 1). The e f f e c t of different receptor f l u i d s on cinnamyl anthranilate absorption was determined by comparing values obtained at the end of 5 days (Table I ) . Only 12.7% of the compound absorbed i n vivo i n the 5-day period was obtained i n a corresponding i n v i t r o experiment using normal saline receptor f l u i d and s p l i t - t h i c k n e s s rat skin. A 1.5> solution of the
In Dermal Exposure Related to Pesticide Use; Honeycutt, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.
Downloaded by COLUMBIA UNIV on March 9, 2013 | http://pubs.acs.org Publication Date: February 25, 1985 | doi: 10.1021/bk-1985-0273.ch003
3.
BRONAUGH
In
Vitro Methods
for
Percutaneous
35
Absorption
nonionic surfactant oleth 20 (Volpo 20, Croda Inc., New York, NY) enhanced by 3-fold the skin permeability to cinnamyl anthranilate; the cortisone permeation determined simultaneously i n the dual-label experiment was not altered. When full-thickness rat skin was used, no increase i n cinnamyl anthranilate absorption was obtained with the oleth 20, indicating the importance of removing most of the dermal t i s s u e . The optimal concentration of oleth 20 was 6%. A 5-fold increase i n cinnamyl anthranilate permeation was obtained without a l t e r i n g the permeation of the cortisone c o n t r o l . Rabbit serum and bovine serum albumin had no effect on the i n t e g r i t y of the barrier, but they were also less e f f e c t i v e i n increasing cinnamyl anthranilate absorption than oleth 20. A methanol-water solution and 6% octoxynol 9 (Triton X-100, Rohm and Haas Co., Philadelphia, PA) were equal to or superior to 6% oleth 20, but s i g n i f i c a n t damage to the skin was indicated by the increased cortisone permeation. A 6% solution of poloxamer 188 i n the receptor resulted i n s l i g h t enhancement of both cinnamyl anthranilate and cortisone permeation. Cinnamyl anthranilate absorption was evaluated i n the flow-through c e l l using saline or a solution of the nonionic surfactant as the receptor f l u i d (7.). With normal saline receptor
Table I. Effect of Diffusion C e l l Conditions on the Absorption of Cinnamyl Anthranilate (I) (Cortisone C o n t r o l ) I, % Applied Dose Absorbed (5 days)
Receptor Fluid
6
Normal saline ( 4 ) 1.5% Oleth 20 ( 4 ) Normal saline (4) 1.5% Oleth 20 (10) 6% Oleth 20 (8) 20% Oleth 20 (8) Rabbit serum (4) 3% Bovine serum albumin (4) 50:50 Methanolwater (4) 1.5* Octoxynol 9 (4) 6% Octoxynol 9 (4) 6% Poloxamer 188 (4) D
Cortisone Permeability Constant Χ 10 (cm/h)
5.0 5.4 5.8 15.5 27.9 18.3 8.8 12.1
+ + + + + + + +
0.3 0.9 0.4 1.2° 1.8° 1.8° 0.6 1.2
7.1 6.1 7.0 9.3 6.8 5.4
27.1
+ 2.0°
17.2
C
C
17.9 + 1.1° 38.4 + 2.9 7.3 + 1.3 C
a
5
0.7
3.»
-
+ + + + + +
0.5 0.5 0.9 0.9 0.8 0.2
+ 0.2
C
10.8 + 0.5 14.5 + 1.3 9.8 + 0.6C
a
C
C
Values are the mean + S.E. of the number of determinations i n parentheses. For most experiments, a 350-um section from the surface of whole rat skin was prepared with a dermatome. Compounds were applied to the skin i n a petrolatum vehicle. In vivo absorption of I was 45.6%. Whole skin. S i g n i f i c a n t increase when compared to results from saline (dermatome section) by one-tailed Student's t - t e s t , Ρ
