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Quantification of Sodium Lauryl Sulfate Penetration into the Skin and Underlying Tissues after Topical Application—Pharmacological and Toxicological Implications
Quantification of Sodium Lauryl Sulfate Penetration into the Skin and Underlying Tissues after Topical Application-Pharmacological and Toxicological Implications SUNlTA PATIL, PARMINDER SINGH,
KHALEDSARASOUR,
AND
HOWARD MAIBACH~
Received January 30, 1995, from the Department of Demafohgy, School of Medicine, Surge 110, Universify of California, San Francisco, CA 94143. Accepted for publication July 18, 1995@. ~
Sodium lauryl sulfate (SLS)is known to penetrate skin and cause cutaneous irritation. Some of these effects have been well-defined using bioengineering techniques. In this study, the ability of SLS to penetrate skin was quantified in a hairless rat model. In addition, local deep tissue penetration and systemic exposure to SLS were also evaluated to assess the toxic potential of topically applied SLS. SLS was observed to penetrate directly to a depth of about 5-6 mm below the applied site. Systemic redistribution was predominantly responsible in determining concentrations of SLS in tissues deeper than 5-6 mm. Epidermal concentrations of SLS after application of 1% (34 mM) aqueous SLS solution for 24 h were above the threshold levels which are known to evoke typical skin irritation responses. Deeper underlying tissues including dermis, subcutaneous, and muscle may also be exposed to high levels of SLS. Topically applied SLS was also observed in blood and contralateral tissues but the observed levels were not likely to elicit any systemic side effects at these doses. Traces of SLS were observed in tissues 7 days after single 24 h application of SLS, which supports the prolonged barrier disruption data generated using conventional bioengineering techniques. Cumulative treatment of SLS significantly increased the concentration of this compound in the underlying epidermis. The known preferential affinity of SLS for skin lipids and proteins was further confirmed by both in vitro and in vivo results. However, in vitro studies failed to predict the underlying tissue toxicity of SLS under the patch site when compared to the in vivo results. Such quantitative pharmacokinetic-pharmacodynamic correlations may be useful predictors for effective use of surfactants as penetration enhancers in cosmetic, pharmaceutical, and industrial applications.
Abstract
Electron microscopy revealed morphological changes in epidermis after SLS treatment.1° In spite of its known irritant potential, the distribution of SLS in skin has not been quantified in vivo. Although most topically applied compounds are cleared by the dermal blood supply, direct deep tissue penetration of many solutes has been detected.'l-I6 The extent, depth and pharmacokinetics of a variety of solutes with diverse physicochemical properties have recently been quantified.11J3The deep tissue penetrant potential of topically applied SLS is unknown. Most of the published studies deal with the local Given l penetration of SLS in the epidermis and d e r m i ~ . ' ~ - ~ that SLS is a common irritant, it becomes imperative to study if deeper tissues below the site of topically applied SLS are also exposed to high levels of SLS. This study quantifies the depth of penetration of topically applied SLS in a hairless rat model by varying the duration and mode (single vs repeated) of SLS application. The plasma concentrations and contralateral tissue concentrations were also determined to evaluate the extent of systemic exposure of topically applied SLS. The data were also interpreted to provide a quantitative estimation of SLS levels likely to elicit an irritant response in skin and underlying tissues. An in vitro test model was also utilized to compare the penetration kinetics of SLS with in uivo skin patch test results.
Materials and Methods
Chemicals and Instrument-[35SlSodium lauryl sulfate was purchased from Amersham Co., tissue solubilizer (Soluene-350 and Insta-flour) for organic samples was obtained from Packard Instrument Co., Meridan CT, and the cocktail (Universol) for inorganic samples from was from ICN. Sodium lauryl sulfate was obtained from Introduction Sigma Chemical Co., St. Louis, MO (purity > 99%). All other reagents used were of analytical grade. A liquid scintillation counter (Packard The surface active nature of sodium lauryl sulfate (SLS) Instrument Co., Model Tricarb-1500) was used to detect the radiomakes it a useful component for a multitude of diverse activity in the samples. applications: (i) component of various consumer products like In Vivo Studies-Animals-Hairless rats (350-400 g) were obshampoos, cosmetics, toothpastes, carpet cleaners, and detertained from the Charles River Laboratory. The animals were housed gents, (ii) as a biological tool to increase intestinal absorption, under standard laboratory conditions (25.0 f 0.5 "C, relative humidity 55-75%) and supplied with normal pellet diet and water ad lib. The as a cytolytic agent, as a virus-dissociating agent in electroexperiments had been approved by the Committee for Animal phoretic studies, and for solubilizing lipid membranes and (iii) Research, University of California, San Francisco. in the electroplating industry and also in many other indusPatch Test Treatment-Standard patches (Large Hilltop Chambers, trial products.' Due to its widespread and inadvertent topical Cincinnati, OH; inner diameter 1.5 cm) were applied on the dorsum exposure, the skin is extensively exposed to SLS. SLS impairs of the anesthetized animals. The patches were secured in place with the skin barrier function by removing intercellular hydrophoa n adhesive tape (3M Transpore Surgical tape) and a n elastic bic lipids leading t o an increase in transepidermal water bandage. The animals were divided into three study groups with each ~ o s s . ~SLS - ~ also binds extensively to intracellular keratin, group receiving one of the following treatments: (a)single application which explains some of its irritant effects like tightness and of 1%aqueous SLS solution (300 pL) for 6 h, (b) single application of roughening of kin.^^^ 1%aqueous SLS solution (300 pL) for 24 h, (c) Two applications of 1%aqueous SLS solution (300 pL) for 24 h each; the time interval Many of the cutaneous side effects associated with topical between the two applications was 1 week. SLS use of SLS have been studied using bioengineering Dermal Penetration and Local Tissue Uptake Studies-After dosing increases transepidermal water loss, induces local vasodilaanesthesia, the animals were allowed to recover and then ~ tation and decreases the water retention properties of ~ k i n . ~ 3 , under placed in separate cages. At predetermined times (6 h or 24 h postdosing) the animals were lightly anesthetized using anesthetic Abstract published in Advance ACS Abstracts, August 15, 1995. ether, and blood was withdrawn from the tail vein into heparinized @
1240 / Journal of Pharmaceutical Sciences Vol. 84, No. 10, October 1995
0022-3549/9%3184-1240$09.00/0
0 1995, American Chemical Sociev and American Pharmaceutical Association
tubes. The blood samples were centrifuged immediately a t 2000 rpm to separate the plasma. A known amount of plasma was placed in the scintillation vials and the scintillant added to assess the SLS activity in the plasma. After withdrawal of the blood, the animals were immediately sacrificed by an overdose of anesthetic ether, the patches were removed carefully, and the test area was swabbed dry three or four times with tissue paper. A portion of the test area was tape stripped (3M Transpore Surgical tape) 15 times to remove the stratum corneum. The scintillant was added to the tape stripped samples and counted for radioactivity. The skin was excised and epidermis and dermis separated by heat splitting at 60 "C in a water bath. The underlying subcutaneous tissue, muscle, deep muscle, and fat layers were dissected surgically. A similar procedure was adopted to dissect the tissues from the contralateral side (i.e. from the corresponding untreated site on the other lateral half of the animal). To avoid radioactivity contamination, surgical instruments were thoroughly cleaned before each tissue excision. Furthermore, wipes were taken from the cleaned instruments and counted for radioactivity to ascertain that there was no contamination from one site to another. The tissues were then weighed, put in the vials containing tissue solubilizer, and kept in a n incubator at 45 "C overnight to dissolve the tissue. Cocktail for tissue samples was added to these vials and the radioactivity counted in the scintillation counter. Background radioactivity was determined for plasma and various tissues in one control animal. In Vitro Studies-Skin preparation-Whole thickness rat skin including the subcutaneous fat layer was excised from euthanized animals and used for the in vitro permeation studies. Skin pieces of approximately 1.5 cm2 were immersed in buffered solution a t 37 "C before mounting them on the diffusion cell. Permeation Studies-Vertical, Marzulli-Bronaugh type flow through diffusion cells (Laboratory Glass Apparatus Inc.) consisting of two water-jacketed cylindrical half-cells, each having a volume of 2.8 mL ~ ~ skin s ~ ~was mounted on and a diameter of 1 cm2, were ~ s e d . The the diffusion cells with the epidermis facing the donor compartment. A constant temperature was maintained in the cells by circulating water a t 37 "C. The fluid in the receptor cells was constantly stirred a t about 300 rpm by Teflon-coated magnetic bars. The donor compartment received 100 pL of 1% SLS solution containing the radiolabeled SLS (1pCi/cell). The receiver compartment was continuously pumped with PBS (phosphate-buffered saline, 7.4 pH) at a steady flow rate of 2 mL/h. The donor compartment was partially covered with aluminium foil to avoid evaporation of the donor fluid. The samples were collected on a fraction collector over a period of 48 h. Tape stripping (15times) and separation of the dermis, subcutaneous, and the fat layer were performed after completion of the skin permeation studies. The method used for separation, dissection and processing of the tissues was similar to the in vivo studies. Partition Coeficient Studies-Tissues (epidermis, dermis, subcutaneous, muscle, deep muscle, fat) were incubated with 1mL of SLS for 24 h (with a known amount of radioactivity) at room temperature. After 24 h (assuming a pseudo-equilibrium state) the SLS solution was removed and counted for radioactivity. The partition coefficient was expressed as a ratio of the SLS concentration in tissue to the SLS concentration in the aqueous solution. D a t a Analysis-The cumulative amount of SLS penetrating excised skin was plotted against time. The apparent steady-state flux was obtained by linear regression of the linear asymptote. Dividing the steady-state flux by the initial concentration yielded the apparent permeability coefficient. The lag time was estimated by extrapolating the straight line portion of the cumulative solute penetrating-time profile to the x-axis. The intercept was taken as the lag time. The dpm values of the tissues (aRer correction for background counts) were converted to the fraction of initial applied concentration and expressed as micromoles per gram of the tissue. Statistical comparisons were made using paired or unpaired Student's t test where appropriate. The level of significance was taken as p = 0.05.
Results and Discussion Surfactants can directly affect the stratum corneum barrier leading to a prolonged alteration of skin barrier proper tie^.^^^^^^ The SLS concentrations producing a 50% decrease in cell
T
5l l
0
10
20
time
30
4 0
5 0
(hours)
Figure 1-Cumulative amount of SLS penetrating excised rat skin (in vitro) with time. Values are expressed as the mean rt SD (n = 3-4). Significant permeation is observed only after 24 h. Table 1-Distribution of SLS (as Percentage of Applied Dose) in the Stratum Corneum after Various (in Vitro and in Vivo) Topical Treatments
Yo SLS in Tape Strips
Yo SLS in Tape Strips
Treatment
nos. 1-5
nos. 6-15
Treatment
nos. 1-5
nos. 6-15
6h 24 h Cumulative
17.9 19.3 22.0
1.3 2.3 2.5
In vitroa Control cumulativebsc
0.85 0.43
0.6 0.2
a 48 h study. SLS concentrations 1 week after a single 24 h application (prior to a second 24 h treatment). Results from one animal only.
viability are reported to be linked to critical micellar concentration (cmc)(8.0-8.2 mM in distilled water), and cell viability is concentration dependent above the cmc.25,26SLS application to guinea pig skin produced no damage at a concentration of 0.1%,partial dermal necrosis at 1.0%(34 mM), and total necrosis at Since 1% SLS solution is known t o elicit typical cutaneous irritant responses and is commonly used in various s t ~ d i e s ,this ~ , ~concentration ~ was chosen for the present work. Figure 1 shows the cumulative amount of SLS penetrating excised full thickness rat skin with time. A mass balance study revealed 93% recovery of SLS a t the end of the experiment. A steady state flux of 0.506 f 0.131 nmol/cm2/h and an apparent permeability coefficient of 3.90 x cds was obtained when 1%SLS was applied to excised rat skin. and 2.0 x cds Permeability coefficients of 3.0 x have earlier been reported for human epidermisls and neonatal rat stratum corneum,28respectively. The higher permeability coefficient of SLS observed in this study is probably because the previous experiments were not conducted long enough to reach steady state and SLS is undoubtedly changing the barrier property of the skin (by damaging the skin) over longer durations.29 Furthermore, SLS is known to act as permeation e n h a n ~ e r ,and ~ ~ its , ~ permeability coefficient has been reported to be time dependent. The distribution of SLS (as percent of applied dose) after various treatments (both in vivo and in vitro) into different skin compartments, i.e. stratum corneum tape strips (1-5 and 6-15), is shown in Table 1, while the values for (epidermis dermis), subcutaneous tissue, and fat after 24 h in vivo and 48 h in vitro treatments are shown in Table 2. A concentration gradient of SLS within the stratum corneum was observed for all in vivo treatments (Figure 2). The percent SLS in the first five strips (sum of 1-5) and the remaining ten strips (sum of 6-15) was comparable between 6 and 24 h treatments (p > 0.05) and also between 24 h and cumulative treatments (p > 0.05). This suggests that the stratum corneum uptake of SLS has reached a steady state within 6
+
Journal of Pharmaceutical Sciences / 1241 Vol. 84, No. 10, October 1995
+ cumulative (two 24h treatments) -0- 6h
dermis
sub cut
dermis
sub cut
m u s c l e d. muscle
fat
1 <
2
1
4
3
6
5
tape
9
8
7
strip
10 1 1 1 2 13 1 4 1 5
number
Figure 2-SLS distribution in stratum corneum (in vivo) expressed as percent of applied dose. Values are expressed as the mean k SD ( n = 3-4). No notable difference was observed after various treatments. 10 1
% 0
100
1
---t 24h(ln vivo)
* 4Bh (in vltro) I
I
T
-
U
-.-Q
m u s c l e d. muscle
fat
0. 0.
m
6
10- 1 -
ae 10' 0
1
2
3
4
5
6
tape
7
8
strip
9
1 0 1 1 1 2 1 3 1 4 1 5
number
. 5
Figure 3-SLS distribution in stratum corneum expressed as percent of applied dose. Values are expressed as the mean f SD (n = 3-4). Significant difference in SLS concentration is observed after in vivo and in vitro treatments.
h after application. In contrast, the percent of applied SLS observed in stratum corneum during in vitro treatment was significantly less as compared to in vivo concentrations (Figure 3 and Table 1). Since the penetration-enhancing effects of SLS may be observed a t later times after SLS application as discussed above, the prolonged 48 h in vitro study allows barrier disruption properties of SLS to manifest in such a way that SLS easily penetrates the stratum corneum and the viable tissue now becomes the transport limiting step. Higher levels of SLS in the lower layers of skin as compared to stratum corneum have been observed in earlier studies, and the effect is more pronounced for 48 h as compared to 24 h SLS application.21 The higher levels of SLS in dermis (4.4%) in vitro as compared to stratum corneum (1.42%)is also consistent with the results of Fullerton et a1.,21who also observed higher levels of SLS in the skin (epidermis dermis) as compared to stratum corneum. In deeper skin layers such as dermis, subcutaneous tissue, and underlying fat, the percent of SLS accumulated was higher for in vitro studies as compared to in vivo application of SLS (Table 2). This reflects the lack of available viable dermal blood supply in vitro, which results in SLS accumulation in skin and associated structures. A functioning dermal blood supply is available at the epidermal-dermal junction in vivo, which is responsible for the dermal clearance of the compounds once they bypass the stratum corneum barrier. The in vitro experiments utilized skin having dermis and underlying fat which would probably hold (and bind) SLS during its passage through the skin and into the receiver fluid. Black and Howes31 have reported higher percutaneous pen-
Q
0
E 2
d e r m i s sub cut
fat
m u s c l e d . muscle
Tissue
Figure 4-Tissue distribution of SLS after topical application for (A) 6 h, (B)24 h, and (C) cumulative. Key: (0)patch tissue, (0)contralateral tissue, (-) blood; subcut = subcutaneous tissue, d. muscle = deep muscle. Values are expressed as the mean f SD (n = 3-4). Direct SLS penetration to an approximate depth of underlying muscle can be noted. SLS concentrations at the contralateral site are comparable to systemic levels. Table 2-Concentrations of SLS in Underlying Tissues after 24 h in Vivo and 48 h in Vitro Treatments SLS Concn hmollg of tissue)
+
1242 / Journal of Pharmaceutical Sciences Vol. 84, No. 10, October 1995
,0-4
cn
a
Tissue
in Vivoa
in Vitrob
Dermis Subcutaneolls Fat
0.4110 f0.070 0.0344 f 0.002 0.0002 k 0.000
1.60 f 1.OO 1.50 k 1.OO 3.30 f 0.02
Mean f SD ( n = 3-4),
Mean f SD (n = 6).
etration rates of SLS in vivo after 24 h than can be predicted by 24 h in vitro studies. The ability of in vitro models to predict in vivo availability and related toxicity of compounds may therefore be limited.32x33It should however be noted that the in vitro experiments were conducted for 48 h as compared t o 24 h in vivo study. Figure 4A--C shows the in vivo tissue concentration (expressed as a fraction of the initial concentration) vs depth profiles of SLS for various treatments in rats. For epidermal
--t. 24h
loo
1
+ cumulative * 6h
\
10' 1 -
E
. u l
-aJ v)
10-2-
0
E
rr 10' 3-
10-
dermis
subcut
fat
muscle
d.muscle
Tissue Figure 5-Underlying patch tissue distribution of SLS after topical application. Key: subcut = subcutaneous tissue, d. muscle = deep muscle. Values are expressed as the mean f SD ( n = 3-4)Refer to the text for details. Table 3-In Vivo Concentrations of SLS in the Epidermis below the Patch Site after Different Topical Treatmentsa Concentration(,umol/g of tissue)
a
treatment
Patch Site
Contralateral
6h 24 h Cumulative
4.7f1.2 6.3 f 1.8 43.9 1.2
0.03 k 0.01 0.06 ? 0.02 0.50 f 0.03
*
Mean f SD ( n = 3 4 ) .
Table 4-Partition Coefficents of 35S-LabeledSLS in Various Tissues in Vitro Tissue
Partition Coefficient Tissue:Water
Tissue
Partition Coefficient Tissue:Water
Epidermis Dermis Subcutaneous
448.06 7.31 10.46
Fat Muscle Deep muscle
9.78 113.17 94.04
concentrations below the application site, refer to Table 3. For 6 h application, the concentrations of SLS in tissues below the applied site were always higher than plasma concentrations and contralateral tissue concentrations up to the depth of underlying muscle, suggesting direct penetration to an approximate depth of 5-6 mm below the applied site (Figure 4A). The concentrations of SLS in deep muscle were less than plasma concentration and comparable to contralateral deep muscle concentration, suggesting systemic redistribution of SLS to tissues deeper than 5-6 mm below the applied site (Figure 4A) Similar results were obtained for 24 h (except the fat) and cumulative application of SLS (Figure 4B,C). The plasma levels of SLS after various treatments were selfconsistent, indicating pseudo-steady-state conditions. The in vivo concentrations of SLS in underlying epidermis, subcutaneous tissue, fat, and deep muscle were not statisti-
cally different after 6 and 24 h treatments ( p 0.05) while for dermis (p < 0.002) and muscle ( p < 0.01) the concentrations at 24 h were slightly higher compared t o the 6 h application time. The in vivo cumulative application of SLS produced 8 times higher levels of SLS in epidermis relative to 24 h application (p < 0.007). The concentrations of SLS in underlying dermis, subcutaneous tissue, and muscle were significantly less after cumulative treatment as compared to 24 h treatment (Figure 5). This may be due to the increased clearance of SLS from these tissues after repeated application of SLS. Studies using LDV (laser Doppler velocimetry) have demonstrated that there is an increase in the local blood flow at the site of SLS app1i~ation.l~ A 2-fold increase in the local blood flow was noted after cumulative treatment.24 However, the relative effects on different tissues below the applied site after single and repeated application of SLS are unknown and are the subject of a future study in our laboratory. The fat and the deep muscle showed comparable values after 24 h and repeated SLS treatments (p > 0.05). The direct penetration of compounds (in vivo) after topical application is determined both by physicochemical characteristics of the compounds as well as physiological variables (such as tissue blood f l o ~ ) l l J ~ Solutes . which are predominantly ionized at the dermal pH of 7.4 penetrate to an approximate depth of 3-4 mm below the applied site while more lipophilic solutes may penetrate as deeply as 1 cm below the applied site.12J4 SLS molecule possesses both polar and non polar groups. The direct penetration (as distinct from systemic redistribution) of SLS to an approximate depth of 5-6 mm below a topically applied site is therefore consistent with its relatively amphophilic nature.l In order to explain high levels of SLS in contralateral epidermis (in vivo) as compared to other contralateral tissues, SLS partitioning studies were performed for various tissues in vitro, and the results are shown in Table 4. A relatively higher partition coefficient value for epidermis was obtained as compared t o other tissues, suggesting maximum preferential affinity for SLS in this cutaneous layer. Higher levels of SLS in epideqmis are consistent with its preferential affinity for skin lipids as well as Table 5 shows in vivo results from only one rat where SLS was applied on the skin for 24 h and SLS concentrations in tissues measured 7 days later (i.e. prior to the second application in a cumulative treatment). Residual SLS was observed in all tissues with maximum again being in the epidermis of both the treated and untreated site. Studies using bioengineering techniques on human volunteers have also shown that the skin barrier does not heal even 1 week after a single 24 h exposure to SLS8 Even longer effects (3-6 weeks) of SLS on skin have recently been reported.39 The deleterious effects of SLS on skin using bioengineering methods are well d o c ~ m e n t e d . ~The ~ ~ ,effects ~ , ~ ~ are known to be concentration dependentz6 and SLS is known to be cytotoxic a t its cmc, which is around 8 mM or 0.24% SLSZ5 The cumulative and 24 h treatments in this study produced epidermal concentrations of SLS close to or above its cmc. The underlying dermis and other tissues had concentrations well
Table 5-In vivo SLS Concentration (as Percentage of Applied Dose) in the Tissues 1 week after a Single 24 h Treatment*
Yo SLS
a
Yo
SLS
Tissue
Patch Site
ContralateralSite
Tissue
Patch Site
ContralateralSite
Epidermis Dermis Subcutaneous Muscle
4.3 0.01 0.005 0.009
0.019 0.0006 0.003 0.006
Deep muscle Fat Blood Tape Strips
0.006 0.004 NS 0.43% (nos. 1-5)
0.004 0.003 NS 0.23% (nos. 6-15)
Results from one animal only.
Journal of Pharmaceutical Sciences / 1243 Vol. 84, No. 10, October 1995
below the cmc. The blood and contralateral tissue concentrations of SLS were also substantially below the toxic concentrations. The present results suggest that treatment with 1% SLS for 24 h and repeated applications can produce irritant effects in the underlying epidermis, while the deeper tissues attain concentrations that may elicit only mild irritation. Our data therefore for the first time quantitatively reinforce the much published qualitative data by showing high levels of SLS in epidermis after topical application, which can lead to barrier disruption and other physiological and biochemical changes in the skin. Also important is the systemic absorption and redistribution of SLS throughout the body after topical treatment, which could be responsible for the retention of this compound in the skin for a longer time.
Conclusion SLS concentrations in skin and deeper underlying tissues were quantified in vivo in a rat model. Topically applied SLS can penetrate directly to a depth of about 5-6 mm below the applied site. Epidermis may be exposed to high concentrations of SLS after 1%application of SLS. Tissues deeper than skin may also be exposed to high levels of SLS. The concentrations of SLS in deeper underlying tissues ( 2 5 mm) are determined predominantly by systemic redistribution. Topically applied SLS also appears in blood and other tissues, but the levels are well below concentrations known to evoke systemic side effects. Traces of SLS persist in tissues 7 days after single 24 h exposure of SLS, which is consistent with the barrier disruption data generated using bioengineering methods.6 The known preferential affinity of SLS for skin structures was further confirmed by both in vitro and in vivo results from this study. Furthermore, the in vitro model was not predictive of in vivo data in terms of the extent of exposure of the underlying tissues. The quantification of SLS penetration below the applied site and the extent of systemic exposure provide valuable information and insight for the appropriate use of SLS in the cosmetic and pharmaceutical industry. The results of these studies, which demonstrate the extent of absorption, distribution, and accumulation of SLS in tissues will also be useful in helping to quantitate the potential for irritation and pharmacological consequences of topical application.
References and Notes 1. Singer, M. M.; Tjeerdema, R. S. In Reviews of Environmental Contamination and Toxicology, Springer-Verlag New York, Inc., 1994; Vol. 133, pp 95-149. 2. Van der Valk, P. G. M.; Nater, J . P.; Bleumink, E. J . Znuest. Dermatol. 1984,82, 291-293. 3. Froebe, C. L.; Simion, F. A,; Rhein, L. D.; Cagan, R. H.; Kligman, A. M. Dermatologica, 1990, 181: 277-283. 4. Smith, E.; Maibach, H. I. In Percutaneous Penetration Enhancers; CRC Press: Boca Raton, FL, in press. 5. Faucher, J . A,; Goddard, E. D. J. SOC.Cosmet. Chem. 1978,29, 323-337.
1244 / Journal of Pharmaceutical Sciences Vol. 84, No. 10, October 1995
6. Leveque, J. L.; Rigal, J.; Saint-Leger, D.; Billy, D. Skin Pharmacol. 1993, 6, 111-115. 7. Lee, C. H.; Maibach, H. I. Contact Dermatitis. In press. 8. Patil. S.: Sineh. P.:. Maibach. H. I. Znt. J . Pharm. 1994.110 (2). 147-'155. 9. Wilhelm, K. P.; Surber, C.; Maibach, H. I. Arch. Derm. Res. 1989, 281,293-295. 10. Willis, C. M.; Stephens, C. J.; Wilkinson, J . D. J . Znuest. Dermatol. 1989, 93, 695-699. 11. Singh, P.; Roberts, M. J . Pharmacohinet. Biopharm. 1993,21, 337-372. 12. Singh, P.; Roberts, M. J . Pharmacol. Exp. Ther. 1994,268,144151. 13. Singh, P.; Roberts, M. J . Pharm. Sci. 1994, 83, 774-782. 14. Singh P.; Roberts, M. J . Pharm. Pharmacol. In press. 15. Marty, J . P.; Guy, R. H.; Maibach, H. I. In Percutaneous Absorption: Mechanisms and Methodology, Drug Delivery, 2nd ed.; Bronaugh, R. L., Maibach, H. I., Eds.; Marcel Dekker: New York, 1989; pp 511-529. 16. Guy, R.; Maibach, H. J . Pharm. Sci. 1983, 72, 1375-1380. 17. Blank, I.; Gould, E. J . Znuest. Dermatol. 1959, 33, 327-335. 18. Howes, D. J . SOC.Cosmet. Chem. 1975,26, 47-63. 19. Agner, T;. Serup, J . J. Invest. Dermatol. 1990, 95, 543-547. 20. Loden, M. J. SOC.Cosmet. Chem. 1990,41, 227-233. 21. Fullerton, A,; Broby-Johansen, U.; Agner, T. Contact Dermatitis 1994, 30, 222-225. 22. Bronaugh:, R. L.; Maibach, H. I., Eds. I n Vitro Percutaneous Absorption: Principles,, Fundamentals and Applications; CRC Press: Boston, 1991. 23. Buyuktimkin S.;Buyuktimkin N.; Rytting, J . H. Pharm. Res. 1993,10, 1632-1637. 24. Freeman, S.; Maibach, H. I. J . Am. Acad. Dermatol. 1988, 19, 496-502. 25. Williams, R. J.; Phillips, J . N.; Mysels, K. J . Trans. Farraday SOC. 1955, 51, 729-737. 26. Bloom, E.; Sznitowska, J.; Polansky, Z. D.; Maibach, H. I. Dermatology 1994, 188, 63-268. 27. Gisslen, H.; Magnusson, B. Acta Derm.-Vereneol. 1966, 46, 269-274. 28. Faucher, J . A,; Goddard, E. D.; Kulkarni, R. D. J . A m . Oil Chem. SOC.1979, 56, 776. 29. Faucher, J. A,; Goddard, E. D. J . SOC.Cosmet. Chem. 1978,29, 339-352. 30. Wilhelm , K. P.; Surber, C.; Maibach, H. I. J . Znuest. Dermatol. 1991, 97 (51, 927-932. 31. Black, J . G.; Howes, D. J . SOC.Cosmet. Chem. 1979, 30, 157163. 32. Bartek, M.; LaBudde, J. A.; Maibach, H. I. J . Invest. Dermatol. 1972, 58, 114-123. 33. Bronaugh. R. L.; Maibach, H. I., Eds. In I n Vitro Percutaneous Absorution. Princiwles. Fundamentals and Audications: CRC *. Press. Boston. 19b1. ' 34. Imokawa, G.; Sumura, K.; Katsumi, M. J . Am. 011 Chem. SOC. 1975,52, 490-493. 35. Walters. K. A,: Walker., J.:, Oleinik. " , 0. J . Pharm. Pharmacol. 1988, 40, 525:529. 36. Rhein, J. D.; Robbins, C. R.; Fernee, K.; Cantore, R. J . SOC. Cosmet. Chem. 1986,37, 125-139. 37. Bartnik, F. G. In Anionic surfactants: Biochemistry, toxicology, dermatology, 2nd ed.; Ch. Gloxhuber, K. Kunstler, Eds.; Marcel Dekker: New York, 1992, 1-42. 38. Kalamanzon, E., Zlotkin, E.; Cohen, R.; Barenholz, Y. Biochim. Biophys. Acta 1992,1103, 148-156. 39. Widmer, J.; Elsner, P.; Burg, G. Contact Dermatitis 1994, 30, 35-39. 40. CuaiA. B.; Wilhelm, K. P.; Maibach, H. I. Br. J . Dermatol. 1990, 123 (51, 607-613.
JS950039F
KHALEDSARASOUR,
AND
HOWARD MAIBACH~
Received January 30, 1995, from the Department of Demafohgy, School of Medicine, Surge 110, Universify of California, San Francisco, CA 94143. Accepted for publication July 18, 1995@. ~
Sodium lauryl sulfate (SLS)is known to penetrate skin and cause cutaneous irritation. Some of these effects have been well-defined using bioengineering techniques. In this study, the ability of SLS to penetrate skin was quantified in a hairless rat model. In addition, local deep tissue penetration and systemic exposure to SLS were also evaluated to assess the toxic potential of topically applied SLS. SLS was observed to penetrate directly to a depth of about 5-6 mm below the applied site. Systemic redistribution was predominantly responsible in determining concentrations of SLS in tissues deeper than 5-6 mm. Epidermal concentrations of SLS after application of 1% (34 mM) aqueous SLS solution for 24 h were above the threshold levels which are known to evoke typical skin irritation responses. Deeper underlying tissues including dermis, subcutaneous, and muscle may also be exposed to high levels of SLS. Topically applied SLS was also observed in blood and contralateral tissues but the observed levels were not likely to elicit any systemic side effects at these doses. Traces of SLS were observed in tissues 7 days after single 24 h application of SLS, which supports the prolonged barrier disruption data generated using conventional bioengineering techniques. Cumulative treatment of SLS significantly increased the concentration of this compound in the underlying epidermis. The known preferential affinity of SLS for skin lipids and proteins was further confirmed by both in vitro and in vivo results. However, in vitro studies failed to predict the underlying tissue toxicity of SLS under the patch site when compared to the in vivo results. Such quantitative pharmacokinetic-pharmacodynamic correlations may be useful predictors for effective use of surfactants as penetration enhancers in cosmetic, pharmaceutical, and industrial applications.
Abstract
Electron microscopy revealed morphological changes in epidermis after SLS treatment.1° In spite of its known irritant potential, the distribution of SLS in skin has not been quantified in vivo. Although most topically applied compounds are cleared by the dermal blood supply, direct deep tissue penetration of many solutes has been detected.'l-I6 The extent, depth and pharmacokinetics of a variety of solutes with diverse physicochemical properties have recently been quantified.11J3The deep tissue penetrant potential of topically applied SLS is unknown. Most of the published studies deal with the local Given l penetration of SLS in the epidermis and d e r m i ~ . ' ~ - ~ that SLS is a common irritant, it becomes imperative to study if deeper tissues below the site of topically applied SLS are also exposed to high levels of SLS. This study quantifies the depth of penetration of topically applied SLS in a hairless rat model by varying the duration and mode (single vs repeated) of SLS application. The plasma concentrations and contralateral tissue concentrations were also determined to evaluate the extent of systemic exposure of topically applied SLS. The data were also interpreted to provide a quantitative estimation of SLS levels likely to elicit an irritant response in skin and underlying tissues. An in vitro test model was also utilized to compare the penetration kinetics of SLS with in uivo skin patch test results.
Materials and Methods
Chemicals and Instrument-[35SlSodium lauryl sulfate was purchased from Amersham Co., tissue solubilizer (Soluene-350 and Insta-flour) for organic samples was obtained from Packard Instrument Co., Meridan CT, and the cocktail (Universol) for inorganic samples from was from ICN. Sodium lauryl sulfate was obtained from Introduction Sigma Chemical Co., St. Louis, MO (purity > 99%). All other reagents used were of analytical grade. A liquid scintillation counter (Packard The surface active nature of sodium lauryl sulfate (SLS) Instrument Co., Model Tricarb-1500) was used to detect the radiomakes it a useful component for a multitude of diverse activity in the samples. applications: (i) component of various consumer products like In Vivo Studies-Animals-Hairless rats (350-400 g) were obshampoos, cosmetics, toothpastes, carpet cleaners, and detertained from the Charles River Laboratory. The animals were housed gents, (ii) as a biological tool to increase intestinal absorption, under standard laboratory conditions (25.0 f 0.5 "C, relative humidity 55-75%) and supplied with normal pellet diet and water ad lib. The as a cytolytic agent, as a virus-dissociating agent in electroexperiments had been approved by the Committee for Animal phoretic studies, and for solubilizing lipid membranes and (iii) Research, University of California, San Francisco. in the electroplating industry and also in many other indusPatch Test Treatment-Standard patches (Large Hilltop Chambers, trial products.' Due to its widespread and inadvertent topical Cincinnati, OH; inner diameter 1.5 cm) were applied on the dorsum exposure, the skin is extensively exposed to SLS. SLS impairs of the anesthetized animals. The patches were secured in place with the skin barrier function by removing intercellular hydrophoa n adhesive tape (3M Transpore Surgical tape) and a n elastic bic lipids leading t o an increase in transepidermal water bandage. The animals were divided into three study groups with each ~ o s s . ~SLS - ~ also binds extensively to intracellular keratin, group receiving one of the following treatments: (a)single application which explains some of its irritant effects like tightness and of 1%aqueous SLS solution (300 pL) for 6 h, (b) single application of roughening of kin.^^^ 1%aqueous SLS solution (300 pL) for 24 h, (c) Two applications of 1%aqueous SLS solution (300 pL) for 24 h each; the time interval Many of the cutaneous side effects associated with topical between the two applications was 1 week. SLS use of SLS have been studied using bioengineering Dermal Penetration and Local Tissue Uptake Studies-After dosing increases transepidermal water loss, induces local vasodilaanesthesia, the animals were allowed to recover and then ~ tation and decreases the water retention properties of ~ k i n . ~ 3 , under placed in separate cages. At predetermined times (6 h or 24 h postdosing) the animals were lightly anesthetized using anesthetic Abstract published in Advance ACS Abstracts, August 15, 1995. ether, and blood was withdrawn from the tail vein into heparinized @
1240 / Journal of Pharmaceutical Sciences Vol. 84, No. 10, October 1995
0022-3549/9%3184-1240$09.00/0
0 1995, American Chemical Sociev and American Pharmaceutical Association
tubes. The blood samples were centrifuged immediately a t 2000 rpm to separate the plasma. A known amount of plasma was placed in the scintillation vials and the scintillant added to assess the SLS activity in the plasma. After withdrawal of the blood, the animals were immediately sacrificed by an overdose of anesthetic ether, the patches were removed carefully, and the test area was swabbed dry three or four times with tissue paper. A portion of the test area was tape stripped (3M Transpore Surgical tape) 15 times to remove the stratum corneum. The scintillant was added to the tape stripped samples and counted for radioactivity. The skin was excised and epidermis and dermis separated by heat splitting at 60 "C in a water bath. The underlying subcutaneous tissue, muscle, deep muscle, and fat layers were dissected surgically. A similar procedure was adopted to dissect the tissues from the contralateral side (i.e. from the corresponding untreated site on the other lateral half of the animal). To avoid radioactivity contamination, surgical instruments were thoroughly cleaned before each tissue excision. Furthermore, wipes were taken from the cleaned instruments and counted for radioactivity to ascertain that there was no contamination from one site to another. The tissues were then weighed, put in the vials containing tissue solubilizer, and kept in a n incubator at 45 "C overnight to dissolve the tissue. Cocktail for tissue samples was added to these vials and the radioactivity counted in the scintillation counter. Background radioactivity was determined for plasma and various tissues in one control animal. In Vitro Studies-Skin preparation-Whole thickness rat skin including the subcutaneous fat layer was excised from euthanized animals and used for the in vitro permeation studies. Skin pieces of approximately 1.5 cm2 were immersed in buffered solution a t 37 "C before mounting them on the diffusion cell. Permeation Studies-Vertical, Marzulli-Bronaugh type flow through diffusion cells (Laboratory Glass Apparatus Inc.) consisting of two water-jacketed cylindrical half-cells, each having a volume of 2.8 mL ~ ~ skin s ~ ~was mounted on and a diameter of 1 cm2, were ~ s e d . The the diffusion cells with the epidermis facing the donor compartment. A constant temperature was maintained in the cells by circulating water a t 37 "C. The fluid in the receptor cells was constantly stirred a t about 300 rpm by Teflon-coated magnetic bars. The donor compartment received 100 pL of 1% SLS solution containing the radiolabeled SLS (1pCi/cell). The receiver compartment was continuously pumped with PBS (phosphate-buffered saline, 7.4 pH) at a steady flow rate of 2 mL/h. The donor compartment was partially covered with aluminium foil to avoid evaporation of the donor fluid. The samples were collected on a fraction collector over a period of 48 h. Tape stripping (15times) and separation of the dermis, subcutaneous, and the fat layer were performed after completion of the skin permeation studies. The method used for separation, dissection and processing of the tissues was similar to the in vivo studies. Partition Coeficient Studies-Tissues (epidermis, dermis, subcutaneous, muscle, deep muscle, fat) were incubated with 1mL of SLS for 24 h (with a known amount of radioactivity) at room temperature. After 24 h (assuming a pseudo-equilibrium state) the SLS solution was removed and counted for radioactivity. The partition coefficient was expressed as a ratio of the SLS concentration in tissue to the SLS concentration in the aqueous solution. D a t a Analysis-The cumulative amount of SLS penetrating excised skin was plotted against time. The apparent steady-state flux was obtained by linear regression of the linear asymptote. Dividing the steady-state flux by the initial concentration yielded the apparent permeability coefficient. The lag time was estimated by extrapolating the straight line portion of the cumulative solute penetrating-time profile to the x-axis. The intercept was taken as the lag time. The dpm values of the tissues (aRer correction for background counts) were converted to the fraction of initial applied concentration and expressed as micromoles per gram of the tissue. Statistical comparisons were made using paired or unpaired Student's t test where appropriate. The level of significance was taken as p = 0.05.
Results and Discussion Surfactants can directly affect the stratum corneum barrier leading to a prolonged alteration of skin barrier proper tie^.^^^^^^ The SLS concentrations producing a 50% decrease in cell
T
5l l
0
10
20
time
30
4 0
5 0
(hours)
Figure 1-Cumulative amount of SLS penetrating excised rat skin (in vitro) with time. Values are expressed as the mean rt SD (n = 3-4). Significant permeation is observed only after 24 h. Table 1-Distribution of SLS (as Percentage of Applied Dose) in the Stratum Corneum after Various (in Vitro and in Vivo) Topical Treatments
Yo SLS in Tape Strips
Yo SLS in Tape Strips
Treatment
nos. 1-5
nos. 6-15
Treatment
nos. 1-5
nos. 6-15
6h 24 h Cumulative
17.9 19.3 22.0
1.3 2.3 2.5
In vitroa Control cumulativebsc
0.85 0.43
0.6 0.2
a 48 h study. SLS concentrations 1 week after a single 24 h application (prior to a second 24 h treatment). Results from one animal only.
viability are reported to be linked to critical micellar concentration (cmc)(8.0-8.2 mM in distilled water), and cell viability is concentration dependent above the cmc.25,26SLS application to guinea pig skin produced no damage at a concentration of 0.1%,partial dermal necrosis at 1.0%(34 mM), and total necrosis at Since 1% SLS solution is known t o elicit typical cutaneous irritant responses and is commonly used in various s t ~ d i e s ,this ~ , ~concentration ~ was chosen for the present work. Figure 1 shows the cumulative amount of SLS penetrating excised full thickness rat skin with time. A mass balance study revealed 93% recovery of SLS a t the end of the experiment. A steady state flux of 0.506 f 0.131 nmol/cm2/h and an apparent permeability coefficient of 3.90 x cds was obtained when 1%SLS was applied to excised rat skin. and 2.0 x cds Permeability coefficients of 3.0 x have earlier been reported for human epidermisls and neonatal rat stratum corneum,28respectively. The higher permeability coefficient of SLS observed in this study is probably because the previous experiments were not conducted long enough to reach steady state and SLS is undoubtedly changing the barrier property of the skin (by damaging the skin) over longer durations.29 Furthermore, SLS is known to act as permeation e n h a n ~ e r ,and ~ ~ its , ~ permeability coefficient has been reported to be time dependent. The distribution of SLS (as percent of applied dose) after various treatments (both in vivo and in vitro) into different skin compartments, i.e. stratum corneum tape strips (1-5 and 6-15), is shown in Table 1, while the values for (epidermis dermis), subcutaneous tissue, and fat after 24 h in vivo and 48 h in vitro treatments are shown in Table 2. A concentration gradient of SLS within the stratum corneum was observed for all in vivo treatments (Figure 2). The percent SLS in the first five strips (sum of 1-5) and the remaining ten strips (sum of 6-15) was comparable between 6 and 24 h treatments (p > 0.05) and also between 24 h and cumulative treatments (p > 0.05). This suggests that the stratum corneum uptake of SLS has reached a steady state within 6
+
Journal of Pharmaceutical Sciences / 1241 Vol. 84, No. 10, October 1995
+ cumulative (two 24h treatments) -0- 6h
dermis
sub cut
dermis
sub cut
m u s c l e d. muscle
fat
1 <
2
1
4
3
6
5
tape
9
8
7
strip
10 1 1 1 2 13 1 4 1 5
number
Figure 2-SLS distribution in stratum corneum (in vivo) expressed as percent of applied dose. Values are expressed as the mean k SD ( n = 3-4). No notable difference was observed after various treatments. 10 1
% 0
100
1
---t 24h(ln vivo)
* 4Bh (in vltro) I
I
T
-
U
-.-Q
m u s c l e d. muscle
fat
0. 0.
m
6
10- 1 -
ae 10' 0
1
2
3
4
5
6
tape
7
8
strip
9
1 0 1 1 1 2 1 3 1 4 1 5
number
. 5
Figure 3-SLS distribution in stratum corneum expressed as percent of applied dose. Values are expressed as the mean f SD (n = 3-4). Significant difference in SLS concentration is observed after in vivo and in vitro treatments.
h after application. In contrast, the percent of applied SLS observed in stratum corneum during in vitro treatment was significantly less as compared to in vivo concentrations (Figure 3 and Table 1). Since the penetration-enhancing effects of SLS may be observed a t later times after SLS application as discussed above, the prolonged 48 h in vitro study allows barrier disruption properties of SLS to manifest in such a way that SLS easily penetrates the stratum corneum and the viable tissue now becomes the transport limiting step. Higher levels of SLS in the lower layers of skin as compared to stratum corneum have been observed in earlier studies, and the effect is more pronounced for 48 h as compared to 24 h SLS application.21 The higher levels of SLS in dermis (4.4%) in vitro as compared to stratum corneum (1.42%)is also consistent with the results of Fullerton et a1.,21who also observed higher levels of SLS in the skin (epidermis dermis) as compared to stratum corneum. In deeper skin layers such as dermis, subcutaneous tissue, and underlying fat, the percent of SLS accumulated was higher for in vitro studies as compared to in vivo application of SLS (Table 2). This reflects the lack of available viable dermal blood supply in vitro, which results in SLS accumulation in skin and associated structures. A functioning dermal blood supply is available at the epidermal-dermal junction in vivo, which is responsible for the dermal clearance of the compounds once they bypass the stratum corneum barrier. The in vitro experiments utilized skin having dermis and underlying fat which would probably hold (and bind) SLS during its passage through the skin and into the receiver fluid. Black and Howes31 have reported higher percutaneous pen-
Q
0
E 2
d e r m i s sub cut
fat
m u s c l e d . muscle
Tissue
Figure 4-Tissue distribution of SLS after topical application for (A) 6 h, (B)24 h, and (C) cumulative. Key: (0)patch tissue, (0)contralateral tissue, (-) blood; subcut = subcutaneous tissue, d. muscle = deep muscle. Values are expressed as the mean f SD (n = 3-4). Direct SLS penetration to an approximate depth of underlying muscle can be noted. SLS concentrations at the contralateral site are comparable to systemic levels. Table 2-Concentrations of SLS in Underlying Tissues after 24 h in Vivo and 48 h in Vitro Treatments SLS Concn hmollg of tissue)
+
1242 / Journal of Pharmaceutical Sciences Vol. 84, No. 10, October 1995
,0-4
cn
a
Tissue
in Vivoa
in Vitrob
Dermis Subcutaneolls Fat
0.4110 f0.070 0.0344 f 0.002 0.0002 k 0.000
1.60 f 1.OO 1.50 k 1.OO 3.30 f 0.02
Mean f SD ( n = 3-4),
Mean f SD (n = 6).
etration rates of SLS in vivo after 24 h than can be predicted by 24 h in vitro studies. The ability of in vitro models to predict in vivo availability and related toxicity of compounds may therefore be limited.32x33It should however be noted that the in vitro experiments were conducted for 48 h as compared t o 24 h in vivo study. Figure 4A--C shows the in vivo tissue concentration (expressed as a fraction of the initial concentration) vs depth profiles of SLS for various treatments in rats. For epidermal
--t. 24h
loo
1
+ cumulative * 6h
\
10' 1 -
E
. u l
-aJ v)
10-2-
0
E
rr 10' 3-
10-
dermis
subcut
fat
muscle
d.muscle
Tissue Figure 5-Underlying patch tissue distribution of SLS after topical application. Key: subcut = subcutaneous tissue, d. muscle = deep muscle. Values are expressed as the mean f SD ( n = 3-4)Refer to the text for details. Table 3-In Vivo Concentrations of SLS in the Epidermis below the Patch Site after Different Topical Treatmentsa Concentration(,umol/g of tissue)
a
treatment
Patch Site
Contralateral
6h 24 h Cumulative
4.7f1.2 6.3 f 1.8 43.9 1.2
0.03 k 0.01 0.06 ? 0.02 0.50 f 0.03
*
Mean f SD ( n = 3 4 ) .
Table 4-Partition Coefficents of 35S-LabeledSLS in Various Tissues in Vitro Tissue
Partition Coefficient Tissue:Water
Tissue
Partition Coefficient Tissue:Water
Epidermis Dermis Subcutaneous
448.06 7.31 10.46
Fat Muscle Deep muscle
9.78 113.17 94.04
concentrations below the application site, refer to Table 3. For 6 h application, the concentrations of SLS in tissues below the applied site were always higher than plasma concentrations and contralateral tissue concentrations up to the depth of underlying muscle, suggesting direct penetration to an approximate depth of 5-6 mm below the applied site (Figure 4A). The concentrations of SLS in deep muscle were less than plasma concentration and comparable to contralateral deep muscle concentration, suggesting systemic redistribution of SLS to tissues deeper than 5-6 mm below the applied site (Figure 4A) Similar results were obtained for 24 h (except the fat) and cumulative application of SLS (Figure 4B,C). The plasma levels of SLS after various treatments were selfconsistent, indicating pseudo-steady-state conditions. The in vivo concentrations of SLS in underlying epidermis, subcutaneous tissue, fat, and deep muscle were not statisti-
cally different after 6 and 24 h treatments ( p 0.05) while for dermis (p < 0.002) and muscle ( p < 0.01) the concentrations at 24 h were slightly higher compared t o the 6 h application time. The in vivo cumulative application of SLS produced 8 times higher levels of SLS in epidermis relative to 24 h application (p < 0.007). The concentrations of SLS in underlying dermis, subcutaneous tissue, and muscle were significantly less after cumulative treatment as compared to 24 h treatment (Figure 5). This may be due to the increased clearance of SLS from these tissues after repeated application of SLS. Studies using LDV (laser Doppler velocimetry) have demonstrated that there is an increase in the local blood flow at the site of SLS app1i~ation.l~ A 2-fold increase in the local blood flow was noted after cumulative treatment.24 However, the relative effects on different tissues below the applied site after single and repeated application of SLS are unknown and are the subject of a future study in our laboratory. The fat and the deep muscle showed comparable values after 24 h and repeated SLS treatments (p > 0.05). The direct penetration of compounds (in vivo) after topical application is determined both by physicochemical characteristics of the compounds as well as physiological variables (such as tissue blood f l o ~ ) l l J ~ Solutes . which are predominantly ionized at the dermal pH of 7.4 penetrate to an approximate depth of 3-4 mm below the applied site while more lipophilic solutes may penetrate as deeply as 1 cm below the applied site.12J4 SLS molecule possesses both polar and non polar groups. The direct penetration (as distinct from systemic redistribution) of SLS to an approximate depth of 5-6 mm below a topically applied site is therefore consistent with its relatively amphophilic nature.l In order to explain high levels of SLS in contralateral epidermis (in vivo) as compared to other contralateral tissues, SLS partitioning studies were performed for various tissues in vitro, and the results are shown in Table 4. A relatively higher partition coefficient value for epidermis was obtained as compared t o other tissues, suggesting maximum preferential affinity for SLS in this cutaneous layer. Higher levels of SLS in epideqmis are consistent with its preferential affinity for skin lipids as well as Table 5 shows in vivo results from only one rat where SLS was applied on the skin for 24 h and SLS concentrations in tissues measured 7 days later (i.e. prior to the second application in a cumulative treatment). Residual SLS was observed in all tissues with maximum again being in the epidermis of both the treated and untreated site. Studies using bioengineering techniques on human volunteers have also shown that the skin barrier does not heal even 1 week after a single 24 h exposure to SLS8 Even longer effects (3-6 weeks) of SLS on skin have recently been reported.39 The deleterious effects of SLS on skin using bioengineering methods are well d o c ~ m e n t e d . ~The ~ ~ ,effects ~ , ~ ~ are known to be concentration dependentz6 and SLS is known to be cytotoxic a t its cmc, which is around 8 mM or 0.24% SLSZ5 The cumulative and 24 h treatments in this study produced epidermal concentrations of SLS close to or above its cmc. The underlying dermis and other tissues had concentrations well
Table 5-In vivo SLS Concentration (as Percentage of Applied Dose) in the Tissues 1 week after a Single 24 h Treatment*
Yo SLS
a
Yo
SLS
Tissue
Patch Site
ContralateralSite
Tissue
Patch Site
ContralateralSite
Epidermis Dermis Subcutaneous Muscle
4.3 0.01 0.005 0.009
0.019 0.0006 0.003 0.006
Deep muscle Fat Blood Tape Strips
0.006 0.004 NS 0.43% (nos. 1-5)
0.004 0.003 NS 0.23% (nos. 6-15)
Results from one animal only.
Journal of Pharmaceutical Sciences / 1243 Vol. 84, No. 10, October 1995
below the cmc. The blood and contralateral tissue concentrations of SLS were also substantially below the toxic concentrations. The present results suggest that treatment with 1% SLS for 24 h and repeated applications can produce irritant effects in the underlying epidermis, while the deeper tissues attain concentrations that may elicit only mild irritation. Our data therefore for the first time quantitatively reinforce the much published qualitative data by showing high levels of SLS in epidermis after topical application, which can lead to barrier disruption and other physiological and biochemical changes in the skin. Also important is the systemic absorption and redistribution of SLS throughout the body after topical treatment, which could be responsible for the retention of this compound in the skin for a longer time.
Conclusion SLS concentrations in skin and deeper underlying tissues were quantified in vivo in a rat model. Topically applied SLS can penetrate directly to a depth of about 5-6 mm below the applied site. Epidermis may be exposed to high concentrations of SLS after 1%application of SLS. Tissues deeper than skin may also be exposed to high levels of SLS. The concentrations of SLS in deeper underlying tissues ( 2 5 mm) are determined predominantly by systemic redistribution. Topically applied SLS also appears in blood and other tissues, but the levels are well below concentrations known to evoke systemic side effects. Traces of SLS persist in tissues 7 days after single 24 h exposure of SLS, which is consistent with the barrier disruption data generated using bioengineering methods.6 The known preferential affinity of SLS for skin structures was further confirmed by both in vitro and in vivo results from this study. Furthermore, the in vitro model was not predictive of in vivo data in terms of the extent of exposure of the underlying tissues. The quantification of SLS penetration below the applied site and the extent of systemic exposure provide valuable information and insight for the appropriate use of SLS in the cosmetic and pharmaceutical industry. The results of these studies, which demonstrate the extent of absorption, distribution, and accumulation of SLS in tissues will also be useful in helping to quantitate the potential for irritation and pharmacological consequences of topical application.
References and Notes 1. Singer, M. M.; Tjeerdema, R. S. In Reviews of Environmental Contamination and Toxicology, Springer-Verlag New York, Inc., 1994; Vol. 133, pp 95-149. 2. Van der Valk, P. G. M.; Nater, J . P.; Bleumink, E. J . Znuest. Dermatol. 1984,82, 291-293. 3. Froebe, C. L.; Simion, F. A,; Rhein, L. D.; Cagan, R. H.; Kligman, A. M. Dermatologica, 1990, 181: 277-283. 4. Smith, E.; Maibach, H. I. In Percutaneous Penetration Enhancers; CRC Press: Boca Raton, FL, in press. 5. Faucher, J . A,; Goddard, E. D. J. SOC.Cosmet. Chem. 1978,29, 323-337.
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6. Leveque, J. L.; Rigal, J.; Saint-Leger, D.; Billy, D. Skin Pharmacol. 1993, 6, 111-115. 7. Lee, C. H.; Maibach, H. I. Contact Dermatitis. In press. 8. Patil. S.: Sineh. P.:. Maibach. H. I. Znt. J . Pharm. 1994.110 (2). 147-'155. 9. Wilhelm, K. P.; Surber, C.; Maibach, H. I. Arch. Derm. Res. 1989, 281,293-295. 10. Willis, C. M.; Stephens, C. J.; Wilkinson, J . D. J . Znuest. Dermatol. 1989, 93, 695-699. 11. Singh, P.; Roberts, M. J . Pharmacohinet. Biopharm. 1993,21, 337-372. 12. Singh, P.; Roberts, M. J . Pharmacol. Exp. Ther. 1994,268,144151. 13. Singh, P.; Roberts, M. J . Pharm. Sci. 1994, 83, 774-782. 14. Singh P.; Roberts, M. J . Pharm. Pharmacol. In press. 15. Marty, J . P.; Guy, R. H.; Maibach, H. I. In Percutaneous Absorption: Mechanisms and Methodology, Drug Delivery, 2nd ed.; Bronaugh, R. L., Maibach, H. I., Eds.; Marcel Dekker: New York, 1989; pp 511-529. 16. Guy, R.; Maibach, H. J . Pharm. Sci. 1983, 72, 1375-1380. 17. Blank, I.; Gould, E. J . Znuest. Dermatol. 1959, 33, 327-335. 18. Howes, D. J . SOC.Cosmet. Chem. 1975,26, 47-63. 19. Agner, T;. Serup, J . J. Invest. Dermatol. 1990, 95, 543-547. 20. Loden, M. J. SOC.Cosmet. Chem. 1990,41, 227-233. 21. Fullerton, A,; Broby-Johansen, U.; Agner, T. Contact Dermatitis 1994, 30, 222-225. 22. Bronaugh:, R. L.; Maibach, H. I., Eds. I n Vitro Percutaneous Absorption: Principles,, Fundamentals and Applications; CRC Press: Boston, 1991. 23. Buyuktimkin S.;Buyuktimkin N.; Rytting, J . H. Pharm. Res. 1993,10, 1632-1637. 24. Freeman, S.; Maibach, H. I. J . Am. Acad. Dermatol. 1988, 19, 496-502. 25. Williams, R. J.; Phillips, J . N.; Mysels, K. J . Trans. Farraday SOC. 1955, 51, 729-737. 26. Bloom, E.; Sznitowska, J.; Polansky, Z. D.; Maibach, H. I. Dermatology 1994, 188, 63-268. 27. Gisslen, H.; Magnusson, B. Acta Derm.-Vereneol. 1966, 46, 269-274. 28. Faucher, J . A,; Goddard, E. D.; Kulkarni, R. D. J . A m . Oil Chem. SOC.1979, 56, 776. 29. Faucher, J. A,; Goddard, E. D. J . SOC.Cosmet. Chem. 1978,29, 339-352. 30. Wilhelm , K. P.; Surber, C.; Maibach, H. I. J . Znuest. Dermatol. 1991, 97 (51, 927-932. 31. Black, J . G.; Howes, D. J . SOC.Cosmet. Chem. 1979, 30, 157163. 32. Bartek, M.; LaBudde, J. A.; Maibach, H. I. J . Invest. Dermatol. 1972, 58, 114-123. 33. Bronaugh. R. L.; Maibach, H. I., Eds. In I n Vitro Percutaneous Absorution. Princiwles. Fundamentals and Audications: CRC *. Press. Boston. 19b1. ' 34. Imokawa, G.; Sumura, K.; Katsumi, M. J . Am. 011 Chem. SOC. 1975,52, 490-493. 35. Walters. K. A,: Walker., J.:, Oleinik. " , 0. J . Pharm. Pharmacol. 1988, 40, 525:529. 36. Rhein, J. D.; Robbins, C. R.; Fernee, K.; Cantore, R. J . SOC. Cosmet. Chem. 1986,37, 125-139. 37. Bartnik, F. G. In Anionic surfactants: Biochemistry, toxicology, dermatology, 2nd ed.; Ch. Gloxhuber, K. Kunstler, Eds.; Marcel Dekker: New York, 1992, 1-42. 38. Kalamanzon, E., Zlotkin, E.; Cohen, R.; Barenholz, Y. Biochim. Biophys. Acta 1992,1103, 148-156. 39. Widmer, J.; Elsner, P.; Burg, G. Contact Dermatitis 1994, 30, 35-39. 40. CuaiA. B.; Wilhelm, K. P.; Maibach, H. I. Br. J . Dermatol. 1990, 123 (51, 607-613.
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