TOXICOLOGYANDAPPLIEDPHARMACOLOGY
Percutaneous
Absorption
35,381-391(1976)
of Hexachlorophene
in the Rat’
HARRY S. NAKAUE~AND DONALDR. BUHLER Department of Agricultural Chemistry and Environmental Health Sciences Center, Oregon State University, Corvallis, Oregon 97331 Received August 8,1975; accepted September 26,1975
PercutaneousAbsorption of Hexachlorophenein the Rat. NAKAUE, H. S., BUHLER, D. R. (1976). Toxicol. Appl. Pharmacol. 35, 381-391. [“Clhexachlorophenewasapplied to intact, clipped skinsof adult rats in four different solventsand an aqueousdetergentsolution. As much as 55% of the appliedradioactivity wasabsorbedthrough the skin in a 24-hr period. Dimethyl sulfoxide enhancedabsorption of the germicideto the greatest extent and aqueoussodiumlauryl sulfate(1 ‘A, w/v) wasthe leasteffective. With both adult and weanlingrats, the first measurableamount of radioactivity appearedin the venous blood in lessthan 1.5 hr, and maximum values were attained 12 hr after topical application of [14C]hexachlorophene.Approximately 27% of the appliedradioactivity remainedboundto the skin after 4 and 24 hr and wasnot removed by washingwith acetone. Distribution of radioactivity in rats 8 and 24 hr after topical application of [14C]hexachlorophenewas similar in weanling and adult rats. Most of the absorbedradioactivity appearedin the plasma,liver, smallintestine, AND
caecum, stomach, kidney, urine, and feces. The chlorinated antibacterial agent, hexachlorophene, has been employed in a variety of soap and cosmetic products. Use of such preparations provides a residual antibacterial effect through the deposition and retention of small amounts of hexachlorophene on the skin (Compeau, 1960; Manowitz and Johnston, 1967; Taber et al., 1971). Following topical exposure to preparations containing hexachlorophene, significant amounts of the bisphenol have been detected in the blood of human infants (Curley et al., 1971; Abbott et al., 1972) and adults (Feldman and Maibach, 1970; Butcher et al., 1973; Warner et al., 1973). Because of its toxicity in animals (Gaines et al., 1973; Kimmel ei al., 1974), particularly the associated cerebral edema (Kimbrough and Gaines, 1971; Nakaue et al., 1973), use of hexachlorophene as a topical bactericide has been greatly curtailed. Percutaneous absorption of hexachlorophene has also been reported in the rat (Carrol et al., 1967), pig (Taylor et al., 1972), and guinea pig (Black et al., 1974). In the present research, we have investigated the influence of various solvents and of age on the percutaneous absorption of [‘4C]hexachlorophene by the rat. The tissue distribution and excretion of dermally absorbed hexachlorophene is also reported. 1 This work was supportedby grantsNo. FD 00041and ES00210,U.S. PublicHealth Service, National Institutes of Health. Manuscript issued as Technical Paper No. 4026 from the Oregon Agricultural Experiment Station. z Present address: Department of Poultry Science, Oregon State University, Corvallis, Oregon 97331. 381 Copyright Q 1976 by Academic Press, Inc. All rights of reproduction Printed in Great Britain
in any form reserved.
382
NAKAUE
AND
BUHLER
METHODS
Chemicals.Hexachlorophene [2,2’-methylenebis (3,4,6-trichlorophenol)], USP3 was recrystallized prior to use [Methylene-14C]-hexachlorophene (sp act, 2.71 mCi/mmol) was obtained from the Mallinckrodt Chemical Co., St. Louis, Missouri, and was shown to be chemically and radiochemically pure by appropriate tic and glc analyses (Gandolfi and Buhler, 1974). Reagent grade chemicals and solvents were employed in all experiments. Penetration of [14C]hexachlorophenethrough the skin. Random-bred Wistar albino rats from a closed colony were used to study the penetration of [14C]hexachlorophene through the skin by the technique of O’Brien and Dannelley (1965). Groups of three or four weanling male (60-83 g) or adult male (250-300 g) or female (206-240 g) rats were lightly anesthetized with ethyl ether or by a 25 mg/kg ip injection of sodium pentobarbital. The ventral pelage from the front to the hind legs was clipped, and the animals were carefully shaved, avoiding damage to the skin. Approximately 20 circular skin areas (38.5 mm’) were then marked on the skin of each rat by means of a rubber stamp. Three-microliter aliquots containing 0.92-l .02 pg of [14C]hexachlorophene in the indicated solvents were applied in a random pattern to at least two marked zones on each of the test animals. During the experimental period, the animals were restrained and protected from air currents in an enclosure. Rats were exposed to the radioactive drug for different time periods up to 24 hr with the application times being adjusted so that all the exposure periods ended simultaneously. All of the marked skin areas were then removed at the same time with a surgical scissors and placed in individual counting vials. Zero-time exposure samples were prepared by removing untreated skin sections from the rats, placing the tissues in counting vials and immediately applying 3-~1 aliquots of the appropriate [14C]hexachlorophene solution directly to the skin. A single circular area (38.5 mm’) was marked on the stomachs of unanesthetized l- or 2-day-old male newborn rats (5.6-7.1 g). At various times, the marked skin areas ,of three animals were then treated with 3 ~1 of an acetone solution containing 0.95 pg of [14C]hexachlorophene. The treated skin areas were protected with masking tape, the adhesive areas over the marked zone being covered loosely with a second piece of tape applied like a large Band-Aid, and the animals were returned to their mothers. Zerotime exposure samples were prepared by removing marked skin sections from the backs of treated animals and applying 3-yl aliquots of the [14C]hexachlorophene directly to the skin segments. To determine the amount of extractable and bound radioactivity in the skin segments, 3 ~1 of an acetone solution containing 1.02 ,ug of [14C]hexachlorophene was applied at various times to four marked areas (38.5 mm’) on each of three female rats. Two of the skin areas for each exposure period were removed from each animal, washed with 2 ml of acetone and the washings and washed skin segments then counted. The remaining two skin sections were removed and counted directly. Percutaneous absorption of [‘4C]hexachlorophene. Three 240-258-g adult male rats were surgically prepared by insertion of cannulas (6 x 290 mm) in the posterior vena cava. One week post-surgery, the shaved and marked areas (255 mm”) on each animal were exposed to 40-~1 aliquots of an acetone solution containing 47.4 pg of [r4C]hexa3 A gift of the Givaudan Corporation,
Clifton, New Jersey.
PERCUTANEOUS
ABSORPTION
OF HEXACHLOROPHENE
383
chlorophene. The treated skin areas were then protected by application of concave plastic guards glued to the skin. At O1l&3,6,12, and 24 hr, a 0.5-ml aliquot of blood was withdrawn and added to heparinized tubes, and the plasma and red cells were then separated by centrifugation. Male weanling rats (51-81 g) were shaved, marked skin areas (227 mm2) treated with 25-40+1 aliquots of an acetone solution containing 10.2-16.2 pg of [14C]hexachlorophene, and the treated skin protected with plastic guards. At 1, 1.5,3,6, 12, and 24 hr, groups of three rats were sacrificed. Blood was obtained directly from the posterior vena cava, and the treated skin area was removed and counted. Plasma was obtained from the heparinized blood after centrifugation. Distribution and excretion of percutaneously absorbed [14C]hexachlorophene. The ventral surfaces of groups of three adult (220-233 g) or weanling (55-83 g) male rats were clipped and shaved. The animals were then treated topically over an 840-mm2 (adult) and 227-mm2 (weanling) skin areas with 0.16 mg/kg of [‘4C]hexachlorophene of 35.0-38.2 pg in 100 ~1 (adult) or 11.1-l 3.5 jig in 60 ,uI (weanling) of acetone. The treated areas were protected by concave plastic guards glued to the skin and the animals then placed in individual metabolism cages for collection of urine and feces over a 24-hr period. Following 8 hr of topical exposure, the treated skin was removed from the third group of two adult male rats, the remaining skin sutured together and the animals then returned to their metabolism cages. All three groups of animals were then sacrificed after 24 hr for removal of the various tissues and organs. A fourth group of three male weanling rats was topically treated with the same [‘4C]hexachlorophene dose and sacrificed after 8 hr. A fifth group of three 6.1-6.4-g male newborn rats was treated over a 38.5mm2 areas with 10 ~1 of an acetone solution containing 1.28 mg/kg (8.0 pg) of [r4C]hexg chlorophene. The treated areas were protected with masking tape as described previously and the animals returned to their mothers until sacrificed at 24 hr. Measurement of radioactivity. Skin and tissue samples were solubilized by the procedure of Mahin and Lofberg (1966). Tissue digests and other radioactive solutions were counted in a Packard Model 3375 liquid scintillation spectrometer (Packard Instrument Company, Downers Grove, Illinois) following addition of counting solution containing 500 ml of toluene, 300 ml of Cellosolve and 3 g of PPO. Counting efficiencies were determined by automatic external standardization. Plasma and tissue radioactivities were either calculated as micrograms of hexachlorophene equivalent per gram wet weight or were presented as percentages of the topical dose.
RESULTS Penetration of [‘4C]HexachIorophene
through the Skin
Absorption of [‘4C]hexachlorophene through rat skin apparently occurred at a relatively rapid rate since as much as 53 % of the applied radioactivity disappeared from the skin segments over a 24hr period (Table 1). The rate of penetration of hexachlorophene through the skin of female rats appeared to be slightly slower than that observed with males. Contrary to observations with other compounds (Idson, 1971), absorption of hexachlorophene through the skin did not increase with decreasing age. In fact, skin penetration was quite comparable in adults and weanlings and was appre-
NAKAUEANDBUHLER
384
TABLE 1 INFLUENCE OF AGE AND SEX ON THE PENETRATION OF [“‘CIHEXACHLOROPHENE SKIN
THROUGH RAT
Recoveryof appliedradioactivity (%) Exposure time 0-d 0 4 8 12 16 24
Adult femalesb
Adult fernaleC
98.2 + 2.5 97.2 +_ 2.1 83.4 f 10.0
101.2* 1.5 91.0+ 6.7 94.2zk1.3
68.4 f 5.4 55.1 2 6.8
75.6+ 2.5 61.8& 3.2
Adult malesb 98.5 2 88.6? 76.9 + 67.1f 60.1 + 46.6f
1.7 1.0
1.0 14Sf 14.8 11.8
Weanling malesd
Newborn malese
99.5f 1.8 85.6+ 2.1 77.3 f 11.6
98.1 + 3.4 78.2 + 2.2 70.5 + 14.0
65.5f 23.3 46.8 +_16.1
71.0& 3.8 59.9 + 5.3
a Mean + SD of four animals and four skin segments (38.5 mm2) per animal. b Each skin segment was treated with 3 ~1 of an acetone solution containing 0.92-1.02 pugof [14C]hexachlorophene (23.9-26.5 ng/mm2). The treated skin was uncovered and the animals restrained. c Each skin segment was treated with 3 ~1 of an acetone solution containing 1.02 pg of [‘%]hexachlorophene and 10.2 pg of nonradioactive hexachlorophene (291 ng/rnm’). The treated skin was uncovered and the animals restrained. d Each skin segment was treated with 3 ~1 of an acetone solution containing 1.17 ,ug of [‘%]hexachlorophene (30.4 ng/rnn-?). The treated skin was uncovered and the animals restrained. ’ Three animals and one skin segment per animal. Each skin segment was treated with 3 ~1 of an acetone solution containing 1.02 pg of [%]hexachlorophene (26.5 rig/mm’). The treated skin was protected by loosely fitting non-adhering tape. f Four animals and two skin segments (38.5 mm’) per animal.
got ‘++.T
A I
70 60
::L0
4
8
12 16 20 24 TIME
(hours)
FIG. 1. Recovery of radioactivity from the skin of rats treated with [%]hexacNorophene, mean + SD. Exposure conditions as described in Table 1. (A), Adult females (0-e); and adult males O---O). (B), Weanling males (O-O); and newborn males (o---o).
PERCUTANEOUS
ABSORPTION
385
OF HEXACHLOROPHENE
ciably lower in newborns. Absorption through the skin by hexachlorophene at the concentrations tested seemed to follow first-order kinetics (Fig. 1). The rate of absorption was concentration dependent, as to be expected by Fick’s Law (Blank, 1969), since total absorption of hexachlorophene was substantially inTABLE INFLUENCE
OF VEHICLE
ON
THE PENETRATION
2 OF
THROUGH RAT
[14C]H~~~~~~~~~~~~~~
SKIN
Recovery of applied radioactivity Exposure time
(%)
Dimethyl
(hr)
EthanoI*
Corn 0iI”
Aqueous sodium lauryl suifate (1 %Y,&
0
102.7 + 2.6
99.1 + 2.6 95.6 rt 2.0 97.1 * 0.4
100.5 f 2.5 97.3 f 2.6 98.8 + 0.1
91.4 + 4.0 88.8 +_ 0.8 71.6 _+ 2.3
82.7 + 4.4 66.3 + 1.2
92.3 + 0.4 78.6 _+ 4.7
60.8 + 1.5 45.0 + 1.5
4 8 12 16 24
96.4 91.7 88.9 75.3
+ + + *
0.5 3.9’ 1.1 2.2
suifoxide”
UMean + SD of four adult animals and four skin segments (38.5 mm2) per animal. Each skin segment was treated with 3 ~1 of the indicated solution containing 0.92-1.02 Pg of [WJhexachlorophene (23.9-26.5 ng/mn?). The treated skin was uncovered and the animals restrained. b Adult males. c Adult females. d Three animals and four skin segments (38.5 mm’) per animal. e Four animals and two skin segments (38.5 mm’) per animal. TABLE
3
EXTRACTABLE AND BOUND [14C]H~~~~~~~~~PHENE FROM RAT SKIN AFTER WASHING WITH ACETONE” Percentage Exposure time fW
0 4 8 16 24
of applied
Skin content after washing 13.0 25.9 27.3 28.0 24.8
+ 6.3 f 1.6 + 1.3 + 4.2 _+ 0.7
radioactivity*
Acetone wash 89.5 61.3 59.1 34.7 37.0
+ + * -c +
6.7 11.7 2.2 3.4 11.5
a Each skin segment (38.5 mm*) was treated with 3 ,ul of an acetone solution containing 1.03 fig of [‘*Clhexachlorophene (26.8 ng/mm2). The treated skin was uncovered and the animals restrained. b Mean f SD of two adult female animals and two skin segments per animal.
386
NAKAUE AND BUHLER
creased when a lo-fold greater amount of the bisphenol was applied to the animals (Table 1). The greatest penetration of [14C]hexachlorophene occurred when the bisphenol was dissolved in DMSO and 1 “/o aqueous sodium lauryl sulfate was the poorest vehicle (Table 2). As previously observed with insecticides (O’Brien and Dannelly, 1965), acetone also gave good skin penetration of [r4C]hexachlorophene (Table 1). When an acetone solution of [‘4C]hexachlorophene was applied to the skin and then immediately washed with acetone, 13 % of the applied dose remained bound to the tissue (Table 3). As the exposure period was prolonged, however, a more or less constant 27% of the applied radioactivity was retained on the skin segments, even though appreciable percutaneous absorption of the [14C]hexachlorophene had occurred. PercutaneousAbsorption of [14C]Hexachlorophene
With acetone as the vehicle, radioactivity from topically applied [14C]hexachlorophene appeared in the blood of adult and weanling rats within 1.5 hr after application TABLE
4
PERCUTANEOUS ABSORPTION OF [14C]H~x~CHLOROPHENE THROUGH THE SKIN OF ADULT ANDWEANLING MALERATS
Exposure time (hd 0 1.5 3 6 12 24
Plasma hexachlorophene concentration (rig/g plasma) Adultb
Weanling”
2+ 0 26_+ 0 44 * 22 73 f 16 83+_31 67 _+ 36
2t 0 9F 3 33 + 10 63f 5 87&31 49* 9
n Mean k SD of three blood samples taken from adult or weanling male rats. The treated skin was protected as described in Methods. Plasma radioactivity presented as nanograms of hexachlorophene equivalent/gram of plasma. b Three adult males weighed between 240 and 258 g and were equipped with surgically implanted cannulas. Skin areas (255 mm2) on each animal were treated with 40 ~1 of an acetone solution containing 47.4 pg of [%]hexachlorophene at a dose of 0.184.20 rig/kg (186 rig/mm’). Blood samples were taken at the indicated times. c Eighteen male weanling rats weighed between 51 and 81 g. Skin areas (227 rrm?) on each animal were treated with 24-10 01 of an acetone solution containing 10.2-16.2 @of [%]hexachlorophene at a dose of0.20mg/kg(44.9-71.4ng/mm2). Groups of three rats were sacrificed at the indicated times for blood samples.
PERCUTANEOUS
ABSORPTION
387
OF HEXACHLOROPHENE
(Table 4). Adults and weanlings exposed to somewhat different concentrations of hexachlorophene on the skin (but comparable O.Zmg/kg doses) showed similar plasma radioactivity concentrations (Table 4). Maximum plasma values of 83 and 87 ng of hexachlorophene equivalent/g of plasma, respectively, were reached at I2 hr. Distribution
and Excretion of Percutaneously
Absorbed [‘4C]Hexachlorophene
Percutaneous absorption of [14C]hexachlorophene by rats was increased when a larger area of the skin was treated with the radioactive bisphenol (Table 5). Absorption of hexachlorophene occurred quite rapidly since 59 % of the applied dose penetrated TABLE EXCRETION
OF PERCU~ANKKJSLY
5
ABSORBED
[WIHEXACHL~ROPHENE
IN MALE
RATS
Percentage of applied radioactivity” Adultb Fraction Treated skin Amount absorbed (by difference) Urine O-12 hr 12-24 hr Feces O-12 hr 12-24 hr
8-Hr exposure”
Weanling’
24-Hr exposure
24-Hr exposure
27.48 4 2.49
18.02 + 3.61
12.52 _+ 2.49
81.98 + 3.61
0.47 + 0.57
1.32 + 0.19 1.49 _+ 0.53
1.33 _+ 0.33 2.92 f 0.60
0.64 + 0.57 8.34 + 3.99
2.54+ 2.11 12.21 _+ 10.63
3.83 + 2.92 31.64 &- 4.~4
40.38
+_ 11.36
59.51 + 11.36
0.64 + 0.61
d Mean f SD of three male rats. The treated skin wasprotected as describedin Methods. bAdult male rats (220-233 g). Each skin segment (840 mm*) wastreated with 100 ~1of an acetone solution containing 35.0-38.2 fig of [‘%]hexachlorophene at a dose of 0.16 mg/kg (43.2 ng/mn?). cWeanling male rats (75-83 g). Each skin segment (227 mm’) wastreated with 60 ~1of an acetone solution containing 11.1-13.5 pg of [‘4C]hexachloropheneat a doseof 0.16 mg/kg (62.1ng/mm2). d Treated
skin
area removed at 8 hr and the remaining skin sutured together.
through the skin of adult males within 8 hr. Perhaps because the radioactive bisphenol was applied at a higher concentration over a smaller skin area in weanling rats, absorption from the skin was somewhat higher in these animals than was observed in the adult rats. In addition, the weanling rats excreted a higher percentage of the applied dose than did the adult rats. Most of the radioactivity from percutaneously absorbed [L4C]hexachlorophene was excreted in the feces. Total recoveries of the topically applied material were 2.6 and 4.3 % of the dose in urine and 14.8 and 35.5 % in feces of adult and weanling males, respectively. Tissue distribution of the percutaneously absorbed [14C]hexachlorophene was similar in adult, weanling, and newborn rats (Table 6). Higheskxadioactivity counts were found in liver, kidney, plasma, and the intestinal tract and contents. In addition, appreciable
12.1+ 2.4 194 III 80 74.9 + 1.6 106 f 12 20.5 &- 1.3 2.7 -+ 0.4 8.8 & 1.1 7.2 + 0.9
3.1 * 0.2
5.9 + 1.6
5.3 + 6.1
2.1 + 0.0 83.9 3~71.8 30.6_+15.4 37.4 + 20.9 4.0f 0.6 1.4+ 0.6 1.4+ 0.1
7.7k 1.6 42.5 f. 2.0 4.7+ 1.4
24-Hr exposure
2.4k 1.1 10.0 + 0.6 5.4 f 3.5
8-Hr exposuree
[14C]H~~~~~~~~~~~~~~
IN MALE
with 10 pl of an acetone solution containing
with 60 pl of an acetone solution containing
Animals sacrificed at 24 hr.
3.7 + 2.2 49.1 -t 3.5
20.3 + 8.9 76.3 _+11.9 37.4 f 15.3 62.5 -t 16.6 41.3 + 9.8 35.5 t 9.6 11.6+ 8.8
1.6_+ 2.2 85.8 + 22.0 9.1 ? 0.6
presented as
-___.
Newborn* __.-. __ 24-Hr exposure
IOOpJ of an acetone solution containing [‘“CJ-
in Methods. Tissue radioactivity
76.8f- 90.4 19.3+ 6.1
0.1
3.3 11.9 2.1 92
409 + 19 30.1+ 2.4
16.8 + 51.1 + 10.3 * 309 + 13.9 +
24-Hr exposure
20.5 + 8.3 163 561 79.8 + 3.5 116 +12 33.7 + 6.2 9.8 + 2.7 13.1+ 6.1
Weanling’
___-
RATS
44.6 + 0.5 430 rt 81 87.9 + 6.7 191 + 19.8 82.0 f 16.5 19.6f 4.2 20.8 + 2.6
21.2 +_ 3.1 106 + 13 23.7If: 18.2 780 +318 83.2 + 41.1
8-Hr exposuref
a Mean+ SD of two or three male rats. Treated skin protected as described nanograms of hexachlorophene equivalent/gram of wet tissue. b Adult male rats (220-233 g). Each skin segment (840 mm? was treated with hexachlorophene at a dose of 0.16 mg/kg (43.2 ng/mm2). c Weanling male rats (65-93 g). Each skin segments (227 mmz) was treated [“‘Clhexachlorophene at a dose of 0.16 mg/kg (62.1 rig/mm’). d Newborn male rats (6.1-6.4 g). Each skin segment (38.5 mmz) was treated [‘%]hexachlorophene at a dose of 1.28 mg/kg (206 rig/mm’). e Treated skin area removed at 8 hr and the remaining skin sutured together. f Animals sacrificed at 8 hr.
Heart Smallintestines (and contents) Kidney Liver Lung Muscle Spleen Stomach(and contents) Testes
Blood cells Plasma Brain Caeca Fat
Tissue
ABSORBED
6
Tissue hexachlorophene concentration (rig/g)
OF PERCUTANEOULSY
Adultb
TISSUE DISTRIBUTION
TABLE
:m iz D s r” B
5
PERCUTANEOUS
ABSORPTION
OF HEXACHLOROPHENE
389
amounts of the percutaneously absorbed hexachlorophene or its metabolites were also found in the stomach of the topically exposed animals, perhaps resulting from coprophagy. DISCUSSION
By means of the “disappearance” technique (O’Brien and Dannelley, 1965; Grass0 and Lansdown, 1972), we have shown that hexachlorophene is readily absorbed through the skin ofadult and weanling rats (Table 1). It was impossible to maintain the newborn rats for a 24-hr period in the absence of the mothers, consequently, it was necessary to protect the hexachlorophene treatment zone with tape to prevent the mother from licking the bisphenol from the skin. Under these circumstances, the partial occlusion of the skin could have enhanced the rate of hexachlorophene absorption. Nevertheless, skin penetration was somewhat less with the newborn animals than that found in the weanlings and adults. These observations are at variance with those in humans for other chemicals where an increased penetration of the skin has been observed in children (Idson, 1971). The reason for the reduced rate of absorption in newborn rats is not known, but the lipid content of rat skin varies inversely with age and the skin of newborn rats contains the highest concentration of lipids (Rothman, 1954). Since addition of cholesterol or other lipids to the skin is known to decrease the penetration of lipidsoluble substances (Rothman, 1954), the high lipid content of newborn rat skin may decrease the rate of hexachlorophene absorption. Conversely, decreasing the lipid content of the skin by defatting also reduces the absorption and increases the retention of various other chemicals (Rothman, 1954) or hexachlorophene (Fahlberg et al., 1948). Under these latter circumstances, the strong binding affinity of hexachlorophene to proteins (Flores and Buhler, 1974) may lead to a greater association of the bisphenol with skin proteins (Compeau, 1960). An appreciable amount of the topically applied [‘4C]hexachlorophene in the present investigation was strongly associated with the skin tissue since it could not be removed by rinsing with a polar solvent (Table 3). Absorption through the skin by hexachlorophene at the concentrations tested apparently occurs via a first-order process (Fig. l), following kinetics similar to those observed with the absorption of the bisphenol via the gastric mucosa (Buhler et al., 1976). Penetration of the insecticides dieldrin and DDT through rat skin also proceeds via similar kinetics (O’Brien and Dannelley, 1965). Feldman and Maibach (1970) found that percutaneous absorption of hexachlorophene in humans was quite low in comparison to a variety of other chemicals. The nature of the vehicle, however, has an important influence on the rate of skin penetration by chemicals. In the present study, greatest absorption ofhexachlorophene wasfound when DMSO was the solvent, acetone gave somewhat lower absorption, and the least absorption occurred with aqueous sodium lauryl sulfate. Percutaneous absorption of a number of drugs or chemicals is enhanced when DMSO (Stoughton and Fritsch, 1964; Feldman and Maibach, 1968) or acetone (O’Brien and Dannelley, 1965) is used as a vehicle. While such solvents increase the permeability of the skin to various chemicals, the mechanism for this effect is somewhat obscure (Idson, 1971). Skin is quite permeable to these solvents alone (Scheuplein and Blank, 1971), and it may be important to note 14*
390
NAKAUE
AND
BUHLER
that hexachlorophene strongly hydrogen bonds to DMSO and acetone (Haque and Buhler, 1972). Such solvents may also modify the conformation and hence permeability of the skin components (Idson, 1971). Although the absorption of a hexachlorophene suspension in water was not tested in the present study, incorporation of sodium lauryl sulfate presumably also increases skin absorption of the bisphenol to some extent. Following skin penetration, percutaneously absorbed [14C]hexachlorophene entered the blood where maximum radioactivity counts occurred at 12 hr (Table 4). By contrast, maximum blood radioactivity appeared 6 hr following oral administration of [‘“Clhexachlorophene to rats (Buhler et al., 1976). The relative distribution of radioactivity in the various tissues and organs of rats following dermal application of [14C]hexachlorophene (Table 6) was quite comparable to that found in rats after intraperitoneal or oral dosing (Buhler et al., 1976). Maximum concentrations of hexachlorophene and its metabolites were found in the gastrointestinal tract, liver, and plasma. As previously observed with intraperitoneally or orally dosed rats (Buhler et al., 1976), most of the radioactivity from percutaneously absorbed [14C]hexachlorophene was excreted in the feces and only small amounts were found in urine of treated rats (Table 5). Weanling rats excreted a substantially higher percentage of radioactivity from the topically applied [‘4C]hexachlorophene than did adult animals. A more rapid rate of hexachlorophene biotransformation and excretion in weanling rats could explain the previously observed lower acute toxicities of hexachlorophene in such animals as compared to adult rats (Nakaue et al., 1973). ACKNOWLEDGMENT The authorswish to thank Dr. F. N. Dost for his guidanceon the rat surgicalprocedures. REFERENCES L. M., BUCKFIELD, P. M., FERRY, D. G., MALCOLM, D. S. AND MCQUEEN, E. G. (1972).Blood levelsof hexachlorophenein neonates.Aust. Paediat. J. 8,246249. BLACK, J. G., SPROTT,W. E., HOWES,D. AND RUTHERFORD, T. (1974). Percutaneousabsorption of hexachlorophene.Toxicology 2, 127-139. BLANK, 1. H. (1969). Transport acrossthe stratum corneum. In Evaluation of Safety of Cosmetics (C. S. Weil and A. Rostenberg,Eds.), pp. 23-29. Toxicol. Appl. Pharmacol. ABBOTT,
Suppl. 3.
BUHLER,D. R., DUST, F. N., RASMUSSEN, M. E. AND GANDOLFI, A. J. (1976).The disposition and biotransformation of hexachlorophenein the rat. Xenobiotica, in press. BUTCHER, H. R., BALLINGER, W. F., GRAVENS, D. L., DEWAR,N. E., LEDLIE,E. F. AND BARTHEL,W. F. (1973).Hexachloropheneconcentrationsin the blood of operating room personnel.Arch. Surg. 107,70-74. CARROLL, F. E., SALAK, W. W., HOWARD,J. M. AND PAIRENT, F. W. (1967).Absorption of antimicrobial agentsacrossexperimentalwounds.Surg. Gynecof. Obstet. 125,974-978. C~MPEAU,G. M. (1960).The adsorptionof dodecylbenzene-sulfonate and hexachlorophene on the skin. J. Amer. Pharm. Assoc. Sci. Ed. 49,574-580. CURLEY, A., HAWK, R. E., KIMBROUGH, R. D., NATHENSON, G. AND FINBERG, L. (1971). Dermal absorptionof hexachlorophenein infants. Lancet 2,296-297. FAHLBERG, W. J., SWAN, J. C., AND SEASTONE, C. V. (1948).Studieson the retention of hexachlorophene(G-l 1) in humanskin. J. Bacterial, 56, 323-328. FELDMANN, R. J. ANDMAIBACH,H. I. (1968).Percutaneouspenetrationin vivo in man. Toilet Goods Ass., 189-203.
PERCUTANEOUSABSORPTION OF HEXACHLOROPHENE
391
R. J. AND MAIBACH, H. I. (1970). Absorption of some organic compounds through the skin in man. J. Invest. Dermatol. 54, 399404. FLORES, G. AND BUHLER, D. R. (1974). Hemolytic properties of hexachlorophene and related chlorinated bisphenols. Biochem. Pharmacol. 23, 1835-1843. GAINES, T. B., KIMBROUGH, R. D. AND LINDER, R. E. (1973). The oral and dermal toxicity of hexachlorophene in rats. Toxicol. Appl. Pharm. 25, 332-343. GANDOLFI, A. J. AND BUHLER, D. R. (1974). Biliary metabolites and enterohepatic circulation of hexachlorophene in the rat. Xenobiotica 4,693-704. GRASSO, P. AND LANSDOWN, A. B. G. (1972). Methods of measuring, and factors affecting, percutaneous absorption. J. Sot. Cosmet. Chem. 23, 481-521. HAQUE, R. AND BUHLER, D. R. (1972). A proton magnetic resonance study of the interaction of hexachlorophene with amides and polypeptides, J. Amer. Chem. Sot. 94, 1824-1828. IDSON, B. (1971). Biophysical factors in skin penetration. J. Sot. Cosmet. Chem. 22, 615-634. KIMBROUGH, R. D. AND GAINES, T. B. (1971). Hexachlorophene effects on the rat brain. Arch. Environ. Health 23, 114-l 18. KIMMEL, C. A., MOORE, W., HYSELL, D. K. AND STARA, J. F. (1974). Teratogenicity of hexachlorophene in rats. Arch. Environ. Health 28, 43-48. MAHIN, D. T. AND LOFBERG, R. T. (1966). A simplified method of sample preparation for determination of tritium, carbon-14, or sulfur-35 in blood or tissue by liquid scintillation counting. Anal. Biochem. 16, 500-509. MANOWITZ, M. ANDJOHNSTON, V. D. (1967). Deposition of hexachloropheneon the skin. J. Sot. Cosmet.Chem.l&527-536. NAKAUE,H. S., DOST,F. N., ANDBUHLER, D. R. (1973).Studieson the toxicity of hexachlorophenein the rat. Toxicol. Appl. Pharmacol.24,239-249. O’BRIEN,R. D. ANDDANNELLEY, C. E. (1965). Penetration of insecticidesthrough rat skin. J. Agr. Food Chem.13,245-247. ROTHMAN,S. (1954). Physiology and Biochemistry of the Skin, pp. 26-59. University of ChicagoPress,Chicago. SCHEUPLEIN, R. J. ANDBLANK,I. H. (1971).Permeabilityof the skin.Physiol. Rev.51, 702-747. STOUGHTON, R. B. AND FRITSCH,W. (1964). Influence of dimethylsulfoxide (DMSO) on human percutaneousabsorption. Arch. Dermatol. 90,512-517. TABER, D., LAZANAS, J. C., FANCHER, 0. E. ANDCALANDRA,J. C. (1971). The accumulation and persistenceof antibacterial agentsin human skin. J. Sot. Cosmet.Chem.22, 369-377. TAYLOR, T., CHASSEAUD, L. F., DOWN,W. H. ANDMEDD,R. K. (1972).The percutaneousabsorption of hexachloropheneby piglets.Food Cosmet.Toxicol. 10, 857-859. ULSAMER, A. G., MARZULLI,F. N. ANDCOEN,R. W. (1973).Hexachloropheneconcentrations in blood associatedwith the use of products containing hexachlorophene.Food Cosmet. Toxicol. 11, 625-633. FELDMANN,
Percutaneous
Absorption
35,381-391(1976)
of Hexachlorophene
in the Rat’
HARRY S. NAKAUE~AND DONALDR. BUHLER Department of Agricultural Chemistry and Environmental Health Sciences Center, Oregon State University, Corvallis, Oregon 97331 Received August 8,1975; accepted September 26,1975
PercutaneousAbsorption of Hexachlorophenein the Rat. NAKAUE, H. S., BUHLER, D. R. (1976). Toxicol. Appl. Pharmacol. 35, 381-391. [“Clhexachlorophenewasapplied to intact, clipped skinsof adult rats in four different solventsand an aqueousdetergentsolution. As much as 55% of the appliedradioactivity wasabsorbedthrough the skin in a 24-hr period. Dimethyl sulfoxide enhancedabsorption of the germicideto the greatest extent and aqueoussodiumlauryl sulfate(1 ‘A, w/v) wasthe leasteffective. With both adult and weanlingrats, the first measurableamount of radioactivity appearedin the venous blood in lessthan 1.5 hr, and maximum values were attained 12 hr after topical application of [14C]hexachlorophene.Approximately 27% of the appliedradioactivity remainedboundto the skin after 4 and 24 hr and wasnot removed by washingwith acetone. Distribution of radioactivity in rats 8 and 24 hr after topical application of [14C]hexachlorophenewas similar in weanling and adult rats. Most of the absorbedradioactivity appearedin the plasma,liver, smallintestine, AND
caecum, stomach, kidney, urine, and feces. The chlorinated antibacterial agent, hexachlorophene, has been employed in a variety of soap and cosmetic products. Use of such preparations provides a residual antibacterial effect through the deposition and retention of small amounts of hexachlorophene on the skin (Compeau, 1960; Manowitz and Johnston, 1967; Taber et al., 1971). Following topical exposure to preparations containing hexachlorophene, significant amounts of the bisphenol have been detected in the blood of human infants (Curley et al., 1971; Abbott et al., 1972) and adults (Feldman and Maibach, 1970; Butcher et al., 1973; Warner et al., 1973). Because of its toxicity in animals (Gaines et al., 1973; Kimmel ei al., 1974), particularly the associated cerebral edema (Kimbrough and Gaines, 1971; Nakaue et al., 1973), use of hexachlorophene as a topical bactericide has been greatly curtailed. Percutaneous absorption of hexachlorophene has also been reported in the rat (Carrol et al., 1967), pig (Taylor et al., 1972), and guinea pig (Black et al., 1974). In the present research, we have investigated the influence of various solvents and of age on the percutaneous absorption of [‘4C]hexachlorophene by the rat. The tissue distribution and excretion of dermally absorbed hexachlorophene is also reported. 1 This work was supportedby grantsNo. FD 00041and ES00210,U.S. PublicHealth Service, National Institutes of Health. Manuscript issued as Technical Paper No. 4026 from the Oregon Agricultural Experiment Station. z Present address: Department of Poultry Science, Oregon State University, Corvallis, Oregon 97331. 381 Copyright Q 1976 by Academic Press, Inc. All rights of reproduction Printed in Great Britain
in any form reserved.
382
NAKAUE
AND
BUHLER
METHODS
Chemicals.Hexachlorophene [2,2’-methylenebis (3,4,6-trichlorophenol)], USP3 was recrystallized prior to use [Methylene-14C]-hexachlorophene (sp act, 2.71 mCi/mmol) was obtained from the Mallinckrodt Chemical Co., St. Louis, Missouri, and was shown to be chemically and radiochemically pure by appropriate tic and glc analyses (Gandolfi and Buhler, 1974). Reagent grade chemicals and solvents were employed in all experiments. Penetration of [14C]hexachlorophenethrough the skin. Random-bred Wistar albino rats from a closed colony were used to study the penetration of [14C]hexachlorophene through the skin by the technique of O’Brien and Dannelley (1965). Groups of three or four weanling male (60-83 g) or adult male (250-300 g) or female (206-240 g) rats were lightly anesthetized with ethyl ether or by a 25 mg/kg ip injection of sodium pentobarbital. The ventral pelage from the front to the hind legs was clipped, and the animals were carefully shaved, avoiding damage to the skin. Approximately 20 circular skin areas (38.5 mm’) were then marked on the skin of each rat by means of a rubber stamp. Three-microliter aliquots containing 0.92-l .02 pg of [14C]hexachlorophene in the indicated solvents were applied in a random pattern to at least two marked zones on each of the test animals. During the experimental period, the animals were restrained and protected from air currents in an enclosure. Rats were exposed to the radioactive drug for different time periods up to 24 hr with the application times being adjusted so that all the exposure periods ended simultaneously. All of the marked skin areas were then removed at the same time with a surgical scissors and placed in individual counting vials. Zero-time exposure samples were prepared by removing untreated skin sections from the rats, placing the tissues in counting vials and immediately applying 3-~1 aliquots of the appropriate [14C]hexachlorophene solution directly to the skin. A single circular area (38.5 mm’) was marked on the stomachs of unanesthetized l- or 2-day-old male newborn rats (5.6-7.1 g). At various times, the marked skin areas ,of three animals were then treated with 3 ~1 of an acetone solution containing 0.95 pg of [14C]hexachlorophene. The treated skin areas were protected with masking tape, the adhesive areas over the marked zone being covered loosely with a second piece of tape applied like a large Band-Aid, and the animals were returned to their mothers. Zerotime exposure samples were prepared by removing marked skin sections from the backs of treated animals and applying 3-yl aliquots of the [14C]hexachlorophene directly to the skin segments. To determine the amount of extractable and bound radioactivity in the skin segments, 3 ~1 of an acetone solution containing 1.02 ,ug of [14C]hexachlorophene was applied at various times to four marked areas (38.5 mm’) on each of three female rats. Two of the skin areas for each exposure period were removed from each animal, washed with 2 ml of acetone and the washings and washed skin segments then counted. The remaining two skin sections were removed and counted directly. Percutaneous absorption of [‘4C]hexachlorophene. Three 240-258-g adult male rats were surgically prepared by insertion of cannulas (6 x 290 mm) in the posterior vena cava. One week post-surgery, the shaved and marked areas (255 mm”) on each animal were exposed to 40-~1 aliquots of an acetone solution containing 47.4 pg of [r4C]hexa3 A gift of the Givaudan Corporation,
Clifton, New Jersey.
PERCUTANEOUS
ABSORPTION
OF HEXACHLOROPHENE
383
chlorophene. The treated skin areas were then protected by application of concave plastic guards glued to the skin. At O1l&3,6,12, and 24 hr, a 0.5-ml aliquot of blood was withdrawn and added to heparinized tubes, and the plasma and red cells were then separated by centrifugation. Male weanling rats (51-81 g) were shaved, marked skin areas (227 mm2) treated with 25-40+1 aliquots of an acetone solution containing 10.2-16.2 pg of [14C]hexachlorophene, and the treated skin protected with plastic guards. At 1, 1.5,3,6, 12, and 24 hr, groups of three rats were sacrificed. Blood was obtained directly from the posterior vena cava, and the treated skin area was removed and counted. Plasma was obtained from the heparinized blood after centrifugation. Distribution and excretion of percutaneously absorbed [14C]hexachlorophene. The ventral surfaces of groups of three adult (220-233 g) or weanling (55-83 g) male rats were clipped and shaved. The animals were then treated topically over an 840-mm2 (adult) and 227-mm2 (weanling) skin areas with 0.16 mg/kg of [‘4C]hexachlorophene of 35.0-38.2 pg in 100 ~1 (adult) or 11.1-l 3.5 jig in 60 ,uI (weanling) of acetone. The treated areas were protected by concave plastic guards glued to the skin and the animals then placed in individual metabolism cages for collection of urine and feces over a 24-hr period. Following 8 hr of topical exposure, the treated skin was removed from the third group of two adult male rats, the remaining skin sutured together and the animals then returned to their metabolism cages. All three groups of animals were then sacrificed after 24 hr for removal of the various tissues and organs. A fourth group of three male weanling rats was topically treated with the same [‘4C]hexachlorophene dose and sacrificed after 8 hr. A fifth group of three 6.1-6.4-g male newborn rats was treated over a 38.5mm2 areas with 10 ~1 of an acetone solution containing 1.28 mg/kg (8.0 pg) of [r4C]hexg chlorophene. The treated areas were protected with masking tape as described previously and the animals returned to their mothers until sacrificed at 24 hr. Measurement of radioactivity. Skin and tissue samples were solubilized by the procedure of Mahin and Lofberg (1966). Tissue digests and other radioactive solutions were counted in a Packard Model 3375 liquid scintillation spectrometer (Packard Instrument Company, Downers Grove, Illinois) following addition of counting solution containing 500 ml of toluene, 300 ml of Cellosolve and 3 g of PPO. Counting efficiencies were determined by automatic external standardization. Plasma and tissue radioactivities were either calculated as micrograms of hexachlorophene equivalent per gram wet weight or were presented as percentages of the topical dose.
RESULTS Penetration of [‘4C]HexachIorophene
through the Skin
Absorption of [‘4C]hexachlorophene through rat skin apparently occurred at a relatively rapid rate since as much as 53 % of the applied radioactivity disappeared from the skin segments over a 24hr period (Table 1). The rate of penetration of hexachlorophene through the skin of female rats appeared to be slightly slower than that observed with males. Contrary to observations with other compounds (Idson, 1971), absorption of hexachlorophene through the skin did not increase with decreasing age. In fact, skin penetration was quite comparable in adults and weanlings and was appre-
NAKAUEANDBUHLER
384
TABLE 1 INFLUENCE OF AGE AND SEX ON THE PENETRATION OF [“‘CIHEXACHLOROPHENE SKIN
THROUGH RAT
Recoveryof appliedradioactivity (%) Exposure time 0-d 0 4 8 12 16 24
Adult femalesb
Adult fernaleC
98.2 + 2.5 97.2 +_ 2.1 83.4 f 10.0
101.2* 1.5 91.0+ 6.7 94.2zk1.3
68.4 f 5.4 55.1 2 6.8
75.6+ 2.5 61.8& 3.2
Adult malesb 98.5 2 88.6? 76.9 + 67.1f 60.1 + 46.6f
1.7 1.0
1.0 14Sf 14.8 11.8
Weanling malesd
Newborn malese
99.5f 1.8 85.6+ 2.1 77.3 f 11.6
98.1 + 3.4 78.2 + 2.2 70.5 + 14.0
65.5f 23.3 46.8 +_16.1
71.0& 3.8 59.9 + 5.3
a Mean + SD of four animals and four skin segments (38.5 mm2) per animal. b Each skin segment was treated with 3 ~1 of an acetone solution containing 0.92-1.02 pugof [14C]hexachlorophene (23.9-26.5 ng/mm2). The treated skin was uncovered and the animals restrained. c Each skin segment was treated with 3 ~1 of an acetone solution containing 1.02 pg of [‘%]hexachlorophene and 10.2 pg of nonradioactive hexachlorophene (291 ng/rnm’). The treated skin was uncovered and the animals restrained. d Each skin segment was treated with 3 ~1 of an acetone solution containing 1.17 ,ug of [‘%]hexachlorophene (30.4 ng/rnn-?). The treated skin was uncovered and the animals restrained. ’ Three animals and one skin segment per animal. Each skin segment was treated with 3 ~1 of an acetone solution containing 1.02 pg of [%]hexachlorophene (26.5 rig/mm’). The treated skin was protected by loosely fitting non-adhering tape. f Four animals and two skin segments (38.5 mm’) per animal.
got ‘++.T
A I
70 60
::L0
4
8
12 16 20 24 TIME
(hours)
FIG. 1. Recovery of radioactivity from the skin of rats treated with [%]hexacNorophene, mean + SD. Exposure conditions as described in Table 1. (A), Adult females (0-e); and adult males O---O). (B), Weanling males (O-O); and newborn males (o---o).
PERCUTANEOUS
ABSORPTION
385
OF HEXACHLOROPHENE
ciably lower in newborns. Absorption through the skin by hexachlorophene at the concentrations tested seemed to follow first-order kinetics (Fig. 1). The rate of absorption was concentration dependent, as to be expected by Fick’s Law (Blank, 1969), since total absorption of hexachlorophene was substantially inTABLE INFLUENCE
OF VEHICLE
ON
THE PENETRATION
2 OF
THROUGH RAT
[14C]H~~~~~~~~~~~~~~
SKIN
Recovery of applied radioactivity Exposure time
(%)
Dimethyl
(hr)
EthanoI*
Corn 0iI”
Aqueous sodium lauryl suifate (1 %Y,&
0
102.7 + 2.6
99.1 + 2.6 95.6 rt 2.0 97.1 * 0.4
100.5 f 2.5 97.3 f 2.6 98.8 + 0.1
91.4 + 4.0 88.8 +_ 0.8 71.6 _+ 2.3
82.7 + 4.4 66.3 + 1.2
92.3 + 0.4 78.6 _+ 4.7
60.8 + 1.5 45.0 + 1.5
4 8 12 16 24
96.4 91.7 88.9 75.3
+ + + *
0.5 3.9’ 1.1 2.2
suifoxide”
UMean + SD of four adult animals and four skin segments (38.5 mm2) per animal. Each skin segment was treated with 3 ~1 of the indicated solution containing 0.92-1.02 Pg of [WJhexachlorophene (23.9-26.5 ng/mn?). The treated skin was uncovered and the animals restrained. b Adult males. c Adult females. d Three animals and four skin segments (38.5 mm’) per animal. e Four animals and two skin segments (38.5 mm’) per animal. TABLE
3
EXTRACTABLE AND BOUND [14C]H~~~~~~~~~PHENE FROM RAT SKIN AFTER WASHING WITH ACETONE” Percentage Exposure time fW
0 4 8 16 24
of applied
Skin content after washing 13.0 25.9 27.3 28.0 24.8
+ 6.3 f 1.6 + 1.3 + 4.2 _+ 0.7
radioactivity*
Acetone wash 89.5 61.3 59.1 34.7 37.0
+ + * -c +
6.7 11.7 2.2 3.4 11.5
a Each skin segment (38.5 mm*) was treated with 3 ,ul of an acetone solution containing 1.03 fig of [‘*Clhexachlorophene (26.8 ng/mm2). The treated skin was uncovered and the animals restrained. b Mean f SD of two adult female animals and two skin segments per animal.
386
NAKAUE AND BUHLER
creased when a lo-fold greater amount of the bisphenol was applied to the animals (Table 1). The greatest penetration of [14C]hexachlorophene occurred when the bisphenol was dissolved in DMSO and 1 “/o aqueous sodium lauryl sulfate was the poorest vehicle (Table 2). As previously observed with insecticides (O’Brien and Dannelly, 1965), acetone also gave good skin penetration of [r4C]hexachlorophene (Table 1). When an acetone solution of [‘4C]hexachlorophene was applied to the skin and then immediately washed with acetone, 13 % of the applied dose remained bound to the tissue (Table 3). As the exposure period was prolonged, however, a more or less constant 27% of the applied radioactivity was retained on the skin segments, even though appreciable percutaneous absorption of the [14C]hexachlorophene had occurred. PercutaneousAbsorption of [14C]Hexachlorophene
With acetone as the vehicle, radioactivity from topically applied [14C]hexachlorophene appeared in the blood of adult and weanling rats within 1.5 hr after application TABLE
4
PERCUTANEOUS ABSORPTION OF [14C]H~x~CHLOROPHENE THROUGH THE SKIN OF ADULT ANDWEANLING MALERATS
Exposure time (hd 0 1.5 3 6 12 24
Plasma hexachlorophene concentration (rig/g plasma) Adultb
Weanling”
2+ 0 26_+ 0 44 * 22 73 f 16 83+_31 67 _+ 36
2t 0 9F 3 33 + 10 63f 5 87&31 49* 9
n Mean k SD of three blood samples taken from adult or weanling male rats. The treated skin was protected as described in Methods. Plasma radioactivity presented as nanograms of hexachlorophene equivalent/gram of plasma. b Three adult males weighed between 240 and 258 g and were equipped with surgically implanted cannulas. Skin areas (255 mm2) on each animal were treated with 40 ~1 of an acetone solution containing 47.4 pg of [%]hexachlorophene at a dose of 0.184.20 rig/kg (186 rig/mm’). Blood samples were taken at the indicated times. c Eighteen male weanling rats weighed between 51 and 81 g. Skin areas (227 rrm?) on each animal were treated with 24-10 01 of an acetone solution containing 10.2-16.2 @of [%]hexachlorophene at a dose of0.20mg/kg(44.9-71.4ng/mm2). Groups of three rats were sacrificed at the indicated times for blood samples.
PERCUTANEOUS
ABSORPTION
387
OF HEXACHLOROPHENE
(Table 4). Adults and weanlings exposed to somewhat different concentrations of hexachlorophene on the skin (but comparable O.Zmg/kg doses) showed similar plasma radioactivity concentrations (Table 4). Maximum plasma values of 83 and 87 ng of hexachlorophene equivalent/g of plasma, respectively, were reached at I2 hr. Distribution
and Excretion of Percutaneously
Absorbed [‘4C]Hexachlorophene
Percutaneous absorption of [14C]hexachlorophene by rats was increased when a larger area of the skin was treated with the radioactive bisphenol (Table 5). Absorption of hexachlorophene occurred quite rapidly since 59 % of the applied dose penetrated TABLE EXCRETION
OF PERCU~ANKKJSLY
5
ABSORBED
[WIHEXACHL~ROPHENE
IN MALE
RATS
Percentage of applied radioactivity” Adultb Fraction Treated skin Amount absorbed (by difference) Urine O-12 hr 12-24 hr Feces O-12 hr 12-24 hr
8-Hr exposure”
Weanling’
24-Hr exposure
24-Hr exposure
27.48 4 2.49
18.02 + 3.61
12.52 _+ 2.49
81.98 + 3.61
0.47 + 0.57
1.32 + 0.19 1.49 _+ 0.53
1.33 _+ 0.33 2.92 f 0.60
0.64 + 0.57 8.34 + 3.99
2.54+ 2.11 12.21 _+ 10.63
3.83 + 2.92 31.64 &- 4.~4
40.38
+_ 11.36
59.51 + 11.36
0.64 + 0.61
d Mean f SD of three male rats. The treated skin wasprotected as describedin Methods. bAdult male rats (220-233 g). Each skin segment (840 mm*) wastreated with 100 ~1of an acetone solution containing 35.0-38.2 fig of [‘%]hexachlorophene at a dose of 0.16 mg/kg (43.2 ng/mn?). cWeanling male rats (75-83 g). Each skin segment (227 mm’) wastreated with 60 ~1of an acetone solution containing 11.1-13.5 pg of [‘4C]hexachloropheneat a doseof 0.16 mg/kg (62.1ng/mm2). d Treated
skin
area removed at 8 hr and the remaining skin sutured together.
through the skin of adult males within 8 hr. Perhaps because the radioactive bisphenol was applied at a higher concentration over a smaller skin area in weanling rats, absorption from the skin was somewhat higher in these animals than was observed in the adult rats. In addition, the weanling rats excreted a higher percentage of the applied dose than did the adult rats. Most of the radioactivity from percutaneously absorbed [L4C]hexachlorophene was excreted in the feces. Total recoveries of the topically applied material were 2.6 and 4.3 % of the dose in urine and 14.8 and 35.5 % in feces of adult and weanling males, respectively. Tissue distribution of the percutaneously absorbed [14C]hexachlorophene was similar in adult, weanling, and newborn rats (Table 6). Higheskxadioactivity counts were found in liver, kidney, plasma, and the intestinal tract and contents. In addition, appreciable
12.1+ 2.4 194 III 80 74.9 + 1.6 106 f 12 20.5 &- 1.3 2.7 -+ 0.4 8.8 & 1.1 7.2 + 0.9
3.1 * 0.2
5.9 + 1.6
5.3 + 6.1
2.1 + 0.0 83.9 3~71.8 30.6_+15.4 37.4 + 20.9 4.0f 0.6 1.4+ 0.6 1.4+ 0.1
7.7k 1.6 42.5 f. 2.0 4.7+ 1.4
24-Hr exposure
2.4k 1.1 10.0 + 0.6 5.4 f 3.5
8-Hr exposuree
[14C]H~~~~~~~~~~~~~~
IN MALE
with 10 pl of an acetone solution containing
with 60 pl of an acetone solution containing
Animals sacrificed at 24 hr.
3.7 + 2.2 49.1 -t 3.5
20.3 + 8.9 76.3 _+11.9 37.4 f 15.3 62.5 -t 16.6 41.3 + 9.8 35.5 t 9.6 11.6+ 8.8
1.6_+ 2.2 85.8 + 22.0 9.1 ? 0.6
presented as
-___.
Newborn* __.-. __ 24-Hr exposure
IOOpJ of an acetone solution containing [‘“CJ-
in Methods. Tissue radioactivity
76.8f- 90.4 19.3+ 6.1
0.1
3.3 11.9 2.1 92
409 + 19 30.1+ 2.4
16.8 + 51.1 + 10.3 * 309 + 13.9 +
24-Hr exposure
20.5 + 8.3 163 561 79.8 + 3.5 116 +12 33.7 + 6.2 9.8 + 2.7 13.1+ 6.1
Weanling’
___-
RATS
44.6 + 0.5 430 rt 81 87.9 + 6.7 191 + 19.8 82.0 f 16.5 19.6f 4.2 20.8 + 2.6
21.2 +_ 3.1 106 + 13 23.7If: 18.2 780 +318 83.2 + 41.1
8-Hr exposuref
a Mean+ SD of two or three male rats. Treated skin protected as described nanograms of hexachlorophene equivalent/gram of wet tissue. b Adult male rats (220-233 g). Each skin segment (840 mm? was treated with hexachlorophene at a dose of 0.16 mg/kg (43.2 ng/mm2). c Weanling male rats (65-93 g). Each skin segments (227 mmz) was treated [“‘Clhexachlorophene at a dose of 0.16 mg/kg (62.1 rig/mm’). d Newborn male rats (6.1-6.4 g). Each skin segment (38.5 mmz) was treated [‘%]hexachlorophene at a dose of 1.28 mg/kg (206 rig/mm’). e Treated skin area removed at 8 hr and the remaining skin sutured together. f Animals sacrificed at 8 hr.
Heart Smallintestines (and contents) Kidney Liver Lung Muscle Spleen Stomach(and contents) Testes
Blood cells Plasma Brain Caeca Fat
Tissue
ABSORBED
6
Tissue hexachlorophene concentration (rig/g)
OF PERCUTANEOULSY
Adultb
TISSUE DISTRIBUTION
TABLE
:m iz D s r” B
5
PERCUTANEOUS
ABSORPTION
OF HEXACHLOROPHENE
389
amounts of the percutaneously absorbed hexachlorophene or its metabolites were also found in the stomach of the topically exposed animals, perhaps resulting from coprophagy. DISCUSSION
By means of the “disappearance” technique (O’Brien and Dannelley, 1965; Grass0 and Lansdown, 1972), we have shown that hexachlorophene is readily absorbed through the skin ofadult and weanling rats (Table 1). It was impossible to maintain the newborn rats for a 24-hr period in the absence of the mothers, consequently, it was necessary to protect the hexachlorophene treatment zone with tape to prevent the mother from licking the bisphenol from the skin. Under these circumstances, the partial occlusion of the skin could have enhanced the rate of hexachlorophene absorption. Nevertheless, skin penetration was somewhat less with the newborn animals than that found in the weanlings and adults. These observations are at variance with those in humans for other chemicals where an increased penetration of the skin has been observed in children (Idson, 1971). The reason for the reduced rate of absorption in newborn rats is not known, but the lipid content of rat skin varies inversely with age and the skin of newborn rats contains the highest concentration of lipids (Rothman, 1954). Since addition of cholesterol or other lipids to the skin is known to decrease the penetration of lipidsoluble substances (Rothman, 1954), the high lipid content of newborn rat skin may decrease the rate of hexachlorophene absorption. Conversely, decreasing the lipid content of the skin by defatting also reduces the absorption and increases the retention of various other chemicals (Rothman, 1954) or hexachlorophene (Fahlberg et al., 1948). Under these latter circumstances, the strong binding affinity of hexachlorophene to proteins (Flores and Buhler, 1974) may lead to a greater association of the bisphenol with skin proteins (Compeau, 1960). An appreciable amount of the topically applied [‘4C]hexachlorophene in the present investigation was strongly associated with the skin tissue since it could not be removed by rinsing with a polar solvent (Table 3). Absorption through the skin by hexachlorophene at the concentrations tested apparently occurs via a first-order process (Fig. l), following kinetics similar to those observed with the absorption of the bisphenol via the gastric mucosa (Buhler et al., 1976). Penetration of the insecticides dieldrin and DDT through rat skin also proceeds via similar kinetics (O’Brien and Dannelley, 1965). Feldman and Maibach (1970) found that percutaneous absorption of hexachlorophene in humans was quite low in comparison to a variety of other chemicals. The nature of the vehicle, however, has an important influence on the rate of skin penetration by chemicals. In the present study, greatest absorption ofhexachlorophene wasfound when DMSO was the solvent, acetone gave somewhat lower absorption, and the least absorption occurred with aqueous sodium lauryl sulfate. Percutaneous absorption of a number of drugs or chemicals is enhanced when DMSO (Stoughton and Fritsch, 1964; Feldman and Maibach, 1968) or acetone (O’Brien and Dannelley, 1965) is used as a vehicle. While such solvents increase the permeability of the skin to various chemicals, the mechanism for this effect is somewhat obscure (Idson, 1971). Skin is quite permeable to these solvents alone (Scheuplein and Blank, 1971), and it may be important to note 14*
390
NAKAUE
AND
BUHLER
that hexachlorophene strongly hydrogen bonds to DMSO and acetone (Haque and Buhler, 1972). Such solvents may also modify the conformation and hence permeability of the skin components (Idson, 1971). Although the absorption of a hexachlorophene suspension in water was not tested in the present study, incorporation of sodium lauryl sulfate presumably also increases skin absorption of the bisphenol to some extent. Following skin penetration, percutaneously absorbed [14C]hexachlorophene entered the blood where maximum radioactivity counts occurred at 12 hr (Table 4). By contrast, maximum blood radioactivity appeared 6 hr following oral administration of [‘“Clhexachlorophene to rats (Buhler et al., 1976). The relative distribution of radioactivity in the various tissues and organs of rats following dermal application of [14C]hexachlorophene (Table 6) was quite comparable to that found in rats after intraperitoneal or oral dosing (Buhler et al., 1976). Maximum concentrations of hexachlorophene and its metabolites were found in the gastrointestinal tract, liver, and plasma. As previously observed with intraperitoneally or orally dosed rats (Buhler et al., 1976), most of the radioactivity from percutaneously absorbed [14C]hexachlorophene was excreted in the feces and only small amounts were found in urine of treated rats (Table 5). Weanling rats excreted a substantially higher percentage of radioactivity from the topically applied [‘4C]hexachlorophene than did adult animals. A more rapid rate of hexachlorophene biotransformation and excretion in weanling rats could explain the previously observed lower acute toxicities of hexachlorophene in such animals as compared to adult rats (Nakaue et al., 1973). ACKNOWLEDGMENT The authorswish to thank Dr. F. N. Dost for his guidanceon the rat surgicalprocedures. REFERENCES L. M., BUCKFIELD, P. M., FERRY, D. G., MALCOLM, D. S. AND MCQUEEN, E. G. (1972).Blood levelsof hexachlorophenein neonates.Aust. Paediat. J. 8,246249. BLACK, J. G., SPROTT,W. E., HOWES,D. AND RUTHERFORD, T. (1974). Percutaneousabsorption of hexachlorophene.Toxicology 2, 127-139. BLANK, 1. H. (1969). Transport acrossthe stratum corneum. In Evaluation of Safety of Cosmetics (C. S. Weil and A. Rostenberg,Eds.), pp. 23-29. Toxicol. Appl. Pharmacol. ABBOTT,
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