Toxicology, 3 (1975) 253--264 © Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
SKIN DEPOSITION AND P E N E T R A T I O N OF TRICHLOROCARBANILIDE
J.G. BLACK, D. HOWES and T. RUTHERFORD
Environmental Safety Division, Unilever Research Laboratory, Colworth House, Sharnbrook, Bedford (Great Britain) (Received May l l t h , 1974)
SUMMARY
Studies are reported on the localization and quantitative distribution of 3,4,4,_trichloro[14 C] carbanilide([14 C] TCC) in guinea-pig and human skin, and on the percutaneous absorption of TCC following topical application to guinea pigs. [14C]TCC was applied to guinea-pig skin in various vehicles (conventional, superfatted, 10% non-soap detergent (NSD) and 30% NSD soap suspensions or in N,N-dimethylformamide [DMF] ) and under various conditions (e.g. freshly prepared or equilibrated suspensions; single or multiple washes). Most of the amount of TCC remaining in the skin after rinsing was deposited on the skin surface and only relatively minute amounts actually penetrated through the epidermis into the dermis. Whereas conventional soap facilitated a greater deposition of TCC on the skin surface than NSD, the latter caused greater amounts of TCC to be deposited in the pilosebaceous system and lower dermis than conventional soap. The absence of TCC in the blood and tissues of guinea pigs given topical applications of TCC lends further support to the very low order of percutaneous absorption of TCC. The localisation of TCC followed a similar pattern in human skin as in guinea-pig skin, but the a m o u n t deposited was less and the rate of disappearance was more for human than for guinea-pig skin.
Abbreviations: BBOT, 2,5-bis(5-tertbutylbenzoxazol-2-yl)thiophene; DMF, N,N-dimethylformamide; NSD, non-soap detergent; PEG 400 ML, polyethylene glycol 400 monolaurate; TCC, 3,4,4r-trichlorocarbanUide.
253
INTRODUCTION TCC is included in toilet soap as a bacteriostatic agent designed to reduce the bacterial flora of the skin. Although a large proportion of the initial application of germicide is immediately removed during rinsing it is important to establish the extent to which the germicide remains in the skin and is available for absorption into the body. Autoradiographic and scintillation counting techniques have been employed to determine in guinea pig and man the quantitative distribution of [14C]TCC in various skin layers notably the surface (0--25 p), which comprises the stratum corneum and stubble hair, epidermis and dermis. In certain instances the levels of [14 C] TCC have been determined in blood, excreta and tissues of guinea pigs as an indication of absorption through skin. The importance of various factors that may affect the deposition of TCC has been investigated. These factors include the nature of the soap vehicles, equilibration of soap-germicide formulation before application and the presence of a solubilizer in the formulation, the concentration of TCC in the product, time interval between topical application and determination of TCC in skin and the number of washes. EXPERIMENTAL Materials
[14C-Carbonyl] TCC was synthesized and checked for purity as described by Rutherford and Black [1]. [14 C] TCC with specific activities of 1.5--14.6 pCi/mg was added to an 8% (w/v) aqueous suspension of a conventional soap, superfatted soap, NSD bars containing 10% or 30% of the synthetic detergent sodium alkoyl isethionate, or to DMF (GPR grade; Hopkin and Williams), to give a final germicide concentration of 0.08% (w/v), i.e. equivalent to 1% of the toilet bar. Superfatted soap suspensions 8% (w/v) were also prepared containing the equivalent of 0.1% and 2.0% (w/w) in the soap bar. All aqueous soap suspensions were made up with water containing 60 p p m CaCO3 (6 ° hardness). The TCC sample was usually ground to a fine powder and the soap suspension added. The preparation was gently homogenised in a Potter--Elvehjem homogeniser to give a very fine suspension of particles of TCC in the soap with the minimum of frothing. Equilibration of the TCC-soap suspension was achieved by shaking the preparation at 40 ° for a minimum of 24 h. The state of the TCC in the equilibrated suspension most closely resembles the state of the TCC in the toilet bar. A n i m a l s and t r e a t m e n t
The dorsal hair of groups o f 3 or 5 male Dunkin-Hartley guinea pigs (250 g b o d y weight approx.) was clipped and in some, but not all, experiments the animals were anaesthetised with ether. Aliquots (0.5 ml) of the germicide preparations were pipetted on to a marked area (20 cm 2) of the dorsal skin and rubbed gently for 2 or 10 min with a gloved finger. Another
254
group of 3 guinea pigs received 9 similar washes, 2 per day for 4 days and the ninth wash on the morning of the 5th day. In most experiments the skin was rinsed in situ before excision but in some others the excised skin was frozen on to a glass plate and rinsed [ 1]. The animals were killed either immediately or at 24 and 48 h or 2 and 4 weeks after the rinse. Surviving animals were placed in separate metabolism cages, and fed a pelleted diet and cabbage. The excreta were collected in polythene-lined trays beneath the cages in two 24 hourly lots. At the appropriate time the animals were killed by cervical dislocation and the treated area of skin, the lymph nodes of the groin, the liver, depot fat and heart blood samples taken. Skins were kept frozen until sections parallel to the surface were cut on a freezing microtome for scintillation counting and autoradiography as described by Rutherford and Black [1]. Whole skin 6 m m punch autopsies, in which the epidermis was separated from the dermis after exposure to 0.880 NH4OH for 10 min, were taken for determination of radioactivity by combustion. Livers were homogenised in 60 ml water and the homogenate made up to 100 ml. Three aliquots of 5 ml were taken and dried in vacuo at 40 ° over P 2 0 5 , as were lymph nodes, depot fat and blood samples, before combusting. A combustion train technique was used to determine sample radioactivity. The resulting 14 CO2 was trapped in a scintillation cocktail containing 7.5% (v/v) of ethanolamine and 4 g/1 BBOT. In some experiments the radioactivity of samples was determined by solubilising up to 100 mg of tissue in 1.0 ml of Soluene (Packard Instruments Ltd.) and counting in the manner recom'mended by the manufacturers. The counting efficiency was 70% for the samples which were combusted. For the Soluene-treated samples the efficiency was 65--75% for the tissues and 25% for 100 t~l of blood. The faecal pellets were removed from the collection trays, dried and combusted. The trays were washed with water and the washings made up to a standard volume with ethanol and 1.0 ml counted in 10 ml of a dioxan-based liquid scintillator [2]. Counting efficiences were 70% for urine and faeces. All counting was done on a Packard series 4000 liquid scintillation spectrometer using an internal n-[ 1-14 C] hexadecane standard. S k i n d i s t r i b u t i o n studies o f T C C in h u m a n volunteers
Human volunteers (one per treatment) had their lower backs {approx. 20 cm 2) washed for 2 min either once or 6 times in 3 days with 0.25 ml of an 8% (w/v) conventional soap suspension containing 0.08% (w/v) [14C]TCC either freshly prepared or allowed to equilibrate for 1 week at 40 ° with shaking for 10 min twice daily, or an 8% (w/v) suspension of 10% NSD containing 0.08% (w/v) [14C] TCC (freshly prepared). Pairs of skin biopsies were taken at 10 min, 2 days and 4 days after a single wash and at 10 min, 2 days and 2 weeks after the sixth wash. The biopsies were kept frozen until use. One of the pair was sectioned for autoradiography, and the other was
255
separated into epidermis and dermis by heating for 2 min in a polythene bag in water at 60 ° and combusted as described above. RESULTS
Distribution of TCC in guinea-pig skin Autoradiography of the skin of guinea-pigs washed with [1 4C] TCC in soap suspension or DMF solution showed that the germicide is deposited predominantly on the stratum corneum and that penetration through the epidermis is minimal with very small amounts present in the dermis. From DMF, [14C]TCC was deposited evenly over the skin surface but, despite equilibration of the [14 C] TCC in superfatted soap suspension, the germicide distribution over the skin surface was patchy and clusters of [14C]TCC particles were seen on the skin surface. Differences were also observed among the soap vehicles in respect to autoradiographic localisation of [1 a C] TCC on the skin. Thus [14 C] TCC from soap was deposited on the corneum, around the entrances to hair follicles and penetrated transepidermally. In contrast, [1 a C] TCC from NSD preparations showed follicular and sebaceous gland localisation as well as transepidermal penetration. The quantities of [14C]TCC present in skin from various vehicles are summarised in Table I. The results agree with the data from autoradiography. The greatest proportion of [14C]TCC retained in skin is present on the surface (0--25 p section) and relatively small amounts, TABLE I E F F E C T O F V A R I O U S V E H I C L E S ON [ 1 4 C ] T C C D I S T R I B U T I O N IN G U I N E A PIG SKIN F O L L O W I N G T O P I C A L A P P L I C A T I O N G r o u p s o f 5 a n a e s t h e s i s e d guinea pigs were w a s h e d for 10 min over 20 c m 2 o f c l i p p e d skin w i t h 0.5 ml o f 8% (w/v) s u s p e n s i o n o f freshly p r e p a r e d various soaps and DMF containing 0.08% [ 14 C] TCC. Skins were excised a f t e r application, f r o z e n t o a glass plate and rinsed e x h a u s t i v e l y with tap water. Results in p g / 2 0 c m 2 are means-+ SD for 15 determin a t i o n s (each animal e x a m i n e d in triplicate). Soap bar
Amount applied
Amount in rinsings
(ug)
(ug)
TCC d e p o s i t i o n (pg/20 c m 2 ) 25//sections
Punch autopsy
(0-500 u) Conventional Superfatted 10% NSD 30%NSD
375 + 71.9 272 -+ 14.3 272 + 110.0 3 0 3 + 43.6
285.7 287.7 322.7 195.9
16.9 15.5 12.2 6.7
DMF a
330 +
277.3
53.2 + 6 . 5 5
1.4
+ 3.26 -+ 9.77 -+ 5.10 +0.94
a Final [ 14 C ] TCC c o n c e n t r a t i o n in DMF was 0.068%.
256
Epidermis
Dermis
8.7 + 3.47 10.4 + 3.67 8.8 + 2.86 4.6+0.78
0.47 + 0.14 0.59 + 0.26 0.47 +- 0.13 0.30+0.08
37.72 + 7 . 9 3
2.32+0.74
¢.n
0--25 p section
(0-500 u)
Whole skin
13.5 + 4.05
29.3 + 6.08
8.3 + 1.96
20.4 + 3.22
14.3 + 3.68
25.6 + 4.30
13.8 + 2.90
26.9 + 7.73
6.2 +- 2.04
15.7 +- 4 . 2 6
Freshly prepared
Equilibrated
Freshly prepared
Freshly prepared
Equilibrated
30% N S D
Conventional soap/PEG400 ml
Conventional soap
TCC d e p o s i t i o n ( p g / 2 0 c m 2 )
5.4 -+ 0.93
15,3 + 4.17
Equilibrated
G r o u p s of 5 guinea pigs were w a s h e d for 10 rain over 20 c m 2 o f clipped skin w i t h 0.5 m l of 8% ( w / v ) s u s p e n s i o n of freshly p r e p a r e d or e q u i l i b r a t e d c o n v e n t i o n a l s o a p s u s p e n s i o n w i t h / w i t h o u t solubilizer (0.32% P E G 4 0 0 ml) a n d c o n t a i n i n g 0.08% [ 14 C] TCC or o f freshly p r e p a r e d or e q u i l i b r a t e d 30% NSD soap c o n t a i n i n g 0 . 0 8 % [ 14 C ] TCC. D e t e r m i n a t i o n s m a d e i m m e d i a t e l y a f t e r rinsing t h e skin in situ. Results in p g / 2 0 c m 2 are m e a n s + SD for groups o f 5 a n i m a l s ; e a c h a n i m a l was e x a m i n e d in triplicate.
E F F E C T O F E Q U I L I B R A T I O N O F [ 14 C] TCC F O R M U L A T I O N ON D I S T R I B U T I O N O F T O P I C A L L Y A P P L I E D [ 14 C] TCC T O G U I N E A PIG SKIN
T A B L E II
of the order of 5--7% of the [14 C] TCC deposited in skin, are present in the dermis. Only the 30% NSD bar deposited significantly less [14C]TCC (P = 0.05) than the other soap vehicles. A very high proportion of the applied [14C] TCC was recovered from the rinse water from the soap-washed animals. Effect of equilibrating TCC-soap formulation. The effect of equilibration of [14 C] TCC with conventional or 30% NSD soap can be seen in Table II. TCC deposition in the surface layer and whole skin was reduced by equilibration of TCC-conventional soap but was unaffected by equilibration of TCC/ 30% NSD. Comparing equilibrated preparations, TCC deposition was greater with conventional than with 30% NSD soap (P = 0.05). Effect of solubilizer in TCC formulation. The inclusion of PEG 400 ML in [14 C] TCC/conventional soap preparation had little effect on the deposition of [14 C] TCC in the surface layer (0--25 p) or whole skin (Table II) when the formulation applied was freshly prepared. However, when the formulation applied was equilibrated for 1 week at 40 °, the solubilizer significantly (P = 0.05) increased [14C] TCC deposition on the surface layer (0--25 p section) b u t the increase was not statistically significant in whole skin. Effect of TCC level in formulation. Increasing the [14C] TCC level in the superfatted soap bar from 0.1 to 2.0% i.e. from 0.008 to 0.16% (w/v) in the soap applied to the skin, resulted in linear increases in amounts of TCC deposited in the skin surface, epidermis and dermis (Table III). This was demonstrated by scintillation counting of both 25 p skin slices and skin fractions (epidermis and dermis) after combustion although agreement between the two techniques in these early experiments was not consistently good. Again the dermis contains a relatively small proportion (6--10%) of the total skin TCC. T A B L E III E F F E C T O F [14 C ] T C C L E V E L IN S O A P S U S P E N S I O N ON [ 1 4 C ] T C C D I S T R I B U T I O N IN G U I N E A PIG S K I N F O L L O W I N G T O P I C A L A P P L I C A T I O N G r o u p s of 5 guinea pigs were w a s h e d for 10 rain over 20 c m 2 o f clipped skin w i t h 0.5 m l o f 8% (w/v) o f freshly p r e p a r e d , s u p e r f a t t e d s o a p s u s p e n s i o n c o n t a i n i n g 0 . 0 0 8 - - 0 . 1 6 % [ 1 4 C ] T C C , e q u i v a l e n t t o 0.1--2% in t h e s o a p bar. Results in p g / 2 0 c m 2 are m e a n s -+ SD for 15 d e t e r m i n a t i o n s ( e a c h a n i m a l e x a m i n e d in t r i p l i c a t e ) ; results o f c o m b u s t i o n techn i q u e are m e a n s + SD for 20 d e t e r m i n a t i o n s ( e a c h a n i m a l e x a m i n e d in q u a d r u p l i c a t e ) . Technique
TCC d e p o s i t i o n ( p g / 2 0 c m 2) 0.1%
25 p S e c t i o n s
1.7
1.0% + 0.15
15.5
2.0% + 9.77
25.5
+ 8.10
(0--500 u) Punch autopsy Epidermis Dermis
258
1.2 +- 0.41 0.14 + 0.04
10.4 + 3.67 0 . 5 9 + 0.26
15.9 + 2.87 0.98 + 0.48
It was observed that doubling the volume of TCC solution applied to the area of skin from 0.5 to 1.0 ml had no significant effect on TCC distribution in the skin or on amounts deposited. Effect of post-treatment time. The scintillation counting data in Table IV show that the loss of [14 C] TCC from the skin is n o t very significant up to 48 h after topical application. Even after 2 weeks, [14 C] TCC was still to be seen by autoradiography on the stratum corneum of skin treated with superfatted soap preparations or DMF solutions and in the dermis of DMF-treated skin but not of soap-treated animals. Good agreement was obtained between the skin slicing and skin punch autopsy techniques. Effect of repeated washing. Table IV shows even after nine 2-rain washes with [14 C] TCC in superfatted soap suspension there was no increased deposition of [14 C] TCC compared to a single wash. The results obtained by skin slicing and punch autopsy techniques in respect of [14 C] TCC in whole skin were similar.
Distribution of TCC in human skin Autoradiography of human skin biopsies at 10 min after a single wash with various soap suspensions showed low or very low grain densities in the stratum corneum, epidermis and dermis. No germicide was localised in the pilosebaceous system of 10-min biopsy (i.e. immediately after washing and rinsing) or of whole skin biopsied at 48 h. After 6 washes [14 C] TCC deposition in the stratum corneum was greater from soap than NSD. The degree of localisation in the dermis was similar after one or 6 washes. Very small amounts of [14C]TCC were localised in the pilosebaceous system from the equilibrated TCC/goap and TCC/NSD suspensions. There was no evidence by autoradiography of build-up of [14C]TCC in the skin after 6 washes. At 4 8 h after the 6-wash regime [14C]TCC had disappeared from the skin washed with the NSD suspension and only very small amcunts were present in the skin washed with the equilibrated TCC/soap. Scintillation counting of biopsies (Table V) showed that much smaller amounts of [14 C]TCC were deposited in human skin than in guinea-pig skin, whether the vehicle was the freshly-prepared or equilibrated conventional soap or the freshly-prepared NSD suspension. The decrease in the amount of [14 C] TCC in skin with time was also more rapid in the human than in the guinea-pig. The increase in [14C] TCC in skin with a multiple wash may be exaggerated as the single wash application did not produce the usual lather.
Percutaneous absorption of TCC in guinea pigs Analysis of tissues o f guinea pigs treated for 10 min with [ 1 4 C ] T C C at 0.08% (w/v} in conventional soap, superfatted soap, 10% or 30% NSD formulations revealed mean levels of < 0.01 pg [14C]TCC/g of lymph nodes, liver and blood. None of the samples tested gave more than 10 cpm above background (25---30 cpm) and thus no detectable amounts of [14C]TCC (0.001--0.002 ppm) were able to penetrate through the skin. Even using 0.016% (w/v) of [14C]TCC in a superfatted soap formulation or 0.08%
259
O
b~
Immediately 2 weeks 4 weeks
DMF (single a p p l i c a t i o n )
Immediately
50.8 6.4 2.5
18.7
± + +
+
48.7 + 6.94 + 2.1 +
Immediately 2 weeks 4 weeks
Soap ( n i n e washes)
36.8 19.0 22.1
0.75(5) 1.45(5) 1.44(5)
5.44(3)
4.70(5) 1.98(5) 0.72(5)
+ 15.25(3) + 11.32(3) + 7.28(3)
25/1 sections ( 0 - - 5 0 0 U)
TCC d e p o s i t i o n ( p g / 2 0 c m 2)
Immediately 24 h 48 h
Soap (single wash)
Time after treatment
43.3 ± 7.2 + 2.1 +
13.7 ±
5.4 (5) 2.2 (5) 1.5 (5)
5.65(3)
38.6 + 6.3 (5) 5.6 + 2.1 (5) 3.1 +- 1.3 (5)
34.0 + 1 9 . 3 6 ( 3 ) 13.3 + 9 . 0 4 ( 3 ) 19.0 -+ 8 . 8 6 ( 3 )
Epidermis
Punch autopsy
4.5 + 1.1 (5) 0.8 ± 0.2 (5) 0.2 ± 0.1 (5)
2.2 + 0 . 9 5 ( 3 )
7.1 ± 4.6 (5) 0.7 +- 0.2 (5) 0.4 + 0.2 (5)
5.9 +- 4 . 4 5 ( 3 ) 2.9 + 2 . 7 1 ( 3 ) 1.3 -+ 0 . 6 2 ( 3 )
Dermis
47.8 + 8.0 + 2.3 ±
16.9 +
45.7 ± 6.3 + 3.5 +
5.67(5) 2.4 (5) 1.58(5)
5.43(3)
7.33(5) 2.29(5) 1.39(5)
41.9 + 2 1 . 7 1 ( 3 ) 18.5 + 1 1 . 2 0 ( 3 ) 21.4 + 1 0 . 0 9 ( 3 )
Total
G r o u p s of 5 guinea pigs were w a s h e d for 2 m i n o n c e or 9 t i m e s (twice daily) over 20 c m 2 clipped skin w i t h 0.5 m l o f a n 8% ( w / v ) of a n e q u i l i b r a t e d s u p e r f a t t e d s o a p s u s p e n s i o n or D M F c o n t a i n i n g 0.08% ( w / v ) [ 1 4 C ] T C C . T h e skin was r i n s e d w i t h t a p w a t e r b e f o r e excision. Results, in pg per 20 c m 2 , are m e a n + SD for e a c h group.
E F F E C T O F T I M E ON [ 1 4 C ] T C C D E P O S I T I O N IN G U I N E A P I G SKIN
TABLE IV
b~
0.007 0.005 0.012
10 rain
0.167 0.021
0.188
Multiple wash
Epidermis Dermis
Summation
2 day
0.005
0.103
< 0.001
< 0.001 0.001
14 day
< 0.001
0.312
0.257 0.055
10 rain
0.086
0.072 0.014
Summation
< 0.001 < 0.001
0.005 < 0.001
0.084 0.019
Epidermis Dermis
0.060
0.052 0.008
2 day
0.002
0.002 < 0.001
2 day
10 rain
4 day
2 day
10 min
Single wash
Conventional soap equilibrated
Conventional soap freshly prepared
TCC deposition (pg/cm:)
< 0.001
< 0.001 < 0.001
14 day
< 0.001
< 0.001
SKIN DEPOSITION AND P E N E T R A T I O N OF TRICHLOROCARBANILIDE
J.G. BLACK, D. HOWES and T. RUTHERFORD
Environmental Safety Division, Unilever Research Laboratory, Colworth House, Sharnbrook, Bedford (Great Britain) (Received May l l t h , 1974)
SUMMARY
Studies are reported on the localization and quantitative distribution of 3,4,4,_trichloro[14 C] carbanilide([14 C] TCC) in guinea-pig and human skin, and on the percutaneous absorption of TCC following topical application to guinea pigs. [14C]TCC was applied to guinea-pig skin in various vehicles (conventional, superfatted, 10% non-soap detergent (NSD) and 30% NSD soap suspensions or in N,N-dimethylformamide [DMF] ) and under various conditions (e.g. freshly prepared or equilibrated suspensions; single or multiple washes). Most of the amount of TCC remaining in the skin after rinsing was deposited on the skin surface and only relatively minute amounts actually penetrated through the epidermis into the dermis. Whereas conventional soap facilitated a greater deposition of TCC on the skin surface than NSD, the latter caused greater amounts of TCC to be deposited in the pilosebaceous system and lower dermis than conventional soap. The absence of TCC in the blood and tissues of guinea pigs given topical applications of TCC lends further support to the very low order of percutaneous absorption of TCC. The localisation of TCC followed a similar pattern in human skin as in guinea-pig skin, but the a m o u n t deposited was less and the rate of disappearance was more for human than for guinea-pig skin.
Abbreviations: BBOT, 2,5-bis(5-tertbutylbenzoxazol-2-yl)thiophene; DMF, N,N-dimethylformamide; NSD, non-soap detergent; PEG 400 ML, polyethylene glycol 400 monolaurate; TCC, 3,4,4r-trichlorocarbanUide.
253
INTRODUCTION TCC is included in toilet soap as a bacteriostatic agent designed to reduce the bacterial flora of the skin. Although a large proportion of the initial application of germicide is immediately removed during rinsing it is important to establish the extent to which the germicide remains in the skin and is available for absorption into the body. Autoradiographic and scintillation counting techniques have been employed to determine in guinea pig and man the quantitative distribution of [14C]TCC in various skin layers notably the surface (0--25 p), which comprises the stratum corneum and stubble hair, epidermis and dermis. In certain instances the levels of [14 C] TCC have been determined in blood, excreta and tissues of guinea pigs as an indication of absorption through skin. The importance of various factors that may affect the deposition of TCC has been investigated. These factors include the nature of the soap vehicles, equilibration of soap-germicide formulation before application and the presence of a solubilizer in the formulation, the concentration of TCC in the product, time interval between topical application and determination of TCC in skin and the number of washes. EXPERIMENTAL Materials
[14C-Carbonyl] TCC was synthesized and checked for purity as described by Rutherford and Black [1]. [14 C] TCC with specific activities of 1.5--14.6 pCi/mg was added to an 8% (w/v) aqueous suspension of a conventional soap, superfatted soap, NSD bars containing 10% or 30% of the synthetic detergent sodium alkoyl isethionate, or to DMF (GPR grade; Hopkin and Williams), to give a final germicide concentration of 0.08% (w/v), i.e. equivalent to 1% of the toilet bar. Superfatted soap suspensions 8% (w/v) were also prepared containing the equivalent of 0.1% and 2.0% (w/w) in the soap bar. All aqueous soap suspensions were made up with water containing 60 p p m CaCO3 (6 ° hardness). The TCC sample was usually ground to a fine powder and the soap suspension added. The preparation was gently homogenised in a Potter--Elvehjem homogeniser to give a very fine suspension of particles of TCC in the soap with the minimum of frothing. Equilibration of the TCC-soap suspension was achieved by shaking the preparation at 40 ° for a minimum of 24 h. The state of the TCC in the equilibrated suspension most closely resembles the state of the TCC in the toilet bar. A n i m a l s and t r e a t m e n t
The dorsal hair of groups o f 3 or 5 male Dunkin-Hartley guinea pigs (250 g b o d y weight approx.) was clipped and in some, but not all, experiments the animals were anaesthetised with ether. Aliquots (0.5 ml) of the germicide preparations were pipetted on to a marked area (20 cm 2) of the dorsal skin and rubbed gently for 2 or 10 min with a gloved finger. Another
254
group of 3 guinea pigs received 9 similar washes, 2 per day for 4 days and the ninth wash on the morning of the 5th day. In most experiments the skin was rinsed in situ before excision but in some others the excised skin was frozen on to a glass plate and rinsed [ 1]. The animals were killed either immediately or at 24 and 48 h or 2 and 4 weeks after the rinse. Surviving animals were placed in separate metabolism cages, and fed a pelleted diet and cabbage. The excreta were collected in polythene-lined trays beneath the cages in two 24 hourly lots. At the appropriate time the animals were killed by cervical dislocation and the treated area of skin, the lymph nodes of the groin, the liver, depot fat and heart blood samples taken. Skins were kept frozen until sections parallel to the surface were cut on a freezing microtome for scintillation counting and autoradiography as described by Rutherford and Black [1]. Whole skin 6 m m punch autopsies, in which the epidermis was separated from the dermis after exposure to 0.880 NH4OH for 10 min, were taken for determination of radioactivity by combustion. Livers were homogenised in 60 ml water and the homogenate made up to 100 ml. Three aliquots of 5 ml were taken and dried in vacuo at 40 ° over P 2 0 5 , as were lymph nodes, depot fat and blood samples, before combusting. A combustion train technique was used to determine sample radioactivity. The resulting 14 CO2 was trapped in a scintillation cocktail containing 7.5% (v/v) of ethanolamine and 4 g/1 BBOT. In some experiments the radioactivity of samples was determined by solubilising up to 100 mg of tissue in 1.0 ml of Soluene (Packard Instruments Ltd.) and counting in the manner recom'mended by the manufacturers. The counting efficiency was 70% for the samples which were combusted. For the Soluene-treated samples the efficiency was 65--75% for the tissues and 25% for 100 t~l of blood. The faecal pellets were removed from the collection trays, dried and combusted. The trays were washed with water and the washings made up to a standard volume with ethanol and 1.0 ml counted in 10 ml of a dioxan-based liquid scintillator [2]. Counting efficiences were 70% for urine and faeces. All counting was done on a Packard series 4000 liquid scintillation spectrometer using an internal n-[ 1-14 C] hexadecane standard. S k i n d i s t r i b u t i o n studies o f T C C in h u m a n volunteers
Human volunteers (one per treatment) had their lower backs {approx. 20 cm 2) washed for 2 min either once or 6 times in 3 days with 0.25 ml of an 8% (w/v) conventional soap suspension containing 0.08% (w/v) [14C]TCC either freshly prepared or allowed to equilibrate for 1 week at 40 ° with shaking for 10 min twice daily, or an 8% (w/v) suspension of 10% NSD containing 0.08% (w/v) [14C] TCC (freshly prepared). Pairs of skin biopsies were taken at 10 min, 2 days and 4 days after a single wash and at 10 min, 2 days and 2 weeks after the sixth wash. The biopsies were kept frozen until use. One of the pair was sectioned for autoradiography, and the other was
255
separated into epidermis and dermis by heating for 2 min in a polythene bag in water at 60 ° and combusted as described above. RESULTS
Distribution of TCC in guinea-pig skin Autoradiography of the skin of guinea-pigs washed with [1 4C] TCC in soap suspension or DMF solution showed that the germicide is deposited predominantly on the stratum corneum and that penetration through the epidermis is minimal with very small amounts present in the dermis. From DMF, [14C]TCC was deposited evenly over the skin surface but, despite equilibration of the [14 C] TCC in superfatted soap suspension, the germicide distribution over the skin surface was patchy and clusters of [14C]TCC particles were seen on the skin surface. Differences were also observed among the soap vehicles in respect to autoradiographic localisation of [1 a C] TCC on the skin. Thus [14 C] TCC from soap was deposited on the corneum, around the entrances to hair follicles and penetrated transepidermally. In contrast, [1 a C] TCC from NSD preparations showed follicular and sebaceous gland localisation as well as transepidermal penetration. The quantities of [14C]TCC present in skin from various vehicles are summarised in Table I. The results agree with the data from autoradiography. The greatest proportion of [14C]TCC retained in skin is present on the surface (0--25 p section) and relatively small amounts, TABLE I E F F E C T O F V A R I O U S V E H I C L E S ON [ 1 4 C ] T C C D I S T R I B U T I O N IN G U I N E A PIG SKIN F O L L O W I N G T O P I C A L A P P L I C A T I O N G r o u p s o f 5 a n a e s t h e s i s e d guinea pigs were w a s h e d for 10 min over 20 c m 2 o f c l i p p e d skin w i t h 0.5 ml o f 8% (w/v) s u s p e n s i o n o f freshly p r e p a r e d various soaps and DMF containing 0.08% [ 14 C] TCC. Skins were excised a f t e r application, f r o z e n t o a glass plate and rinsed e x h a u s t i v e l y with tap water. Results in p g / 2 0 c m 2 are means-+ SD for 15 determin a t i o n s (each animal e x a m i n e d in triplicate). Soap bar
Amount applied
Amount in rinsings
(ug)
(ug)
TCC d e p o s i t i o n (pg/20 c m 2 ) 25//sections
Punch autopsy
(0-500 u) Conventional Superfatted 10% NSD 30%NSD
375 + 71.9 272 -+ 14.3 272 + 110.0 3 0 3 + 43.6
285.7 287.7 322.7 195.9
16.9 15.5 12.2 6.7
DMF a
330 +
277.3
53.2 + 6 . 5 5
1.4
+ 3.26 -+ 9.77 -+ 5.10 +0.94
a Final [ 14 C ] TCC c o n c e n t r a t i o n in DMF was 0.068%.
256
Epidermis
Dermis
8.7 + 3.47 10.4 + 3.67 8.8 + 2.86 4.6+0.78
0.47 + 0.14 0.59 + 0.26 0.47 +- 0.13 0.30+0.08
37.72 + 7 . 9 3
2.32+0.74
¢.n
0--25 p section
(0-500 u)
Whole skin
13.5 + 4.05
29.3 + 6.08
8.3 + 1.96
20.4 + 3.22
14.3 + 3.68
25.6 + 4.30
13.8 + 2.90
26.9 + 7.73
6.2 +- 2.04
15.7 +- 4 . 2 6
Freshly prepared
Equilibrated
Freshly prepared
Freshly prepared
Equilibrated
30% N S D
Conventional soap/PEG400 ml
Conventional soap
TCC d e p o s i t i o n ( p g / 2 0 c m 2 )
5.4 -+ 0.93
15,3 + 4.17
Equilibrated
G r o u p s of 5 guinea pigs were w a s h e d for 10 rain over 20 c m 2 o f clipped skin w i t h 0.5 m l of 8% ( w / v ) s u s p e n s i o n of freshly p r e p a r e d or e q u i l i b r a t e d c o n v e n t i o n a l s o a p s u s p e n s i o n w i t h / w i t h o u t solubilizer (0.32% P E G 4 0 0 ml) a n d c o n t a i n i n g 0.08% [ 14 C] TCC or o f freshly p r e p a r e d or e q u i l i b r a t e d 30% NSD soap c o n t a i n i n g 0 . 0 8 % [ 14 C ] TCC. D e t e r m i n a t i o n s m a d e i m m e d i a t e l y a f t e r rinsing t h e skin in situ. Results in p g / 2 0 c m 2 are m e a n s + SD for groups o f 5 a n i m a l s ; e a c h a n i m a l was e x a m i n e d in triplicate.
E F F E C T O F E Q U I L I B R A T I O N O F [ 14 C] TCC F O R M U L A T I O N ON D I S T R I B U T I O N O F T O P I C A L L Y A P P L I E D [ 14 C] TCC T O G U I N E A PIG SKIN
T A B L E II
of the order of 5--7% of the [14 C] TCC deposited in skin, are present in the dermis. Only the 30% NSD bar deposited significantly less [14C]TCC (P = 0.05) than the other soap vehicles. A very high proportion of the applied [14C] TCC was recovered from the rinse water from the soap-washed animals. Effect of equilibrating TCC-soap formulation. The effect of equilibration of [14 C] TCC with conventional or 30% NSD soap can be seen in Table II. TCC deposition in the surface layer and whole skin was reduced by equilibration of TCC-conventional soap but was unaffected by equilibration of TCC/ 30% NSD. Comparing equilibrated preparations, TCC deposition was greater with conventional than with 30% NSD soap (P = 0.05). Effect of solubilizer in TCC formulation. The inclusion of PEG 400 ML in [14 C] TCC/conventional soap preparation had little effect on the deposition of [14 C] TCC in the surface layer (0--25 p) or whole skin (Table II) when the formulation applied was freshly prepared. However, when the formulation applied was equilibrated for 1 week at 40 °, the solubilizer significantly (P = 0.05) increased [14C] TCC deposition on the surface layer (0--25 p section) b u t the increase was not statistically significant in whole skin. Effect of TCC level in formulation. Increasing the [14C] TCC level in the superfatted soap bar from 0.1 to 2.0% i.e. from 0.008 to 0.16% (w/v) in the soap applied to the skin, resulted in linear increases in amounts of TCC deposited in the skin surface, epidermis and dermis (Table III). This was demonstrated by scintillation counting of both 25 p skin slices and skin fractions (epidermis and dermis) after combustion although agreement between the two techniques in these early experiments was not consistently good. Again the dermis contains a relatively small proportion (6--10%) of the total skin TCC. T A B L E III E F F E C T O F [14 C ] T C C L E V E L IN S O A P S U S P E N S I O N ON [ 1 4 C ] T C C D I S T R I B U T I O N IN G U I N E A PIG S K I N F O L L O W I N G T O P I C A L A P P L I C A T I O N G r o u p s of 5 guinea pigs were w a s h e d for 10 rain over 20 c m 2 o f clipped skin w i t h 0.5 m l o f 8% (w/v) o f freshly p r e p a r e d , s u p e r f a t t e d s o a p s u s p e n s i o n c o n t a i n i n g 0 . 0 0 8 - - 0 . 1 6 % [ 1 4 C ] T C C , e q u i v a l e n t t o 0.1--2% in t h e s o a p bar. Results in p g / 2 0 c m 2 are m e a n s -+ SD for 15 d e t e r m i n a t i o n s ( e a c h a n i m a l e x a m i n e d in t r i p l i c a t e ) ; results o f c o m b u s t i o n techn i q u e are m e a n s + SD for 20 d e t e r m i n a t i o n s ( e a c h a n i m a l e x a m i n e d in q u a d r u p l i c a t e ) . Technique
TCC d e p o s i t i o n ( p g / 2 0 c m 2) 0.1%
25 p S e c t i o n s
1.7
1.0% + 0.15
15.5
2.0% + 9.77
25.5
+ 8.10
(0--500 u) Punch autopsy Epidermis Dermis
258
1.2 +- 0.41 0.14 + 0.04
10.4 + 3.67 0 . 5 9 + 0.26
15.9 + 2.87 0.98 + 0.48
It was observed that doubling the volume of TCC solution applied to the area of skin from 0.5 to 1.0 ml had no significant effect on TCC distribution in the skin or on amounts deposited. Effect of post-treatment time. The scintillation counting data in Table IV show that the loss of [14 C] TCC from the skin is n o t very significant up to 48 h after topical application. Even after 2 weeks, [14 C] TCC was still to be seen by autoradiography on the stratum corneum of skin treated with superfatted soap preparations or DMF solutions and in the dermis of DMF-treated skin but not of soap-treated animals. Good agreement was obtained between the skin slicing and skin punch autopsy techniques. Effect of repeated washing. Table IV shows even after nine 2-rain washes with [14 C] TCC in superfatted soap suspension there was no increased deposition of [14 C] TCC compared to a single wash. The results obtained by skin slicing and punch autopsy techniques in respect of [14 C] TCC in whole skin were similar.
Distribution of TCC in human skin Autoradiography of human skin biopsies at 10 min after a single wash with various soap suspensions showed low or very low grain densities in the stratum corneum, epidermis and dermis. No germicide was localised in the pilosebaceous system of 10-min biopsy (i.e. immediately after washing and rinsing) or of whole skin biopsied at 48 h. After 6 washes [14 C] TCC deposition in the stratum corneum was greater from soap than NSD. The degree of localisation in the dermis was similar after one or 6 washes. Very small amounts of [14C]TCC were localised in the pilosebaceous system from the equilibrated TCC/goap and TCC/NSD suspensions. There was no evidence by autoradiography of build-up of [14C]TCC in the skin after 6 washes. At 4 8 h after the 6-wash regime [14C]TCC had disappeared from the skin washed with the NSD suspension and only very small amcunts were present in the skin washed with the equilibrated TCC/soap. Scintillation counting of biopsies (Table V) showed that much smaller amounts of [14 C]TCC were deposited in human skin than in guinea-pig skin, whether the vehicle was the freshly-prepared or equilibrated conventional soap or the freshly-prepared NSD suspension. The decrease in the amount of [14 C] TCC in skin with time was also more rapid in the human than in the guinea-pig. The increase in [14C] TCC in skin with a multiple wash may be exaggerated as the single wash application did not produce the usual lather.
Percutaneous absorption of TCC in guinea pigs Analysis of tissues o f guinea pigs treated for 10 min with [ 1 4 C ] T C C at 0.08% (w/v} in conventional soap, superfatted soap, 10% or 30% NSD formulations revealed mean levels of < 0.01 pg [14C]TCC/g of lymph nodes, liver and blood. None of the samples tested gave more than 10 cpm above background (25---30 cpm) and thus no detectable amounts of [14C]TCC (0.001--0.002 ppm) were able to penetrate through the skin. Even using 0.016% (w/v) of [14C]TCC in a superfatted soap formulation or 0.08%
259
O
b~
Immediately 2 weeks 4 weeks
DMF (single a p p l i c a t i o n )
Immediately
50.8 6.4 2.5
18.7
± + +
+
48.7 + 6.94 + 2.1 +
Immediately 2 weeks 4 weeks
Soap ( n i n e washes)
36.8 19.0 22.1
0.75(5) 1.45(5) 1.44(5)
5.44(3)
4.70(5) 1.98(5) 0.72(5)
+ 15.25(3) + 11.32(3) + 7.28(3)
25/1 sections ( 0 - - 5 0 0 U)
TCC d e p o s i t i o n ( p g / 2 0 c m 2)
Immediately 24 h 48 h
Soap (single wash)
Time after treatment
43.3 ± 7.2 + 2.1 +
13.7 ±
5.4 (5) 2.2 (5) 1.5 (5)
5.65(3)
38.6 + 6.3 (5) 5.6 + 2.1 (5) 3.1 +- 1.3 (5)
34.0 + 1 9 . 3 6 ( 3 ) 13.3 + 9 . 0 4 ( 3 ) 19.0 -+ 8 . 8 6 ( 3 )
Epidermis
Punch autopsy
4.5 + 1.1 (5) 0.8 ± 0.2 (5) 0.2 ± 0.1 (5)
2.2 + 0 . 9 5 ( 3 )
7.1 ± 4.6 (5) 0.7 +- 0.2 (5) 0.4 + 0.2 (5)
5.9 +- 4 . 4 5 ( 3 ) 2.9 + 2 . 7 1 ( 3 ) 1.3 -+ 0 . 6 2 ( 3 )
Dermis
47.8 + 8.0 + 2.3 ±
16.9 +
45.7 ± 6.3 + 3.5 +
5.67(5) 2.4 (5) 1.58(5)
5.43(3)
7.33(5) 2.29(5) 1.39(5)
41.9 + 2 1 . 7 1 ( 3 ) 18.5 + 1 1 . 2 0 ( 3 ) 21.4 + 1 0 . 0 9 ( 3 )
Total
G r o u p s of 5 guinea pigs were w a s h e d for 2 m i n o n c e or 9 t i m e s (twice daily) over 20 c m 2 clipped skin w i t h 0.5 m l o f a n 8% ( w / v ) of a n e q u i l i b r a t e d s u p e r f a t t e d s o a p s u s p e n s i o n or D M F c o n t a i n i n g 0.08% ( w / v ) [ 1 4 C ] T C C . T h e skin was r i n s e d w i t h t a p w a t e r b e f o r e excision. Results, in pg per 20 c m 2 , are m e a n + SD for e a c h group.
E F F E C T O F T I M E ON [ 1 4 C ] T C C D E P O S I T I O N IN G U I N E A P I G SKIN
TABLE IV
b~
0.007 0.005 0.012
10 rain
0.167 0.021
0.188
Multiple wash
Epidermis Dermis
Summation
2 day
0.005
0.103
< 0.001
< 0.001 0.001
14 day
< 0.001
0.312
0.257 0.055
10 rain
0.086
0.072 0.014
Summation
< 0.001 < 0.001
0.005 < 0.001
0.084 0.019
Epidermis Dermis
0.060
0.052 0.008
2 day
0.002
0.002 < 0.001
2 day
10 rain
4 day
2 day
10 min
Single wash
Conventional soap equilibrated
Conventional soap freshly prepared
TCC deposition (pg/cm:)
< 0.001
< 0.001 < 0.001
14 day
< 0.001
< 0.001
