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In vitro dermal absorption of pesticides: VI. In vivo and in vitro comparison of the organochlorine insecticide DDT in rat, guinea pig, pig, human and tissue-cultured skin
~
Toxic'. in Vitro Vol. 8, No. 6, pp. 1225-1232, 1994
Pergamon
0887-2333(94)00172-3
Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0887-2333/94 $7.00 + 0.00
IN VITRO DERMAL ABSORPTION OF PESTICIDES: VI. IN VIVO A N D IN VITRO COMPARISON OF THE ORGANOCHLORINE INSECTICIDE DDT IN RAT, GUINEA PIG, PIG, H U M A N A N D TISSUE-CULTURED SKIN R. P. MOODy, B. NADEAU and I. CHU Health Canada, Environmental Health Centre, Tunney's Pasture, Ottawa, Ontario KIA 0L2, Canada (Received 23 December 1993; revisions received 6 April 1994)
Abstract--Invitro dermal absorption tests were conducted with the ~4C-ring-labelled
pesticide, l,l,ltrichloro-2,2-bis(4-chlorophenyl)ethane (DDT) dissolved in acetone and applied to dermatomed skin (0.5 mm) of a number of species at dose rates of 16-27/~g/cm2. Skin absorption was determined for 48 hr after exposure using in vitro flow-through cells. Skin absorption was calculated from the sum of the percentage recovery of '4C activity in the receiver solution added to the percentage recovery for the methanol washes of the skin at 48 hr and the skin digest samples. Two receiver solutions, Ringer's saline (used with Moody aluminium cells), and Hanks' HEPES buffered saline with 4% serum albumin (used with Bronaugh flow-through cells) were used. Listed in decreasing order, the total percentage in vitro dermal absorptions obtained by 48 hr after exposure for the five skin types were: 42 ___2.6% [hairless guinea pig; Hanks' receiver (HR)], 34 + 10.5% (rat; HR), 28 +_ 13.2% [Testskin; Ringer's receiver (RR)], 28 _ 2.9% (human; HR), 22 + 3.3% (Testskin; HR), 18 + 6.2% (pig; RR) and 14 _ 2.1% (pig; HR). The percentage t4C activity recovered in soapy water rinses of the skin specimens at 24 hr, and for methanol skin washes and skin digests at 48 hr, and of ~4C-labelled volatiles collected in air traps are reported. Data obtained with pig and Testskin for DDT using the Moody flow-through permeation cell was compared with that obtained using the Bronaugh cell. Significantly greater (P < 0.05) percentage recovery was obtained for the soap washes at 24 hr of the skin following the Bronaugh procedure than was obtained with the Moody method. Comparative in vivo studies demonstrated urinary recovery was 2 _ 0.5 and 15 + 1.7% for rats (dose rate; 6/~g/cm 2) and guinea pigs (dose rate: 9/tg/cmZ), respectively. Total faecal recovery was 20 + 1.9 and 44 _ 2.75% for rats and guinea pigs, respectively. Analysis of tissue taken at autopsy 14 days after dosing demonstrated total tissue recovery of 51 _ 5.6% in rats but of only 3 _ 0.7% in guinea pigs. Including the '4C activity extracted from the skin removed from the dose site at 14 days after exposure, the total recovery of dermally absorbed residues was 73 + 5.9 and 62 + 4.1% for rats and guinea pigs, respectively. Recovery of '4C from soapy water skin washes conducted at 24 hr after exposure was 3 + 1.4 and 14 ___1.8% for rats and guinea pigs, respectively, and this was significantly less than that obtained by both the Bronaugh and Moody in vitro procedures. Skin patch recovery was 24% for both rats and guinea pigs. In summary, the in vitro data underestimed the degree of dermal absorption observed in vivo for both rats and guinea pigs, and this was thought to be due to an overly vigorous removal of the pesticide skin deposit by the soap washing procedures used in vitro in comparison with the in vivo washing procedure.
INTRODUCTION
1977], which was tested in o u r previous study ( M o o d y et al., 1994), D D T is very lipophilic (log K o w = 6,19; C h i o u et al., 1977). The lipophilicity o f a chemical is
To complete the series o f articles ( M o o d y a n d Martineau, 1990; M o o d y and Ritter, 1992; M o o d y and Nadeau, 1993 and 1994) concerning a crossspecies c o m p a r i s o n of in vitro a n d in vivo dermal a b s o r p t i o n studies with pesticides s p a n n i n g a wide range of lipophilicities, the present study reports data o b t a i n e d for the o r g a n o c h l o r i n e insecticide 1,1,1trichloro-2,2-bis(4-chlorophenyl)ethane (DDT). In contrast to the relatively hydrophilic nature of 2,4dichlorophenoxyacetic acid [2,4-D; log o c t a n o l - w a t e r partition coefficient (log K o w ) = 2.81; Chiou et al., Abbreviations: BSA = bovine serum albumin; GI = gastro-
intestinal; H R = Hanks' receiver fluid; LSC = liquid scintillation counting; PR =perirenal; RR = Ringer's receiver fluid.
generally considered to affect its ability to permeate skin. However, detailed correlations of dermal permeation rates with pesticide lipophilicity have not been reported a l t h o u g h such studies have been conducted with other c o m p o u n d s (such as hair dyes a n d pharmaceuticals) ( B r o n a u g h a n d C o n g d o n , 1984; Hawkins and Reifenrath, 1986). A future report will deal with correlating o u r skin permeation data with Q S A R such as o c t a n o l - w a t e r partition coefficients (log Kow). The present report will serve to d o c u m e n t the D D T data obtained in vitro using b o t h the a l u m i n i u m flow-through M o o d y cell (previously referred to as the A I D A cell), and the teflon flowt h r o u g h B r o n a u g h cell (Bronaugh, 1991; B r o n a u g h
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R.P. MOODYet al.
and Stewart, 1985; Moody and Martineau, 1990). In changing to the use of the Bronaugh cell we have also adopted the Collier and Bronaugh (1991) procedure of using Hanks' medium containing 4% bovine serum albumin (BSA) in place of the Ringer's saline that was used as a receiver solution for the Moody cell (the solution flowing under the skin collecting the chemical permeant). The possible influence of the composition of the receiver solution on dermal flux in vitro will be considered when discussing the in vitro concordance of the data obtained with that obtained in in vivo studies with rats and hairless guinea pigs. MATERIALS AND M E T H O D S
The in vitro procedure using the aluminium (Moody) permeation cell chambers, and methods of skin preparation (e.g. dermatoming), sample collection, liquid scintillation counting (LSC) analysis and calculation of results by electronic spreadsheet were identical to those detailed previously (Moody and Nadeau, 1993). The procedure of Collier and Bronaugh (1991) was used for the teflon (Bronaugh) permeation cell chambers (Bronaugh, 1991). For Moody cell tests, sterile Ringer's solution buffered at pH 7.4 with Trizma and supplemented with glucose (0.8 g/litre), penicillin G (0.06g/litre) and streptomycin sulfate (0.1 g/litre) was used as the receiver solution (Moody and Martineau, 1990). For Bronaugh cell tests, Hanks' HEPES buffered (pH7.4) growth medium containing 4% BSA, NaHCO3 (0.36g/litre) and gentamicin sulfate (50 mg/litre) was used as the receiver solution. Hanks' HEPES buffered receiver solution has been reported to maintain skin viability (Collier and Bronaugh, 1991). Sufficient receiver flow was required to ensure 'sink' conditions in the Moody and Bronaugh cells. The receiver flow rate used in the Moody cell (2.5 ml/hr) was the same as that used in our previous studies. A slower receiver flow rate (1.5 ml/hr) was used for the Bronaugh cells since this rate was sufficient to maintain 'sink' conditions and is the optimal rate given for the Bronaugh procedure (Bronaugh, 1991). The different cross-sectional areas of the two cell chamber types (Moody cell, 0.2 cm2;
Bronaugh cell, 0.64 cm 2) were taken into account for calculating dermal permeation rates. The in vivo procedure was identical to that reported previously (Moody and Nadeau, 1993). To reduce intraspecies variation in the observed data, the skin used in the in vitro studies was from the same rats and guinea pigs used for the in vivo tests. The skin from four animals was used in each in vitro test. The dose application rates and sources of skin specimens used in vitro are reported in Table 1. Testskin ~, a human-derived tissue-cultured skin, was purchased from Organogenesis Inc. (Cambridge, MA, USA). In the in vivo studies DDT was applied to a 4.2-cm 2 area of mid-dorsal skin at a rate of 6.1 and 9.1/tg/cm 2 for male Sprague-Dawley rats and female hairless guinea pigs, respectively. The dose site was protected from surface rub-off by a foam rubber non-occlusive patch (Moody and Nadeau, 1993). As detailed previously, the dosed skin region was rinsed with an aqueous Radiac soap solution (50%) at 24 hr after exposure in both the in vitro and the in vivo studies (Moody and Nadeau, 1993). This soap wash at 24 hr was used to remove the 'unabsorbed' pesticide residues. At 48 hr, when the in vitro studies were terminated, methanol washes of the skin and a subsequent tissue digest were conducted to remove the residual ~4C activity for mass balance purposes. 14C activity persisting in the dosed skin region in vivo was also measured by LSC analysis of digests of skin taken at autopsy 14 days after exposure (Moody and Nadeau, 1993). A two-tailed Student's t-test was used for statistical analysis, and P < 0.05 was taken as the level of significance. Chemicals
~4C-ring-labelled 4,4'-DDT ( > 9 8 % pure; sp. act. 11.8 mCi/mM) was purchased from Sigma Chemical Co. (St Louis, MO, USA). Hanks' balanced salts (modified; Catalogue no. H-2895), HEPES buffer (Catalogue no. H-9136) and bovine serum albumin (Catalogue no. A3425) were also purchased from the Sigma. Radiac soap (Radiacwash) solution was purchased from Atomic Products Corporation, Shirley (NY, USA). Scintillation cocktails and
Table 1. Topical dose application rate for [~4C]DDT in in vitro studies and source information (anatomical site, sex, age) of specimens used Species
Receiver solution*
Dose (/~Ci/cm 2)
Dose (/~g/cm2)
Skin source
Rat Guinea pig Pig Pig Human Testskin Testskin
Hanks' Hanks' Ringer's Hanks' Hanks' Ringer's Hanks"
0.77 0.92 0.80 0.66 0.89 0.55 0.76
23.0 27.1 23.2 19.9 26.3 16.2 22.3
Spragu~Dawley rat back, male, 5 wk old Hairless guinea pig back, female, 9 months old Yorkshire pig back, male, 8-9 wk old Yorkshire pig back, male, 8-9 wk old Caucasian human abdomen, female, 31 yr old Cultured human foreskin, 24br, from supplier Cultured human foreskin, 24 hr, from supplier
*Hanks" receiver solution was used with the Bronaugh cell and Ringer's receiver solution with the Moody cell. Hanks" receiver solution: Hanks" HEPES buffered growth medium containing 4% BSA, NaHCO~ (0.36 g/litre) and gentamicin sulfate (50 mg/litre); Ringer's solution buffered at pH 7.4 with Trizma and supplemented with glucose (0.8 g/litre), penicillin G (0.06 g/litre) and streptomycin sulfate (0.1 g/litre).
In vitro dermal absorption of DDT
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0.16 -Soap Wash
0.14 0.12 0 0 0Q
rc
t
0.1 0.08
l
0.06 0.04 0.02
1
0
0
4
8
12
16
20
24
28
32
36
40
44
48
Time (hr) Fig. 1. In vitro analysis of [~4C]DDT permeating rat skin. The percentage recovery in the receiver solution is shown v. time for each of the four replicate Bronaugh cells ( .~. , cell I; --I--, cell 2; - - : ~ - - , cell 3; [] , cell 4). The onset of the skin wash with Radiac soap and water at 24 hr is shown. organic solvents were obtained and used as described previously (Moody and Nadeau, 1993).
to reach a plateau level that persisted until a second elevation of recovery occurred at 24 hr after exposure (Figs 1 and 2). This peak after the wash at 24 hr was also observed in our previous studies with N , N diethyl-m-toluamide (DEET), diazinon and 2,4-D, however. However, the 'washing-in' effect of the 24-hr soap wash was very pronounced with D D T in the present study. As shown in Fig. 2, this washing-in effect could significantly enhance the degree of exposure experienced by a pesticide applicator who washed the skin with soap at some time after dermal exposure to D D T . It is interesting that following the 'wash-in' peak, the ~4C levels permeating the human skin (Fig. 2) declined to their pre-soap wash levels in
RESULTS AND DISCUSSION
In vitro studies
With the exception of Testskin, which was about 0 . 3 m m thick, the mean skin thickness was 0.5 _+ 0 . 0 1 m m for all test species (Moody and Nadeau, 1993). The results of the present study contrasted with our previous studies in which a rapid decline in the receiver levels of 14C activity occurred after an early peak level. With D D T , the percentage recovery of ~4C activity in the receiver solution tended
0.07
.••ll••Soap
0.06 0.05 t_
0 0 0 0
rc
W ash
0.04 0.03 0.02 0.01 O~ 0
I
I
4
I
I
8
I
I
12
I
I
16
I
I
20
I
I
24
I
I
28
I
I
32
[
I
36
I
I
40
I
I
44
I
48
Time (hr) Fig. 2. In vitro analysis of [~4C]DDT permeating human skin. The percentage recovery in the receiver solution is shown v. time for each of the four replicate Bronaugh cells ( .~. , cell 1; --I--, cell 2; - - ~ : - - , cell 3; [] -, cell 4), The onset of the skin wash with Radiac soap and water at 24 hr is shown.
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R. P. MOODY et al. Table 2. In vitro percentage recovery of radioactivity for different species of animal skin following topical application of [14CIDDT in acetone
Species
Receiver solution (cell type)t
[t4C] recovery (%)*
Rat
Hanks' (B)
Guinea pig
Hanks' (B)
Pig
Ringer's (M)
Pig
Hanks' (B)
Human
Hanks" (B)
Testskin
Ringer's (M)
Testskin
Hanks' (B)
Rad
Was
Dig
W+ D
Air
Total
59,5 (12,22) 54,5 (1,68) 21.9 (2.75) 83.6 (5.03) 65.8 (4.81) 28.4 (20.12) 72.3 (2.86)
13.4 (4.15) 21.2 (1.99) 15.8 (6.33) 9.2 (1.46) 19.0 (3.40) 14.9 (6.55) 11.4 (2.83)
20.2 (6.45) 17.8 (3.61) 1.6 (0.96) 4.0 (0.67) 8.1 (l.02) 12.8 (7.21) 10.3 (0.55)
33.6 (10.48) 39.0 (2.60 17.4 (6.11) 13.2 (2.09) 27.1 (2.88) 27.7 (13.1) 21.7 (3.29)
NS* (NS) NS (NS) 0.3 (0.32) NS (NS) NS (NS) 2.89 (I.23) NS (NS)
95.7 (5.54) 98.7 (2.06) 75.3 (9.62) 100,8 4.34 96.0 (3.85) 93.3 (4.83) 100.1 2.99
*Values are means (SD in parentheses; n = 4). Rad is % ~4C activity recovered in Radiac soap and water washes of the skin at 24 hr; Was is % recovered in methanol washes of the skin at 48 hr; Dig is % recovered in skin digest; W + D is % total recovered by methanol washes and skin digest; Air is % recovered in the air traps; Total is total % recovery (see text for discussion on mass balance). ~'The receiver solution used (Hanks' or Ringer's) is indicated together with the permeation cell type (B = Bronaugh; M = Moody). ~NS = no sample.
only two of the four permeation cells, while in the other two cells the ~4C levels declined only partially and then persisted at a plateau level roughly four times higher than the plateau level attained before soap washing. These results indicate that soap washing could actually elevate the bioavailability of the applied toxicant and thereby increase the risk involved. Since the wash-in peak was also observed in our previous studies in which the Moody cell was used and skin washing was conducted by pumping the soap solution at a slow flow rate (5 ml/min) over the dosed skin surface, it is most likely that this enhanced permeation was due to a surfactant effect of the soap. However, the pronounced wash-in peaks observed in the present study with the Bronaugh cells, suggest that the swabbing skin wash procedure may further enhance this effect by mechanical disruption
of the stratum corneum. Further testing using different soaps and wash procedures is warranted. The percentage recovery and the absorption data are reported in Tables 2 and 3, respectively. The amount of [14C]DDT residue rinsed off the skin by the Radiac soap wash at 24 hr (Table 2, column 3) ranged from a low of 22% in pig skin tested in a Moody cell with Ringer's receiver fluid, to a high of 84%, again with pig skin but using Hanks' receiver fluid and the Bronaugh cell. We also note that for Testskin (Table 2) significantly greater recovery of 14C activity in the soap washings was obtained using the Bronaugh procedure than using the Moody procedure. The value for Testskin (Ringer's, Moody cell) given in Table 2 (28.4 + 20.12%) includes one cell with a value of 59%, which accounts for the high standard deviation. With this cell excluded as an
Table 3. In vitro absorption data for different species of animal skin following topical applications with [~4C]DDT in acetone
Species
Receiver solution (cell type)~:
Rat
Hanks' (B)
Guinea pig
Hanks' (B)
Pig
Ringer's (M)
Pig
Hanks' (B)
Human
Hanks' (B)
Testskin
Ringer's (M)
Testskin
Hanks' (B)
Per* (%)
Per? (%)
Cum (pg/cm 2)
Rp (mg/cmZ/hr)
0.4 (0.04) 2.6 (0.54) 0.2 (0.34) 1.1 (0.23) 0.5 (0.19) 0.2 (0.07) 0.3 (0.02)
33.9 (I 0.49) 41.6 (2.63) 17.6 (6.15) 14.3 (2.07) 27.59 (2.93) 27.9 (13.19) 22.0 (3.30)
0.1 (0.0 I) 0.7 (0.15) 0.0 (0.08) 0.2 (0.05) 0.1 (0.05) 0.0 (0.01 ) 0.1 (0.01)
0.01 (0.002) 0.03 (0.006) 0.02 (0.032) 0.01 (0.002) 0.01 (0.001 ) 0.00 (0.000) 0.01 (0.001)
Values are means (SD in parentheses; n ~ 4). Per* is % skin permeation calculated from the % recovery in the receiver solution; Pert is the % skin permeation calculated from adding the % recovery in receiver solution to the % recovery in the methanol skin washes + skin digest samples 48 hr after exposure (Table 2, column 5): Cure is cumulative absorption in 48 hr; Rp is the maximum rate of skin permeation. ++The receiver solution used (Hanks' or Ringer's) is indicated together with the permeation cell type (B = Bronaugh; M = Moody).
In vitro dermal absorption of DDT
'outlier', the mean value becomes 18 _ 0.8% (n = 3), and increases the difference between the values for the soap wash recovery of J4C for the Moody and Bronaugh procedures. It is possible that the anomalously high value in the one permeation cell was due to a sloughing off of a portion of the treated epidermis during the soap washing procedure. It is not unexpected that the Moody soap washing procedure, which involves a mechanically delivered (syringe pump) gentle rinsing of the treated skin epidermis with soap solution at a constant slow flow rate of 5 ml/min (Moody and Ritter, 1992) would be less vigorous than the Bronaugh method which involves manual washing with cotton-tipped swabs. The friction of the swab on the skin surface would surpass that of a gently flowing stream. Although dermal swabbing is conducted in vivo, the epidermis of the isolated skin at 24 hr after isolation is probably more fragile than the skin in vivo. The effect of different skin washing procedures will be considered further when we discuss the comparison of the in vitro and in vivo data. The amount of ~4C-labelled pesticide not washed off by the Radiac soap and persisting in the skin at 48 hr after exposure varied between species (Table 2, column 6) and ranged from 13 to 39% of the applied dose. The degree of skin storage of lipophiles may depend on the species-specific lipid composition of the stratum corneum and on its total lipid content (Elias, 1983; Harada et al., 1992). In our previous studies, this pesticide 'depot' in human skin was 0.4, 14 and 18% for DEET, diazinon and 2,4-D, respectively, in comparison with 27% for DDT in the present study. The high value for persistence of DDT in skin is consistent with the relatively high lipophilicity of DDT. Air traps were not used with the Bronaugh cells but air-trap data for the Moody cells (Table 2, column 7) indicated that DDT was not volatile under the present conditions, in contrast with, for example, DEET with which we found 34% recovery of ~4C in air traps in tests on Testskin (Moody and Nadeau, 1993) (3% for DDT and Testskin, Table 2). The total mass balance was good with over 93% recovery being obtained in all cases except in the Moody cell study with pig skin in which total recovery was only 75% (Table 2, column 8). In view of the precision of the Radiac soap wash data for pig skin (22 + 2.8%), it is most likely that the low total percentage recovery was due to the administration of a lower dose than intended. The absorption data (Table 3) showed nearly insignificant permeation into the receiver solution (p*, column 3), in contrast with the total permeation (Pt, column 4) calculated by adding the percentage recovery in the skin depots to that for the receiver solutions. In fact this raises the possibility that, at least for lipophilic compounds such as DDT, permeation studies need not be conducted since 'skin depot persistence studies' (e.g. tape stripping studies) would serve the in vitro modelling purpose just as well. This
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also raises concern that for lipophilic compounds like DDT, in vitro methods may not provide a suitable alternative to in vivo animal testing. With the skin depot included as an absorbed residue, the ranking of the skin specimens in order of decreasing permeability (Pt, Table 3, column 4) was: guinea pig, rat, Testskin (Moody cell), human, Testskin (Bronaugh cell), pig (Moody cell), and pig (Bronaugh cell). Excellent Testskin/human skin agreement for skin permeability was evident here for DDT. There were insufficient data in the present study to validate the Moody cell as an alternative to the Bronaugh cell. The results, however, were promising in demonstrating similarly low permeation into the receiver solutions used (Table 3, column 3). Although there was a slight increase in permeation recorded through pig skin into Hanks' receiver with the Bronaugh cell (1.1%), in comparison with the data obtained for the Ringer's receiver (Moody cell, 0.2%), there was similar permeation in the two systems for the Testskin (0.3% with Hanks' receiver in the Bronaugh cell v. 0.2% with Ringer's receiver in the Moody cell, Table 2). These data suggest that in studies with very lipophilic compounds, such as DDT, the presence of BSA may not enhance the dissolution and uptake of the permeant from the skin to the receiver solution. It is quite possible that with the faster flow rate used here for the Moody cell, combined with the slight vortex current established by having the in and out ports of the receiver cells offset, dissolution was optimized in the Moody cell. The cumulative amounts absorbed into the receiver solution and the data for the Rp rate parameter are given in Table 3. The Lag values that are normally presented are not given since, because of the rapid rise to plateau levels of the receiver percentage recoveries (Figs 1 and 2), there were insufficient data for this calculation. In general, the DDT data showed much less cumulative absorption and a slower maximum rate of absorption than that reported previously for DEET (Moody and Nadeau, 1993). For example the DEET data for human skin had a cumulative absorption of 12.4/lg/cm 2 (v. 0.1 pg/cm 2 for DDT) and a maximum permeation rate of 2pg/cm2/hr (v. 0.01 pg/cm2/hr for DDT). In vivo studies
The urinary and faecal recoveries of ~4C activity for both rats and guinea pigs reached maximum values at 48 hr after exposure, with the exception of the guinea pig faecal levels which peaked at 72 hr. For both species, urinary levels declined rapidly to less than 0.1% of the applied dose by 14 days after exposure, while faecal levels declined to less than 1% by 14 days. The absorbed and non-absorbed recoveries of ~4C are presented in Tables 4 and 5, respectively. In contrast to our previous studies with DEET, diazinon and 2,4-D, the faecal percentage recovery for DDT exceeded the urinary recovery (Table 4). This is consistent with general agreement that more
1230
R . P . MOODY et al. Table 4. In viva percentage recovery of absorbed residues of [14C]DDT in rats and hairless guinea pigs 14C recovery (%) in: Dose Species (#g/cm:) Urine Faeces Tissue Total* Rat 6.1 2.1 19.6 50.5 72.1 (0.51) (1.85) (5.63) (5.85) Guinea pig 9.1 15.1 44.3 2.9 62.3 (1.74) (2.69) (0.73) (4.12) Values are means (SD in parentheses; n = 4). *Total is the summed % recoveries of urinary, faecal and tissue samples and represents the total % dermal absorption (see text). Note that the total % dermal absorption including the % recovery in the skin digests (Table 5, column 4) was 72.9 -+ 5.85 and 62.3 _+4.10% for rats and guinea pigs, respectively.
lipophilic c o m p o u n d s t e n d to be excreted to a g r e a t e r e x t e n t by t h e biliary a n d faecal routes. T h e e x c e p t i o n ally large recovery ( 5 1 % ) in the rat tissue s a m p l e s is discussed below. T h e d e r m a l a b s o r p t i o n o f the a p p l i e d D D T d o s e was 73 + 5.9% for rats a n d 62 ___4.1%0 for guinea pigs (see T a b l e 4 f o o t n o t e ) . T h e s e values are m u c h in excess o f the d e r m a l a b s o r p t i o n values r e p o r t e d by Bucks e t al. (1991) in h u m a n s d o s e d o n the f o r e a r m ( 1 0 % ) a n d in rhesus m o n k e y s d o s e d o n t h e a b d o m e n (19%). In the studies by Bucks e t al. (1991), D D T was a p p l i e d in a c e t o n e a n d a s o a p w a s h was c o n d u c t e d at 24 hr. T h e a b s o r p t i o n values for rats a n d guinea pigs in the c u r r e n t s t u d y also exceed the values o f 1.5, 43.4 a n d 4 6 . 3 % given for in viva d e r m a l a b s o r p t i o n o f D D T in m o n k e y s , pigs a n d rabbits, respectively (Bartek a n d L a B u d d e , 1975; also see T a b l e 3 in W a i t e r s a n d R o b e r t s , 1993). A l t h o u g h o u r p r e v i o u s studies have s u g g e s t e d that rat skin is generally m o r e p e r m e a b l e t h a n h u m a n , m o n k e y o r pig skin, in o u r s t u d y with 2,4-D a m i n e we did r e p o r t an e x c e p t i o n w h e r e rat skin was m o r e p e r m e a b l e t h a n r a b b i t skin b u t less p e r m e a b l e t h a n h u m a n skin ( M o o d y e t al., 1990). C o n s i d e r i n g the n o n - a b s o r b e d residues (Table 5), the l o w e r degree o f r e m o v a l o f the J4C residues in skin by the s o a p w a s h at 24 h r in rats in c o m p a r i s o n with guinea pigs is c o n s i s t e n t with the m u c h higher perc e n t a g e recovery o b t a i n e d f r o m the digests o f rat skin ( 3 4 % ) t h a n f r o m the digests o f the guinea pig skin (0%). Simply, the n o n - w a s h a b l e residue was persisting in the skin. T h e excellent total p e r c e n t a g e recoveries r e p o r t e d in Table 5 d e m o n s t r a t e that it is Table 5. In viva percentage recovery of non-absorbed residues of IL4CIDDT in rats and hairless guinea pigs 14C recovery (%) in: Soap Skin Skin wash patch digest Total* 2.8 22.6 34.3 104.3 (1.42) (8.98) (8.09) (3.92) Guinea pig 13.9 23.9 01) 100.0 (I.82) (0.26) ((t.02) (3.67) Values are means (SD in parentheses: ,~ 41. *Total is the summed % recoveries of thc total % recovery in Table 4 (urine + faeces + tissue) in addition to the % recoveries for soap washes of the dose site at 24 hr, the skin patch used to protect the dose site and the % recovered from the digests of the skin tissue removed from the dose site. Species Rat
essential to include t h e skin p a t c h recoveries in the m a s s b a l a n c e analysis. In c o n t r a s t to o u r p r e v i o u s r e p o r t s with D E E T , d i a z i n o n a n d 2,4-D, t h e p r e s e n t s t u d y d e m o n s t r a t e d significant b o d y s t o r a g e o f D D T with a total o f 51 a n d 3 % recovery o f 14C activity being d e t e c t e d in the tissues at 14 days after e x p o s u r e o f rats a n d guinea pig, respectively (Table 4). In rats, r a d i o a c t i v i t y was d e t e c t e d at levels ...<0.01% in only the red b l o o d ceil, spleen a n d b l a d d e r samples, while levels />0.8% were d e t e c t e d in the liver (0.9_+ 1.04%), g a s t r o intestinal (GI) tract (7.4_+ 8.58%), perirenal (PR) w h i t e fat (5.7_+6.62%), i n t e r s c a p u l a r b r o w n fat (0.8 _+ 0.92%), skin external to dose site (8.5_+ 10.15%) a n d in the carcass ( 2 6 . 3 _ + 3 0 . 5 % ) (Table 6). In guinea pigs, r a d i o a c t i v i t y was d e t e c t e d m a i n l y in the liver, G I tract, P R white fat, skin a n d carcass (Table 6). T h e r e a s o n for the o b s e r v e d m u c h increased persistence o f D D T residues in rats in c o m p a r i s o n with guinea pigs is n o t k n o w n but the d a t a suggest that rat tissues in general p r o v i d e a
Table 6. In viva percentage of recovery of ~4C activity in tissue samples taken at autopsy 14 days after exposure to [a4C]DDT E4C recovery (%) Tissue
Rat
Guinea pig
Brain 0.02 _+0.01 0.01 _+0.00 Heart 0.02 + 0.01 0.01 +_0.00 Lungs 0.08 + 0.08 0.01 + 0.01 Kidney 0.18 + 0.22 0.04 + 0.04 Spleen ND ND Liver 0.92 + 1.04 0.22 + 0.23 Diaphragm 0.06 + 0.07 ND GI tract 7.41 + 8.58 0.36 + 0.44 PR white fat 5.74+6.62 0.16 +0.19 Adrenals 0.04 -+ 0.03 ND IBAT 0.79+0.92 0.08_+0.10 Testes 0.03 + 0.02 -Tail 0.07 _+0.06 -Bladder 0.01 _+0.01 ND RBC ND ND Plasma 0.09 _+0.09 0.02 _+0.02 Thymus 0.05 _+0.04 0.01 _+0.01 Stomach 0.18 _+_0.19 0.04 ± 0.05 Skin 8.52 -+ 10.15 0.48 + 0.56 Carcass 26.29 -+ 30.46 1.45 -+ 1.69 ND = non-detectable (<0.005%) GI = gastro-intestinal PR = perirenal IBAT = interscapular brown adipose tissue RBC = red blood cells Values are means+ SD for groups of four male Sprague Dawley rats and four female hairless guinea pigs.
In vitro dermal absorption of DDT
relatively iipophilic 'sink' for such residues. We have previously reported that another organoehlorine insecticide, lindane, was more slowly cleared from rats than from rhesus monkeys following topical application and that body storage of lindane residues was indicated (Moody and Ritter, 1989). In a subsequent study, '4C activity was still detectable in the urine of rats at 72 days after exposure of the tail skin to lindane (Moody et al., 1989). We recommend that further studies are conducted to examine this lipid 'sink' in rats since, unless this happens to approximate to the human condition, the use of the rat model will be limited. In vivo /in vitro comparison
The in vivo data (see footnote to Table 4) demonstrated only slightly higher total dermal absorption of [~4C]DDT for rats (73 _+ 5.9%) than for guinea pigs (62_+4.1%), and this was consistent with similar levels of skin permeability being obtained in vitro for rat skin (34_+ 10.5%) and guinea pig skin (42 ___2.6%) ( p t , Table 3). However, the in vitro data underestimated the dermal absorption observed in vivo. As discussed above with regard to the fact that the Bronaugh soap wash procedure at 24hr was different from that used in the Moody method, it is apparent that an overly vigorous in vitro soap wash relative to that used in vivo could explain the underestimate of dermal absorption obtained by the Bronaugh in vitro procedure carried out here. Simply, the residues washed off the skin at 2 4 h r are not available for absorption, and hence an in vitro skinwashing method that very effectively removes the test compound would lead to an underestimate of in vivo absorption. Therefore, it is recommended that studies are conducted to determine the most appropriate skin washing method to use in vitro so that accurate predictions of percutaneous absorption can be made. In conclusion, the present study has shown that the in vitro procedure underestimated the dermal absorption data obtained in vivo for D D T . Although this underestimate suggested that the in vitro model may not provide a suitable alternative to the in vivo model for lipophilic compounds, good intermodel agreement has recently been observed with another lipophilic compound, benzo[a]pyrene (log Kow = 6.1) in our laboratory (R. P. Moody, unpublished data, 1994), and a general conclusion cannot be made at this time. The ranking of the skin types, listed in decreasing order of permeability to D D T , as determined from the in vitro permeability data were: hairless guinea pig [Hanks' receiver (HR)]>~rat (HR) ~> Testskin [Ringer's receiver (RR)] = human (HR) > T e s t s k i n (HR) ~> pig (RR)/> pig (HR). Future reports will examine the effect of the skin washing procedure on the dermal absorption process. Acknowledgements--We are grateful to Ms Patricia Curry and Ms Dianne Dick for reviewing the manuscript and to Dr Conrad Watters, Ottawa General Hospital, Ottawa,
1231
Ontario, for kindly providing the human skin specimens. We also thank Mr Robert Marr for his excellent technical assistance. REFERENCES
Bartek M. J. and LaBudde J. A. (1975) Percutaneous absorption in vitro. In Animal Models in Dermatology. Edited by H. I. Maibach. pp. 103-112. Churchill-Livingstone, New York. Bronaugh R. L. (1991) A flow-through diffusion cell. In In Vitro Percutaneous Absorption: Principles, Fundamentals, and Applications. Edited by R. L. Bronaugh and H. I. Maibach. pp. 17-23. CRC Press, Boca Raton, FL. Bronaugh R. J. and Congdon E. R. (1984) Percutaneous absorption of hair dyes: correlation with partition coefficients, Journal of Investigative Dermatology 83, 124-127. Bronaugh R. J. and Stewart R. F. (1985) Methods for in vitro percutaneous absorption studies. IV. The flowthrough diffusion cell. Journal of Pharmaceutical Science 74, 64q57. Bucks D. A. W., Wester R. C. and Maibach H. I. (1991) Percutaneous penetration from soil: inhibition or enhancement? In In Vitro Percutaneous Absorption: Principles, Fundamentals, and Applications. Edited by R. L. Bronaugh and H. I. Maibach. pp. 223-230. CRC Press, Boca Raton, FL. Chiou C. T., Freed V. H., Schmedding D. W. and Kohnert R. L. (1977) Partition coefficient and bioaccumulation of selected organic chemicals. Environmental Science and Technology 11, 475-478. Collier S. W. and Bronaugh R. L. (1991) Receptor fluids. In In Vitro Percutaneous Absorption: Principles, Fundamentals, and Applications. Edited by R. L. Bronaugh and H. I, Maibach. pp. 31-49. CRC Press, Boca Raton, FL. Elias P. M. (1983) Epidermal lipids, barrier functions, and desquamation. Journal of Investigative Dermatology 80, 44S-49S. Harada K., Murakami T., Yata N. and Yamamoto S. (1992) Role of intercellular lipids in stratum corneum in the percutaneous permeation of drugs. Journal of Investigative Dermatology 99, 278 282. Hawkins G. S. and Reifenrath W. G. (1986) Influence of skin source, penetration, cell fluid, and partition coefficient on in vitro skin penetration. Journal of Pharmaceutical Science 75, 378-381. Moody R. P., Franklin C. A., Ritter L. and Maibach H. I. (1990) Dermal absorption of the phenoxy herbicides 2,4-D, 2,4-D amine, 2,4-D isooctyl, and 2,4,5-T in rabbits, rats, rhesus monkeys, and humans: a cross-species comparison. Journal of Toxicology and Em,ironmental Health 29, 237-245. Moody R, P., Grayhurst M. and Ritter L. (1989b) Evaluation of the rat tail model for estimating dermal absorption of lindane. Journal o f Toxicology and Environmental Health 2g, 317-326. Moody R. P. and Martineau P. A. (1990) An automated in vitro dermal absorption procedure: 1. Permeation of ~ac-labelled N,N-diethyl-m-toluamide through human skin and effects of short-wave ultraviolet radiation on permeation. Toxicology in Vitro 4, 193-199. Moody R. P. and Nadeau B. (1993) An automated in vitro dermal absorption procedure: III. In vivo and in vitro comparison with the insect repellent N,N-diethyl-m-toluamide in mouse, rat, guinea pig, pig, human and tissuecultured skin. Toxicology in Vitro 7, 167-176. Moody R. P. and Nadeau B. (1994) In vitro dermal absorption of pesticides: IV. In vivo and in vitro comparison with the organophosphorus insecticide diazinon in rat, guinea pig, pig, human and tissue-cultured skin. Toxicology in Vitro 8, 1213-1218.
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R.P. Moot~ et al.
Moody R. P., Nadeau B. and Chu I. (1994) In vitro dermal absorption of pesticides: V. In vivo and in vitro comparison with the herbicide 2,4-dichlorophenoxyacetic acid in rat, guinea pig, pig, human and tissue-cultured skin. Toxicology in Vitro 8, 1219-1224. Moody R. P. and Ritter L. (1992) An automated in vitro dermal absorption procedure: II. Comparative in vivo and in vitro dermal absorption of the herbicide fenoxapropethyl (HOE 33171) in rats. Toxicology in Vitro 6, 53-59.
Moody R. P. and Ritter L. (1989) Dermal absorption of the insecticide lindane (1~, 2~, 3fl, 4a, 5~, 6B-hexachlorocyclohexane) in rats and rhesus monkeys: effect of anatomical site. Journal o f Toxicology and Environmental Health 28, 161-169. Walters K. A. and Roberts M. S. (1993) Veterinary applications of skin penetration enhancers. In Pharmaceutical Skin Penetration Enhancement. Edited by K. A. Waiters and J. Hadgraft. pp. 345-364. Marcel Dekker, New York.
Toxic'. in Vitro Vol. 8, No. 6, pp. 1225-1232, 1994
Pergamon
0887-2333(94)00172-3
Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0887-2333/94 $7.00 + 0.00
IN VITRO DERMAL ABSORPTION OF PESTICIDES: VI. IN VIVO A N D IN VITRO COMPARISON OF THE ORGANOCHLORINE INSECTICIDE DDT IN RAT, GUINEA PIG, PIG, H U M A N A N D TISSUE-CULTURED SKIN R. P. MOODy, B. NADEAU and I. CHU Health Canada, Environmental Health Centre, Tunney's Pasture, Ottawa, Ontario KIA 0L2, Canada (Received 23 December 1993; revisions received 6 April 1994)
Abstract--Invitro dermal absorption tests were conducted with the ~4C-ring-labelled
pesticide, l,l,ltrichloro-2,2-bis(4-chlorophenyl)ethane (DDT) dissolved in acetone and applied to dermatomed skin (0.5 mm) of a number of species at dose rates of 16-27/~g/cm2. Skin absorption was determined for 48 hr after exposure using in vitro flow-through cells. Skin absorption was calculated from the sum of the percentage recovery of '4C activity in the receiver solution added to the percentage recovery for the methanol washes of the skin at 48 hr and the skin digest samples. Two receiver solutions, Ringer's saline (used with Moody aluminium cells), and Hanks' HEPES buffered saline with 4% serum albumin (used with Bronaugh flow-through cells) were used. Listed in decreasing order, the total percentage in vitro dermal absorptions obtained by 48 hr after exposure for the five skin types were: 42 ___2.6% [hairless guinea pig; Hanks' receiver (HR)], 34 + 10.5% (rat; HR), 28 +_ 13.2% [Testskin; Ringer's receiver (RR)], 28 _ 2.9% (human; HR), 22 + 3.3% (Testskin; HR), 18 + 6.2% (pig; RR) and 14 _ 2.1% (pig; HR). The percentage t4C activity recovered in soapy water rinses of the skin specimens at 24 hr, and for methanol skin washes and skin digests at 48 hr, and of ~4C-labelled volatiles collected in air traps are reported. Data obtained with pig and Testskin for DDT using the Moody flow-through permeation cell was compared with that obtained using the Bronaugh cell. Significantly greater (P < 0.05) percentage recovery was obtained for the soap washes at 24 hr of the skin following the Bronaugh procedure than was obtained with the Moody method. Comparative in vivo studies demonstrated urinary recovery was 2 _ 0.5 and 15 + 1.7% for rats (dose rate; 6/~g/cm 2) and guinea pigs (dose rate: 9/tg/cmZ), respectively. Total faecal recovery was 20 + 1.9 and 44 _ 2.75% for rats and guinea pigs, respectively. Analysis of tissue taken at autopsy 14 days after dosing demonstrated total tissue recovery of 51 _ 5.6% in rats but of only 3 _ 0.7% in guinea pigs. Including the '4C activity extracted from the skin removed from the dose site at 14 days after exposure, the total recovery of dermally absorbed residues was 73 + 5.9 and 62 + 4.1% for rats and guinea pigs, respectively. Recovery of '4C from soapy water skin washes conducted at 24 hr after exposure was 3 + 1.4 and 14 ___1.8% for rats and guinea pigs, respectively, and this was significantly less than that obtained by both the Bronaugh and Moody in vitro procedures. Skin patch recovery was 24% for both rats and guinea pigs. In summary, the in vitro data underestimed the degree of dermal absorption observed in vivo for both rats and guinea pigs, and this was thought to be due to an overly vigorous removal of the pesticide skin deposit by the soap washing procedures used in vitro in comparison with the in vivo washing procedure.
INTRODUCTION
1977], which was tested in o u r previous study ( M o o d y et al., 1994), D D T is very lipophilic (log K o w = 6,19; C h i o u et al., 1977). The lipophilicity o f a chemical is
To complete the series o f articles ( M o o d y a n d Martineau, 1990; M o o d y and Ritter, 1992; M o o d y and Nadeau, 1993 and 1994) concerning a crossspecies c o m p a r i s o n of in vitro a n d in vivo dermal a b s o r p t i o n studies with pesticides s p a n n i n g a wide range of lipophilicities, the present study reports data o b t a i n e d for the o r g a n o c h l o r i n e insecticide 1,1,1trichloro-2,2-bis(4-chlorophenyl)ethane (DDT). In contrast to the relatively hydrophilic nature of 2,4dichlorophenoxyacetic acid [2,4-D; log o c t a n o l - w a t e r partition coefficient (log K o w ) = 2.81; Chiou et al., Abbreviations: BSA = bovine serum albumin; GI = gastro-
intestinal; H R = Hanks' receiver fluid; LSC = liquid scintillation counting; PR =perirenal; RR = Ringer's receiver fluid.
generally considered to affect its ability to permeate skin. However, detailed correlations of dermal permeation rates with pesticide lipophilicity have not been reported a l t h o u g h such studies have been conducted with other c o m p o u n d s (such as hair dyes a n d pharmaceuticals) ( B r o n a u g h a n d C o n g d o n , 1984; Hawkins and Reifenrath, 1986). A future report will deal with correlating o u r skin permeation data with Q S A R such as o c t a n o l - w a t e r partition coefficients (log Kow). The present report will serve to d o c u m e n t the D D T data obtained in vitro using b o t h the a l u m i n i u m flow-through M o o d y cell (previously referred to as the A I D A cell), and the teflon flowt h r o u g h B r o n a u g h cell (Bronaugh, 1991; B r o n a u g h
1225
1226
R.P. MOODYet al.
and Stewart, 1985; Moody and Martineau, 1990). In changing to the use of the Bronaugh cell we have also adopted the Collier and Bronaugh (1991) procedure of using Hanks' medium containing 4% bovine serum albumin (BSA) in place of the Ringer's saline that was used as a receiver solution for the Moody cell (the solution flowing under the skin collecting the chemical permeant). The possible influence of the composition of the receiver solution on dermal flux in vitro will be considered when discussing the in vitro concordance of the data obtained with that obtained in in vivo studies with rats and hairless guinea pigs. MATERIALS AND M E T H O D S
The in vitro procedure using the aluminium (Moody) permeation cell chambers, and methods of skin preparation (e.g. dermatoming), sample collection, liquid scintillation counting (LSC) analysis and calculation of results by electronic spreadsheet were identical to those detailed previously (Moody and Nadeau, 1993). The procedure of Collier and Bronaugh (1991) was used for the teflon (Bronaugh) permeation cell chambers (Bronaugh, 1991). For Moody cell tests, sterile Ringer's solution buffered at pH 7.4 with Trizma and supplemented with glucose (0.8 g/litre), penicillin G (0.06g/litre) and streptomycin sulfate (0.1 g/litre) was used as the receiver solution (Moody and Martineau, 1990). For Bronaugh cell tests, Hanks' HEPES buffered (pH7.4) growth medium containing 4% BSA, NaHCO3 (0.36g/litre) and gentamicin sulfate (50 mg/litre) was used as the receiver solution. Hanks' HEPES buffered receiver solution has been reported to maintain skin viability (Collier and Bronaugh, 1991). Sufficient receiver flow was required to ensure 'sink' conditions in the Moody and Bronaugh cells. The receiver flow rate used in the Moody cell (2.5 ml/hr) was the same as that used in our previous studies. A slower receiver flow rate (1.5 ml/hr) was used for the Bronaugh cells since this rate was sufficient to maintain 'sink' conditions and is the optimal rate given for the Bronaugh procedure (Bronaugh, 1991). The different cross-sectional areas of the two cell chamber types (Moody cell, 0.2 cm2;
Bronaugh cell, 0.64 cm 2) were taken into account for calculating dermal permeation rates. The in vivo procedure was identical to that reported previously (Moody and Nadeau, 1993). To reduce intraspecies variation in the observed data, the skin used in the in vitro studies was from the same rats and guinea pigs used for the in vivo tests. The skin from four animals was used in each in vitro test. The dose application rates and sources of skin specimens used in vitro are reported in Table 1. Testskin ~, a human-derived tissue-cultured skin, was purchased from Organogenesis Inc. (Cambridge, MA, USA). In the in vivo studies DDT was applied to a 4.2-cm 2 area of mid-dorsal skin at a rate of 6.1 and 9.1/tg/cm 2 for male Sprague-Dawley rats and female hairless guinea pigs, respectively. The dose site was protected from surface rub-off by a foam rubber non-occlusive patch (Moody and Nadeau, 1993). As detailed previously, the dosed skin region was rinsed with an aqueous Radiac soap solution (50%) at 24 hr after exposure in both the in vitro and the in vivo studies (Moody and Nadeau, 1993). This soap wash at 24 hr was used to remove the 'unabsorbed' pesticide residues. At 48 hr, when the in vitro studies were terminated, methanol washes of the skin and a subsequent tissue digest were conducted to remove the residual ~4C activity for mass balance purposes. 14C activity persisting in the dosed skin region in vivo was also measured by LSC analysis of digests of skin taken at autopsy 14 days after exposure (Moody and Nadeau, 1993). A two-tailed Student's t-test was used for statistical analysis, and P < 0.05 was taken as the level of significance. Chemicals
~4C-ring-labelled 4,4'-DDT ( > 9 8 % pure; sp. act. 11.8 mCi/mM) was purchased from Sigma Chemical Co. (St Louis, MO, USA). Hanks' balanced salts (modified; Catalogue no. H-2895), HEPES buffer (Catalogue no. H-9136) and bovine serum albumin (Catalogue no. A3425) were also purchased from the Sigma. Radiac soap (Radiacwash) solution was purchased from Atomic Products Corporation, Shirley (NY, USA). Scintillation cocktails and
Table 1. Topical dose application rate for [~4C]DDT in in vitro studies and source information (anatomical site, sex, age) of specimens used Species
Receiver solution*
Dose (/~Ci/cm 2)
Dose (/~g/cm2)
Skin source
Rat Guinea pig Pig Pig Human Testskin Testskin
Hanks' Hanks' Ringer's Hanks' Hanks' Ringer's Hanks"
0.77 0.92 0.80 0.66 0.89 0.55 0.76
23.0 27.1 23.2 19.9 26.3 16.2 22.3
Spragu~Dawley rat back, male, 5 wk old Hairless guinea pig back, female, 9 months old Yorkshire pig back, male, 8-9 wk old Yorkshire pig back, male, 8-9 wk old Caucasian human abdomen, female, 31 yr old Cultured human foreskin, 24br, from supplier Cultured human foreskin, 24 hr, from supplier
*Hanks" receiver solution was used with the Bronaugh cell and Ringer's receiver solution with the Moody cell. Hanks" receiver solution: Hanks" HEPES buffered growth medium containing 4% BSA, NaHCO~ (0.36 g/litre) and gentamicin sulfate (50 mg/litre); Ringer's solution buffered at pH 7.4 with Trizma and supplemented with glucose (0.8 g/litre), penicillin G (0.06 g/litre) and streptomycin sulfate (0.1 g/litre).
In vitro dermal absorption of DDT
1227
0.16 -Soap Wash
0.14 0.12 0 0 0Q
rc
t
0.1 0.08
l
0.06 0.04 0.02
1
0
0
4
8
12
16
20
24
28
32
36
40
44
48
Time (hr) Fig. 1. In vitro analysis of [~4C]DDT permeating rat skin. The percentage recovery in the receiver solution is shown v. time for each of the four replicate Bronaugh cells ( .~. , cell I; --I--, cell 2; - - : ~ - - , cell 3; [] , cell 4). The onset of the skin wash with Radiac soap and water at 24 hr is shown. organic solvents were obtained and used as described previously (Moody and Nadeau, 1993).
to reach a plateau level that persisted until a second elevation of recovery occurred at 24 hr after exposure (Figs 1 and 2). This peak after the wash at 24 hr was also observed in our previous studies with N , N diethyl-m-toluamide (DEET), diazinon and 2,4-D, however. However, the 'washing-in' effect of the 24-hr soap wash was very pronounced with D D T in the present study. As shown in Fig. 2, this washing-in effect could significantly enhance the degree of exposure experienced by a pesticide applicator who washed the skin with soap at some time after dermal exposure to D D T . It is interesting that following the 'wash-in' peak, the ~4C levels permeating the human skin (Fig. 2) declined to their pre-soap wash levels in
RESULTS AND DISCUSSION
In vitro studies
With the exception of Testskin, which was about 0 . 3 m m thick, the mean skin thickness was 0.5 _+ 0 . 0 1 m m for all test species (Moody and Nadeau, 1993). The results of the present study contrasted with our previous studies in which a rapid decline in the receiver levels of 14C activity occurred after an early peak level. With D D T , the percentage recovery of ~4C activity in the receiver solution tended
0.07
.••ll••Soap
0.06 0.05 t_
0 0 0 0
rc
W ash
0.04 0.03 0.02 0.01 O~ 0
I
I
4
I
I
8
I
I
12
I
I
16
I
I
20
I
I
24
I
I
28
I
I
32
[
I
36
I
I
40
I
I
44
I
48
Time (hr) Fig. 2. In vitro analysis of [~4C]DDT permeating human skin. The percentage recovery in the receiver solution is shown v. time for each of the four replicate Bronaugh cells ( .~. , cell 1; --I--, cell 2; - - ~ : - - , cell 3; [] -, cell 4), The onset of the skin wash with Radiac soap and water at 24 hr is shown.
1228
R. P. MOODY et al. Table 2. In vitro percentage recovery of radioactivity for different species of animal skin following topical application of [14CIDDT in acetone
Species
Receiver solution (cell type)t
[t4C] recovery (%)*
Rat
Hanks' (B)
Guinea pig
Hanks' (B)
Pig
Ringer's (M)
Pig
Hanks' (B)
Human
Hanks" (B)
Testskin
Ringer's (M)
Testskin
Hanks' (B)
Rad
Was
Dig
W+ D
Air
Total
59,5 (12,22) 54,5 (1,68) 21.9 (2.75) 83.6 (5.03) 65.8 (4.81) 28.4 (20.12) 72.3 (2.86)
13.4 (4.15) 21.2 (1.99) 15.8 (6.33) 9.2 (1.46) 19.0 (3.40) 14.9 (6.55) 11.4 (2.83)
20.2 (6.45) 17.8 (3.61) 1.6 (0.96) 4.0 (0.67) 8.1 (l.02) 12.8 (7.21) 10.3 (0.55)
33.6 (10.48) 39.0 (2.60 17.4 (6.11) 13.2 (2.09) 27.1 (2.88) 27.7 (13.1) 21.7 (3.29)
NS* (NS) NS (NS) 0.3 (0.32) NS (NS) NS (NS) 2.89 (I.23) NS (NS)
95.7 (5.54) 98.7 (2.06) 75.3 (9.62) 100,8 4.34 96.0 (3.85) 93.3 (4.83) 100.1 2.99
*Values are means (SD in parentheses; n = 4). Rad is % ~4C activity recovered in Radiac soap and water washes of the skin at 24 hr; Was is % recovered in methanol washes of the skin at 48 hr; Dig is % recovered in skin digest; W + D is % total recovered by methanol washes and skin digest; Air is % recovered in the air traps; Total is total % recovery (see text for discussion on mass balance). ~'The receiver solution used (Hanks' or Ringer's) is indicated together with the permeation cell type (B = Bronaugh; M = Moody). ~NS = no sample.
only two of the four permeation cells, while in the other two cells the ~4C levels declined only partially and then persisted at a plateau level roughly four times higher than the plateau level attained before soap washing. These results indicate that soap washing could actually elevate the bioavailability of the applied toxicant and thereby increase the risk involved. Since the wash-in peak was also observed in our previous studies in which the Moody cell was used and skin washing was conducted by pumping the soap solution at a slow flow rate (5 ml/min) over the dosed skin surface, it is most likely that this enhanced permeation was due to a surfactant effect of the soap. However, the pronounced wash-in peaks observed in the present study with the Bronaugh cells, suggest that the swabbing skin wash procedure may further enhance this effect by mechanical disruption
of the stratum corneum. Further testing using different soaps and wash procedures is warranted. The percentage recovery and the absorption data are reported in Tables 2 and 3, respectively. The amount of [14C]DDT residue rinsed off the skin by the Radiac soap wash at 24 hr (Table 2, column 3) ranged from a low of 22% in pig skin tested in a Moody cell with Ringer's receiver fluid, to a high of 84%, again with pig skin but using Hanks' receiver fluid and the Bronaugh cell. We also note that for Testskin (Table 2) significantly greater recovery of 14C activity in the soap washings was obtained using the Bronaugh procedure than using the Moody procedure. The value for Testskin (Ringer's, Moody cell) given in Table 2 (28.4 + 20.12%) includes one cell with a value of 59%, which accounts for the high standard deviation. With this cell excluded as an
Table 3. In vitro absorption data for different species of animal skin following topical applications with [~4C]DDT in acetone
Species
Receiver solution (cell type)~:
Rat
Hanks' (B)
Guinea pig
Hanks' (B)
Pig
Ringer's (M)
Pig
Hanks' (B)
Human
Hanks' (B)
Testskin
Ringer's (M)
Testskin
Hanks' (B)
Per* (%)
Per? (%)
Cum (pg/cm 2)
Rp (mg/cmZ/hr)
0.4 (0.04) 2.6 (0.54) 0.2 (0.34) 1.1 (0.23) 0.5 (0.19) 0.2 (0.07) 0.3 (0.02)
33.9 (I 0.49) 41.6 (2.63) 17.6 (6.15) 14.3 (2.07) 27.59 (2.93) 27.9 (13.19) 22.0 (3.30)
0.1 (0.0 I) 0.7 (0.15) 0.0 (0.08) 0.2 (0.05) 0.1 (0.05) 0.0 (0.01 ) 0.1 (0.01)
0.01 (0.002) 0.03 (0.006) 0.02 (0.032) 0.01 (0.002) 0.01 (0.001 ) 0.00 (0.000) 0.01 (0.001)
Values are means (SD in parentheses; n ~ 4). Per* is % skin permeation calculated from the % recovery in the receiver solution; Pert is the % skin permeation calculated from adding the % recovery in receiver solution to the % recovery in the methanol skin washes + skin digest samples 48 hr after exposure (Table 2, column 5): Cure is cumulative absorption in 48 hr; Rp is the maximum rate of skin permeation. ++The receiver solution used (Hanks' or Ringer's) is indicated together with the permeation cell type (B = Bronaugh; M = Moody).
In vitro dermal absorption of DDT
'outlier', the mean value becomes 18 _ 0.8% (n = 3), and increases the difference between the values for the soap wash recovery of J4C for the Moody and Bronaugh procedures. It is possible that the anomalously high value in the one permeation cell was due to a sloughing off of a portion of the treated epidermis during the soap washing procedure. It is not unexpected that the Moody soap washing procedure, which involves a mechanically delivered (syringe pump) gentle rinsing of the treated skin epidermis with soap solution at a constant slow flow rate of 5 ml/min (Moody and Ritter, 1992) would be less vigorous than the Bronaugh method which involves manual washing with cotton-tipped swabs. The friction of the swab on the skin surface would surpass that of a gently flowing stream. Although dermal swabbing is conducted in vivo, the epidermis of the isolated skin at 24 hr after isolation is probably more fragile than the skin in vivo. The effect of different skin washing procedures will be considered further when we discuss the comparison of the in vitro and in vivo data. The amount of ~4C-labelled pesticide not washed off by the Radiac soap and persisting in the skin at 48 hr after exposure varied between species (Table 2, column 6) and ranged from 13 to 39% of the applied dose. The degree of skin storage of lipophiles may depend on the species-specific lipid composition of the stratum corneum and on its total lipid content (Elias, 1983; Harada et al., 1992). In our previous studies, this pesticide 'depot' in human skin was 0.4, 14 and 18% for DEET, diazinon and 2,4-D, respectively, in comparison with 27% for DDT in the present study. The high value for persistence of DDT in skin is consistent with the relatively high lipophilicity of DDT. Air traps were not used with the Bronaugh cells but air-trap data for the Moody cells (Table 2, column 7) indicated that DDT was not volatile under the present conditions, in contrast with, for example, DEET with which we found 34% recovery of ~4C in air traps in tests on Testskin (Moody and Nadeau, 1993) (3% for DDT and Testskin, Table 2). The total mass balance was good with over 93% recovery being obtained in all cases except in the Moody cell study with pig skin in which total recovery was only 75% (Table 2, column 8). In view of the precision of the Radiac soap wash data for pig skin (22 + 2.8%), it is most likely that the low total percentage recovery was due to the administration of a lower dose than intended. The absorption data (Table 3) showed nearly insignificant permeation into the receiver solution (p*, column 3), in contrast with the total permeation (Pt, column 4) calculated by adding the percentage recovery in the skin depots to that for the receiver solutions. In fact this raises the possibility that, at least for lipophilic compounds such as DDT, permeation studies need not be conducted since 'skin depot persistence studies' (e.g. tape stripping studies) would serve the in vitro modelling purpose just as well. This
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also raises concern that for lipophilic compounds like DDT, in vitro methods may not provide a suitable alternative to in vivo animal testing. With the skin depot included as an absorbed residue, the ranking of the skin specimens in order of decreasing permeability (Pt, Table 3, column 4) was: guinea pig, rat, Testskin (Moody cell), human, Testskin (Bronaugh cell), pig (Moody cell), and pig (Bronaugh cell). Excellent Testskin/human skin agreement for skin permeability was evident here for DDT. There were insufficient data in the present study to validate the Moody cell as an alternative to the Bronaugh cell. The results, however, were promising in demonstrating similarly low permeation into the receiver solutions used (Table 3, column 3). Although there was a slight increase in permeation recorded through pig skin into Hanks' receiver with the Bronaugh cell (1.1%), in comparison with the data obtained for the Ringer's receiver (Moody cell, 0.2%), there was similar permeation in the two systems for the Testskin (0.3% with Hanks' receiver in the Bronaugh cell v. 0.2% with Ringer's receiver in the Moody cell, Table 2). These data suggest that in studies with very lipophilic compounds, such as DDT, the presence of BSA may not enhance the dissolution and uptake of the permeant from the skin to the receiver solution. It is quite possible that with the faster flow rate used here for the Moody cell, combined with the slight vortex current established by having the in and out ports of the receiver cells offset, dissolution was optimized in the Moody cell. The cumulative amounts absorbed into the receiver solution and the data for the Rp rate parameter are given in Table 3. The Lag values that are normally presented are not given since, because of the rapid rise to plateau levels of the receiver percentage recoveries (Figs 1 and 2), there were insufficient data for this calculation. In general, the DDT data showed much less cumulative absorption and a slower maximum rate of absorption than that reported previously for DEET (Moody and Nadeau, 1993). For example the DEET data for human skin had a cumulative absorption of 12.4/lg/cm 2 (v. 0.1 pg/cm 2 for DDT) and a maximum permeation rate of 2pg/cm2/hr (v. 0.01 pg/cm2/hr for DDT). In vivo studies
The urinary and faecal recoveries of ~4C activity for both rats and guinea pigs reached maximum values at 48 hr after exposure, with the exception of the guinea pig faecal levels which peaked at 72 hr. For both species, urinary levels declined rapidly to less than 0.1% of the applied dose by 14 days after exposure, while faecal levels declined to less than 1% by 14 days. The absorbed and non-absorbed recoveries of ~4C are presented in Tables 4 and 5, respectively. In contrast to our previous studies with DEET, diazinon and 2,4-D, the faecal percentage recovery for DDT exceeded the urinary recovery (Table 4). This is consistent with general agreement that more
1230
R . P . MOODY et al. Table 4. In viva percentage recovery of absorbed residues of [14C]DDT in rats and hairless guinea pigs 14C recovery (%) in: Dose Species (#g/cm:) Urine Faeces Tissue Total* Rat 6.1 2.1 19.6 50.5 72.1 (0.51) (1.85) (5.63) (5.85) Guinea pig 9.1 15.1 44.3 2.9 62.3 (1.74) (2.69) (0.73) (4.12) Values are means (SD in parentheses; n = 4). *Total is the summed % recoveries of urinary, faecal and tissue samples and represents the total % dermal absorption (see text). Note that the total % dermal absorption including the % recovery in the skin digests (Table 5, column 4) was 72.9 -+ 5.85 and 62.3 _+4.10% for rats and guinea pigs, respectively.
lipophilic c o m p o u n d s t e n d to be excreted to a g r e a t e r e x t e n t by t h e biliary a n d faecal routes. T h e e x c e p t i o n ally large recovery ( 5 1 % ) in the rat tissue s a m p l e s is discussed below. T h e d e r m a l a b s o r p t i o n o f the a p p l i e d D D T d o s e was 73 + 5.9% for rats a n d 62 ___4.1%0 for guinea pigs (see T a b l e 4 f o o t n o t e ) . T h e s e values are m u c h in excess o f the d e r m a l a b s o r p t i o n values r e p o r t e d by Bucks e t al. (1991) in h u m a n s d o s e d o n the f o r e a r m ( 1 0 % ) a n d in rhesus m o n k e y s d o s e d o n t h e a b d o m e n (19%). In the studies by Bucks e t al. (1991), D D T was a p p l i e d in a c e t o n e a n d a s o a p w a s h was c o n d u c t e d at 24 hr. T h e a b s o r p t i o n values for rats a n d guinea pigs in the c u r r e n t s t u d y also exceed the values o f 1.5, 43.4 a n d 4 6 . 3 % given for in viva d e r m a l a b s o r p t i o n o f D D T in m o n k e y s , pigs a n d rabbits, respectively (Bartek a n d L a B u d d e , 1975; also see T a b l e 3 in W a i t e r s a n d R o b e r t s , 1993). A l t h o u g h o u r p r e v i o u s studies have s u g g e s t e d that rat skin is generally m o r e p e r m e a b l e t h a n h u m a n , m o n k e y o r pig skin, in o u r s t u d y with 2,4-D a m i n e we did r e p o r t an e x c e p t i o n w h e r e rat skin was m o r e p e r m e a b l e t h a n r a b b i t skin b u t less p e r m e a b l e t h a n h u m a n skin ( M o o d y e t al., 1990). C o n s i d e r i n g the n o n - a b s o r b e d residues (Table 5), the l o w e r degree o f r e m o v a l o f the J4C residues in skin by the s o a p w a s h at 24 h r in rats in c o m p a r i s o n with guinea pigs is c o n s i s t e n t with the m u c h higher perc e n t a g e recovery o b t a i n e d f r o m the digests o f rat skin ( 3 4 % ) t h a n f r o m the digests o f the guinea pig skin (0%). Simply, the n o n - w a s h a b l e residue was persisting in the skin. T h e excellent total p e r c e n t a g e recoveries r e p o r t e d in Table 5 d e m o n s t r a t e that it is Table 5. In viva percentage recovery of non-absorbed residues of IL4CIDDT in rats and hairless guinea pigs 14C recovery (%) in: Soap Skin Skin wash patch digest Total* 2.8 22.6 34.3 104.3 (1.42) (8.98) (8.09) (3.92) Guinea pig 13.9 23.9 01) 100.0 (I.82) (0.26) ((t.02) (3.67) Values are means (SD in parentheses: ,~ 41. *Total is the summed % recoveries of thc total % recovery in Table 4 (urine + faeces + tissue) in addition to the % recoveries for soap washes of the dose site at 24 hr, the skin patch used to protect the dose site and the % recovered from the digests of the skin tissue removed from the dose site. Species Rat
essential to include t h e skin p a t c h recoveries in the m a s s b a l a n c e analysis. In c o n t r a s t to o u r p r e v i o u s r e p o r t s with D E E T , d i a z i n o n a n d 2,4-D, t h e p r e s e n t s t u d y d e m o n s t r a t e d significant b o d y s t o r a g e o f D D T with a total o f 51 a n d 3 % recovery o f 14C activity being d e t e c t e d in the tissues at 14 days after e x p o s u r e o f rats a n d guinea pig, respectively (Table 4). In rats, r a d i o a c t i v i t y was d e t e c t e d at levels ...<0.01% in only the red b l o o d ceil, spleen a n d b l a d d e r samples, while levels />0.8% were d e t e c t e d in the liver (0.9_+ 1.04%), g a s t r o intestinal (GI) tract (7.4_+ 8.58%), perirenal (PR) w h i t e fat (5.7_+6.62%), i n t e r s c a p u l a r b r o w n fat (0.8 _+ 0.92%), skin external to dose site (8.5_+ 10.15%) a n d in the carcass ( 2 6 . 3 _ + 3 0 . 5 % ) (Table 6). In guinea pigs, r a d i o a c t i v i t y was d e t e c t e d m a i n l y in the liver, G I tract, P R white fat, skin a n d carcass (Table 6). T h e r e a s o n for the o b s e r v e d m u c h increased persistence o f D D T residues in rats in c o m p a r i s o n with guinea pigs is n o t k n o w n but the d a t a suggest that rat tissues in general p r o v i d e a
Table 6. In viva percentage of recovery of ~4C activity in tissue samples taken at autopsy 14 days after exposure to [a4C]DDT E4C recovery (%) Tissue
Rat
Guinea pig
Brain 0.02 _+0.01 0.01 _+0.00 Heart 0.02 + 0.01 0.01 +_0.00 Lungs 0.08 + 0.08 0.01 + 0.01 Kidney 0.18 + 0.22 0.04 + 0.04 Spleen ND ND Liver 0.92 + 1.04 0.22 + 0.23 Diaphragm 0.06 + 0.07 ND GI tract 7.41 + 8.58 0.36 + 0.44 PR white fat 5.74+6.62 0.16 +0.19 Adrenals 0.04 -+ 0.03 ND IBAT 0.79+0.92 0.08_+0.10 Testes 0.03 + 0.02 -Tail 0.07 _+0.06 -Bladder 0.01 _+0.01 ND RBC ND ND Plasma 0.09 _+0.09 0.02 _+0.02 Thymus 0.05 _+0.04 0.01 _+0.01 Stomach 0.18 _+_0.19 0.04 ± 0.05 Skin 8.52 -+ 10.15 0.48 + 0.56 Carcass 26.29 -+ 30.46 1.45 -+ 1.69 ND = non-detectable (<0.005%) GI = gastro-intestinal PR = perirenal IBAT = interscapular brown adipose tissue RBC = red blood cells Values are means+ SD for groups of four male Sprague Dawley rats and four female hairless guinea pigs.
In vitro dermal absorption of DDT
relatively iipophilic 'sink' for such residues. We have previously reported that another organoehlorine insecticide, lindane, was more slowly cleared from rats than from rhesus monkeys following topical application and that body storage of lindane residues was indicated (Moody and Ritter, 1989). In a subsequent study, '4C activity was still detectable in the urine of rats at 72 days after exposure of the tail skin to lindane (Moody et al., 1989). We recommend that further studies are conducted to examine this lipid 'sink' in rats since, unless this happens to approximate to the human condition, the use of the rat model will be limited. In vivo /in vitro comparison
The in vivo data (see footnote to Table 4) demonstrated only slightly higher total dermal absorption of [~4C]DDT for rats (73 _+ 5.9%) than for guinea pigs (62_+4.1%), and this was consistent with similar levels of skin permeability being obtained in vitro for rat skin (34_+ 10.5%) and guinea pig skin (42 ___2.6%) ( p t , Table 3). However, the in vitro data underestimated the dermal absorption observed in vivo. As discussed above with regard to the fact that the Bronaugh soap wash procedure at 24hr was different from that used in the Moody method, it is apparent that an overly vigorous in vitro soap wash relative to that used in vivo could explain the underestimate of dermal absorption obtained by the Bronaugh in vitro procedure carried out here. Simply, the residues washed off the skin at 2 4 h r are not available for absorption, and hence an in vitro skinwashing method that very effectively removes the test compound would lead to an underestimate of in vivo absorption. Therefore, it is recommended that studies are conducted to determine the most appropriate skin washing method to use in vitro so that accurate predictions of percutaneous absorption can be made. In conclusion, the present study has shown that the in vitro procedure underestimated the dermal absorption data obtained in vivo for D D T . Although this underestimate suggested that the in vitro model may not provide a suitable alternative to the in vivo model for lipophilic compounds, good intermodel agreement has recently been observed with another lipophilic compound, benzo[a]pyrene (log Kow = 6.1) in our laboratory (R. P. Moody, unpublished data, 1994), and a general conclusion cannot be made at this time. The ranking of the skin types, listed in decreasing order of permeability to D D T , as determined from the in vitro permeability data were: hairless guinea pig [Hanks' receiver (HR)]>~rat (HR) ~> Testskin [Ringer's receiver (RR)] = human (HR) > T e s t s k i n (HR) ~> pig (RR)/> pig (HR). Future reports will examine the effect of the skin washing procedure on the dermal absorption process. Acknowledgements--We are grateful to Ms Patricia Curry and Ms Dianne Dick for reviewing the manuscript and to Dr Conrad Watters, Ottawa General Hospital, Ottawa,
1231
Ontario, for kindly providing the human skin specimens. We also thank Mr Robert Marr for his excellent technical assistance. REFERENCES
Bartek M. J. and LaBudde J. A. (1975) Percutaneous absorption in vitro. In Animal Models in Dermatology. Edited by H. I. Maibach. pp. 103-112. Churchill-Livingstone, New York. Bronaugh R. L. (1991) A flow-through diffusion cell. In In Vitro Percutaneous Absorption: Principles, Fundamentals, and Applications. Edited by R. L. Bronaugh and H. I. Maibach. pp. 17-23. CRC Press, Boca Raton, FL. Bronaugh R. J. and Congdon E. R. (1984) Percutaneous absorption of hair dyes: correlation with partition coefficients, Journal of Investigative Dermatology 83, 124-127. Bronaugh R. J. and Stewart R. F. (1985) Methods for in vitro percutaneous absorption studies. IV. The flowthrough diffusion cell. Journal of Pharmaceutical Science 74, 64q57. Bucks D. A. W., Wester R. C. and Maibach H. I. (1991) Percutaneous penetration from soil: inhibition or enhancement? In In Vitro Percutaneous Absorption: Principles, Fundamentals, and Applications. Edited by R. L. Bronaugh and H. I. Maibach. pp. 223-230. CRC Press, Boca Raton, FL. Chiou C. T., Freed V. H., Schmedding D. W. and Kohnert R. L. (1977) Partition coefficient and bioaccumulation of selected organic chemicals. Environmental Science and Technology 11, 475-478. Collier S. W. and Bronaugh R. L. (1991) Receptor fluids. In In Vitro Percutaneous Absorption: Principles, Fundamentals, and Applications. Edited by R. L. Bronaugh and H. I, Maibach. pp. 31-49. CRC Press, Boca Raton, FL. Elias P. M. (1983) Epidermal lipids, barrier functions, and desquamation. Journal of Investigative Dermatology 80, 44S-49S. Harada K., Murakami T., Yata N. and Yamamoto S. (1992) Role of intercellular lipids in stratum corneum in the percutaneous permeation of drugs. Journal of Investigative Dermatology 99, 278 282. Hawkins G. S. and Reifenrath W. G. (1986) Influence of skin source, penetration, cell fluid, and partition coefficient on in vitro skin penetration. Journal of Pharmaceutical Science 75, 378-381. Moody R. P., Franklin C. A., Ritter L. and Maibach H. I. (1990) Dermal absorption of the phenoxy herbicides 2,4-D, 2,4-D amine, 2,4-D isooctyl, and 2,4,5-T in rabbits, rats, rhesus monkeys, and humans: a cross-species comparison. Journal of Toxicology and Em,ironmental Health 29, 237-245. Moody R, P., Grayhurst M. and Ritter L. (1989b) Evaluation of the rat tail model for estimating dermal absorption of lindane. Journal o f Toxicology and Environmental Health 2g, 317-326. Moody R. P. and Martineau P. A. (1990) An automated in vitro dermal absorption procedure: 1. Permeation of ~ac-labelled N,N-diethyl-m-toluamide through human skin and effects of short-wave ultraviolet radiation on permeation. Toxicology in Vitro 4, 193-199. Moody R. P. and Nadeau B. (1993) An automated in vitro dermal absorption procedure: III. In vivo and in vitro comparison with the insect repellent N,N-diethyl-m-toluamide in mouse, rat, guinea pig, pig, human and tissuecultured skin. Toxicology in Vitro 7, 167-176. Moody R. P. and Nadeau B. (1994) In vitro dermal absorption of pesticides: IV. In vivo and in vitro comparison with the organophosphorus insecticide diazinon in rat, guinea pig, pig, human and tissue-cultured skin. Toxicology in Vitro 8, 1213-1218.
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Moody R. P., Nadeau B. and Chu I. (1994) In vitro dermal absorption of pesticides: V. In vivo and in vitro comparison with the herbicide 2,4-dichlorophenoxyacetic acid in rat, guinea pig, pig, human and tissue-cultured skin. Toxicology in Vitro 8, 1219-1224. Moody R. P. and Ritter L. (1992) An automated in vitro dermal absorption procedure: II. Comparative in vivo and in vitro dermal absorption of the herbicide fenoxapropethyl (HOE 33171) in rats. Toxicology in Vitro 6, 53-59.
Moody R. P. and Ritter L. (1989) Dermal absorption of the insecticide lindane (1~, 2~, 3fl, 4a, 5~, 6B-hexachlorocyclohexane) in rats and rhesus monkeys: effect of anatomical site. Journal o f Toxicology and Environmental Health 28, 161-169. Walters K. A. and Roberts M. S. (1993) Veterinary applications of skin penetration enhancers. In Pharmaceutical Skin Penetration Enhancement. Edited by K. A. Waiters and J. Hadgraft. pp. 345-364. Marcel Dekker, New York.