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Annals of Occupational Hygiene Advance Access originally published online on October 27, 2004
Annals of Occupational Hygiene 2004 48(8):697-705; doi:10.1093/annhyg/meh070
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© British Occupational Hygiene Society Published by Oxford University Press;

In Vitro Percutaneous Penetration of Five Pesticides—Effects of Molecular Weight and Solubility Characteristics

JESPER BO NIELSEN1,*, FLEMMING NIELSEN1 and JENS AHM SØRENSEN2

1 Environmental Medicine, University of Southern Denmark, Odense, Denmark; 2 Department of Plastic and Reconstructive Surgery, Odense University Hospital, Odense, Denmark

* Author to whom correspondence should be addressed. Tel: +45 65 50 37 64; Fax: + 45 65 91 14 58; E-mail: jbnielsen{at}health.sdu.dk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
This study examined the in vitro percutaneous penetration of five pesticides covering a wide range of solubilities and different molecular weights, which allowed discussion of penetration of single pesticides as well as a comparison between penetration characteristics of different pesticides. The five pesticides were the fungicides methiocarb, pirimicarb and prochloraz; the growth retardant paclobutrazol; and the insecticide dimethoate. An experimental model with static diffusion cells mounted with human breast skin was used, and pesticide concentrations were quantified by high-performance liquid chromatography. Molecular weight as well as solubility affected dermal penetration and skin deposition. Thus, we conclude that for the passage of our test substances from the donor phase, through the skin, into the receptor, a too high as well as a too low lipophilicity may limit the rate and degree of skin penetration. Furthermore, the importance of the skin as a potential reservoir for systemic exposure after exposure has ended was demonstrated. Especially in relation to short-term occupational exposures, an exposure assessment based on penetrated pesticide at the end of a work shift may underestimate the exposure.

Keywords: dimethoate • methiocarb • molecular weight • paclobutrazol • percutaneous penetration • pesticide • pirimicarb • prochloraz • solubility


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Pesticides are among the few substances distributed into our environment with the intent to harm biological systems. The selectivity of pesticides varies and many of the toxicological endpoints that pesticides target also make humans a potential target. The toxicity may involve systemic as well as local effects depending on the route of exposure. The primary occupational exposure to pesticides is dermal (Archibald et al., 1994Go; Benford et al., 1999Go), and occurs during mixing and spraying, and in greenhouses during re-entry activities, i.e. activities related to handling of recently treated plants.

Percutaneous penetration of pesticides has been studied in vivo in rats and pigs and in vitro using animal or human skin samples. Rodent skin will in most cases overestimate the penetration rate of topically applied chemicals (OECD 2000Go; van de Sandt et al., 2004Go). We used an in vitro model with human full-thickness skin and static diffusion cells. The experimental model is described in the most recent OECD guideline (OECD, 2000Go), and has generally had a reasonable good correlation with human in vivo studies (Ramsey et al., 1994Go; OECD, 2000Go). In vitro studies may use different preparations of skin, e.g. full-thickness or dermatomed skin, which will generate skin barriers of different thickness. Skin thickness will affect the experimental results (Van de Sandt et al., 2004Go), and prolonged lag-times might be expected in experiments using full-thickness skin. Which skin preparation generates the most valid data is not obvious, and this is probably one of the reasons why OECD accepts the different experimental approaches in their guidelines (OECD, 2000Go). A recent inter-laboratory comparison of experimental models on percutaneous penetration involving nine European laboratories demonstrated good agreement between data on selected model compounds obtained in the different laboratories, given that comparable experimental procedures were used (van de Sandt et al., 2004Go).

Experimental data on percutaneous penetration rate (flux) is obtained by measurements of concentrations in the receptor chamber over time, and Kp-(permeability coefficient) by dividing the steady-state flux obtained in experiments with infinite dosing with the concentration in the donor chamber. For occupational risk assessment following dermal exposure, the flux or relative absorption (percentage absorption per day of an occupationally relevant dose) is often used to estimate the risk. As evident from Fig. 1, penetration rates are, however, not sufficient to evaluate the toxicity profile of a pesticide after dermal exposure. Thus, two pesticides with significantly different maximal flux, and therefore also different Kps, may cause identical pesticide doses given that their lag-times also differ. Except for in vitro studies with continuous sampling, lag-times are often difficult to measure, and will most often be estimated based on a back extrapolation from the linear part of the penetration curve.



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  		Fig. 1. Theoretical penetration curves for two chemicals (A and B) with identical total penetration after 6 h, but different lag-time and flux.

 
The present study examines the in vitro percutaneous penetration of five pesticides widely used in agriculture and greenhouses. The pesticides cover a wide range of solubilities as well as different molecular weights (Table 1), which allows discussion of penetration characteristics of a single pesticide as well as a comparison between penetration characteristics as influenced by molecular weight and solubility. We have included data on lag-time, steady-state flux (during infinite dosing), and Kp, as well as the apparent deposition of pesticides within the skin membrane.


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  		Table 1. Percutaneous penetration characteristics for five pesticides

 
Deposition in the skin is included, as the amount of pesticide deposited will potentially serve as a reservoir for continued systemic absorption after dermal exposure has ended. Thus, in a set of 19 pesticides tested in an in vivo rat model, absorption from the washed skin continued in 17 of the pesticides tested, although only nine showed increased systemic concentrations (Zendzian, 2003Go).

The five pesticides included in this study were: the fungicides (i) methiocarb and (ii) pirimicarb, both of which are carbamates; (iii) prochloraz; (iv) the growth retardant, paclobutrazol; and (v) the insecticide, dimethoate. Despite their extensive use, the open literature on dermal penetration of these five pesticides is very limited, and risk estimates are often based on extrapolation from high-dose experiments with systemic administration of pesticides and use of a default value of 10 or 100% for dermal absorption. In a previous study using 50% ethanol as receptor fluid, we found lag-times in the range of 10–18 h for methiocarb, pirimicarb and paclobutrazol (Nielsen and Nielsen, 2000Go).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Chemicals
(i) Pirimicarb (2-Dimethylamino-5,6-dimethylpyrimidin-4-yl dimethylcarbamate; CAS No. [23103-98-2]; Mw = 238.3 g/mol); (ii) methiocarb (4-methylthio-3, 5-xylyl methylcarbamate; CAS No. [2032-65-7]; Mw = 225.3 g/mol); (iii) dimethoate (O,O-dimethyl-S-[2-(methylamino)-2-oxoethyl]-phosphordithioate; CAS No. [60-51-5]; Mw = 229.3 g/mol) and (iv) prochloraz (N-propyl-N-[2-(2,4,6-trichlorophenoxy) ethyl]-1H-imidazol-1-carboxamide; CAS No. [67747-09-5]; Mw = 376.7 g/mol) were obtained as reference materials (Dr. Ehrenstorfer, Augsburg, Germany). (v) Paclobutrazol ((2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1H-1,2,4-triazol-1-yl) pentan-3-ol; CAS No. [76738-62-0]; Mw = 293.8 g/mol) was obtained from Riedel-de Häen (Sigma-Aldrich Laborchemikalien Gmbh, Seelze, Germany).

All stock solutions were made in 96% ethanol obtained from Fluka (Fluka Chemie, Buchs, Switzerland). The working solution for dimethoate was made in 0.9% NaCl, whereas other working solutions were made in ethanol:water (50:50).

Water was purified with a Milli-Q system from Millipore Corp. (Billerica, MA, USA). Acetonitril and methanol were of ‘HiPerSolv grade’ and were obtained from BDH (BDH, Poole, England). Bovine serum albumin (BSA) was obtained from Sigma (Sigma-Aldrich Laborchemikalien Gmbh, Steinheim, Germany).

Skin membranes
The human skin samples were obtained from the Department of Plastic and Reconstructive Surgery at the University Hospital in Odense. Skin was sampled from women (20–67 years old) who underwent breast reduction or breast reconstruction. The donors were given complete anonymity, with registration of only the age of the female donors. Skin samples were kept at –20°C for periods not exceeding 12 months. This has been proven to keep the skin fresh without inducing significant change in the water permeability (Bronaugh et al., 1986Go). The skin was allowed to thaw at room temperature 1 h before being gently cleaned with distilled water. Subsequently, subcutaneous fat was removed with a sharp dissection knife while the skin was still partly frozen to ease the preparation without damaging the skin. Skin thickness varied between 0.6 and 0.9 mm. Skin samples from individual donors were equally distributed between experimental groups, and three to four donors were used in each experiment.

Experimental model
A previously described experimental model (Nielsen, 2000Go; Nielsen et al., 2000Go) using in vitro static diffusion cells adapted from Southwell et al. (1994)Go was used. The system consists of two half-cells where the upper cell compartment represents the donor chamber and the lower the receptor chamber. The skin sample dividing the two cells is placed on a metal grid and held in place by a clamp, which at the same time keeps the half-cells together. The cells are kept at a constant temperature (32°C) in a water bath with individual magnetic stirring. This assured a temperature close to 32°C on the skin surface. The median diffusion area was 2.12 cm2/cell. Prior to the experiments, the epidermal site was exposed to ambient laboratory conditions and the dermis to an aqueous solution of 0.9% NaCl and 5% BSA for 18 h. The barrier integrity was evaluated by capacitance measurements (Lutron DM-9023, Acer AB, Sweden) before the exposure to pesticides began, and cells with a capacitance >55 nF were excluded.

Experimental set-up
The number of diffusion cells per group was nine, and the pesticides were applied to the donor chamber in concentrations of 0.2 mM (except dimethoate: 1.0 mM) in a total volume of 600 µl (283 µl/cm2). Pesticides were applied in aqueous solutions containing 0.9% NaCl and 2% ethanol. During the experimental periods, donor and receptor chambers were covered with Parafilm® to avoid evaporation. The skin was exposed to pesticides for 6 or 24 h before the pesticides were removed and the donor chambers washed. Following removal of pesticides from the donor chamber, experiments continued for 42 or 24 h, respectively, reaching a total experimental period of 48 h in all experiments. Samples (1 ml) were taken from the receptor chamber at appropriate intervals during the entire experimental period of 48 h and replaced with fresh receptor fluid (0.9% NaCl and 5% BSA in water), keeping an infinite sink. All five pesticides were soluble in donor as well as receptor fluid at the concentrations relevant for these experiments.

Pesticide analysis
Pesticide concentrations were quantified in the receptor medium by high-performance liquid chromatography (HPLC) after precipitation of proteins with 96% ethanol and centrifugation. In brief, an aliquot of 150 µl receptor medium and 300 µl ethanol was added into a 3 ml centrifugation tube and vortex-mixed for 5 s. The samples were then kept at –20°C for 30 min and centrifuged at 3°C at 1200g for 15 min. A 50 µl aliquot of the supernatant was injected into a Synergi Max-RP 150-4.6 mm column (Phenomenex, Torrance, CA) on the HPLC system using a Kontron 360 auto sampler (Kontron, Milan, Italy). Detection was performed by a Kontron 430 UV-detector and the eluent flow provided by a Merck Hitachi L-6200 intelligent pump (Merck, Darmstadt, Germany). The HPLC system was controlled by Kontron MT-450 software. Pirimicarb, paclobutrazol and methiocarb were eluted with a mobile phase consisting of acetonitrile:water (55:45) using a flow rate of 1.25 ml/min. Pirimicarb was detected at 248 nm, whereas paclobutrazol and methiocarb were detected at 200 nm. Dimethoate was eluted with a mobile phase consisting of acetonitrile : water (40 : 60) at a flow rate of 1 ml/min, and detected at 200 nm. Prochloraz was eluted with a mobile phase consisting of methanol : water (85 : 15) at a flow rate of 1 ml/min and detected at 200 nm. Quantification was based on the peak area of the compound related to a standard curve.

Data processing and statistics
Data are presented as mean values with standard deviation. As a dilution factor is introduced every time a 1 ml sample is removed from the 16.6 ml receptor chamber and replaced by 1 ml fresh receptor, all data on total penetration were corrected for these dilutions. Thus, when calculating total penetration after e.g. 48 h, the pesticide removed during sampling at 6, 12, 24, and 30 h will have to be added to avoid underestimating total penetration. Calculation of penetration rate (flux), lag-time, Kp and total penetration during the experimental period was based upon analysis of pesticides in the receptor chamber. Skin deposition was estimated by subtracting the total penetration to the receptor chamber during 48 h and the total amount of pesticide recovered in the donor wash from the initial amount applied to the donor chamber. The estimated skin deposition will therefore include the small amount of pesticide that potentially may be lost during the experiments. Student's t-test was used for statistical comparisons between groups.

Ethics
The study was approved by the regional ethical review committee.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Dimethoate was the pesticide with the highest solubility in water and also the only pesticide studied that did not penetrate the dermal barrier during the 48 h experimental period (Tables 1 and 2). The donor wash at the end of the 6 h exposure period demonstrated a complete recovery (100.2%) of the pesticide added to the donor, and demonstrated that dimethoate did not even enter the skin barrier during the 6 h exposure (Table 3). During prolonged exposure (24 h), results were identical and only marginal amounts of dimethoate may have entered the skin (Table 4).


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  		Table 2. Percutaneous penetration characteristics for five pesticides

 

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  		Table 3. Total dermal penetration, recovery in donor phase and skin deposition of five pesticides

 

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  		Table 4. Total dermal penetration, recovery in donor phase and skin deposition of five pesticides

 
The lag-time of pirimicarb was below 10 h in both experimental set-ups, with the 6 h experiment showing the shortest lag-time of 5 h (Tables 1 and 2). The maximal fluxes in the two pirimicarb experiments (6 and 24 h exposure) were, however, very similar. The comparable penetration rates during pesticide exposure are also evident from Figs 2 and 3. The two penetration curves for pirimicarb illustrate that its penetration continues with maximal rate for around 6 h after the pesticide has been removed from the donor (Fig. 4). Moreover, very similar penetration rates were also observed during the second phase of the experimental period, after the pesticides had been removed from the donor chamber (Fig. 4). This penetration is due to the pesticide residing in, and subsequently being released from, the skin reservoir to the receptor chamber. Deposition of pirimicarb within the skin reservoir at 48 h was very limited 24 h after pirimicarb had been removed from the donor chamber (4%, Table 4) and absent following 42 h without exposure (Table 3). Basically, the major part of the administered pirimicarb remained in the donor chamber to be recovered during the donor wash (Tables 3 and 4).



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  		Fig. 2. Percutaneous penetration curves of four pesticides during a 48 h experimental period including an initial 24 h exposure to pesticide. Data are given as mean ± SEM (N = 7–9).

 


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  		Fig. 3. Percutaneous penetration curves of four pesticides during a 48 h experimental period including an initial 6 h exposure to pesticide. Data are given as mean ± SEM (N = 7–9).

 


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  		Fig. 4. Percutaneous penetration curves of pirimicarb during a 48 h experimental period with either 6 or 24 h exposure to pirimicarb. Data are given as mean values ± SEM (N = 9).

 
Paclobutrazol had a lag-time between 24 and 30 h (Tables 1 and 2). The maximal flux was significantly (P < 0.05) higher in the experiment with 24 h exposure (0.49 nmol/cm2 h) than in the experiment with 6 h exposure (0.28 nmol/cm2 h) (Tables 1 and 2), which corresponded to the higher total penetration in the experiment with 24 h exposure (Tables 3 and 4). After 6 h exposure, close to two-thirds of the paclobutrazol was still present in the donor chamber (Table 3), whereas the situation at 24 h was changed as only one-third of the paclobutrazol dose was present in the donor chamber (Table 4). These differences were reflected through a larger fraction present in the skin reservoir in experiments with 24 h exposure as compared with 6 h exposure (Table 3). In contrast to the other experiments in this study, a significant fraction (8%) of the paclobutrazol dose was found in the donor wash from 48 h (Table 4).

The lag-time of methiocarb was around 12 h in one experiment and close to 7 h in the second experiment (Tables 1 and 2). The variation in lag-time in the first experiment was, however, large, which reflected large variations between the donors included in this experiment. The maximal flux in the 6 and 24 h exposure experiments were 0.22 and 0.41 nmol/cm2 h, respectively (Tables 1 and 2). The total penetration during 48 h, including the 24 h exposure period was 10.4 nmol, which was 8.7% of the dose applied to the donor chamber (Table 4). In comparison, the total penetration in the experiment with 6 h exposure was 8.5 nmol corresponding to 7.1% of the applied dose (Table 3). The donor wash after 6 h contained 35% of the original dose, which relates to the 52% of the dose (62.6 nmol) that was found in the skin reservoir after 48 h (Table 3). A significantly higher amount of methiocarb was estimated in the skin membrane in the experiment with 24 h exposure to methiocarb, which reflects the longer time period for absorption into the skin and was supported by the significantly lower amount of methiocarb (P < 0.01) remaining in the donor chamber (16 nmol; 13%) after 24 h exposure (Table 4).

Prochloraz had lag-times of 12–18 h with maximal penetrations rates (flux) of 0.11–0.14 nmol/cm2 h (Tables 1 and 2). The long lag-time and the low flux caused a low total penetration during the 48 h experimental period (Tables 3 and 4). The amount of prochloraz remaining in the donor chamber, when exposure was stopped at 6 h, was 48 nmol (40% of the dose) when compared with the significantly lower amount of 5 nmol (5% of the dose) when the exposure with prochloraz was stopped at 24 h (Tables 3 and 4). The significantly lower amount of prochloraz remaining in the donor chamber in the 24 h exposure study corresponded with the significantly higher deposition of prochloraz in the skin reservoir from this experimental group when the experiments were stopped at 48 h (>90% of dose) (Tables 3 and 4).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGEMENTS
 REFERENCES
 
Dermal penetration of a chemical is characterized by the lag-time, the flux (or Kp) and the possible existence of a skin reservoir. The lag-time is defined as the time between application on the skin and its appearance in the blood or lymph. Thus, the lag-time is the summation of the time needed for penetration into the dermal barrier from the epidermal side, transport through the skin membrane (epidermis and dermis) and penetration from the skin compartment and out into the receptor. In in vitro models, this equals the period between application of a chemical on the epidermal side of the skin and the appearance of the chemical in the receptor below the skin membrane and is estimated by back extrapolation from the linear (steady-state) part of the penetration curves. Assuming the penetration process to be passive and the experimental conditions to reflect infinite dose, the maximal flux represents a steady-state situation and will therefore reflect the slowest of the two rate constants for penetration (in or out of the skin compartment). These rate constants depend on molecular size and lipophilicity of the chemical as well as the difference in lipophilicity between the matrix that the chemical is leaving and the one it is approaching. If the chemical in an experiment has a preference for the skin compared with the receptor, a higher concentration gradient may be needed to get appreciable transport, and consequently a reservoir effect may be expected. The lag-time may therefore depend on the existence of a reservoir in the skin barrier, whereas the flux in infinite dose experiments at steady state will be independent of the existence of a reservoir effect. Moreover, the thickness of the skin membrane determines the distance that a chemical will have to diffuse and thus the lag-time.

The present study used a donor matrix of 0.9% NaCl with 2% ethanol and a receptor matrix of 0.9% NaCl with 5% BSA; both fluids were expected to be more hydrophilic than the dermal barrier. We therefore expected the flux, lag-time as well as the skin reservoir of the chemicals tested to depend on the individual lipophilicities.

The present study included three pesticides (methiocarb, pirimicarb and dimethoate) with comparable molecular weights but different solubility characteristics. Despite having the same molecular weight as methiocarb, dimethoate had solubility characteristics, i.e. a 1000-fold higher water solubility, that not only prevented any dermal penetration, but also prevented any appreciable amount of pesticide to even enter the more lipophilic skin compartment (Tables 3 and 4). The distribution of pirimicarb was also pushed strongly away from deposition in the skin compartment (Tables 3 and 4). Pirimicarb did, however, penetrate the skin with considerable speed when compared with methiocarb, despite the higher water solubility of pirimicarb. Thus, 13% of the pirimicarb dose penetrated the dermal barrier in the experiment with 24 h pesticide exposure when compared with only 8.7% of the methiocarb dose (Tables 3 and 4). The high flux and low skin deposition of pirimicarb, when compared with methiocarb, indicate the absence of a longer lasting skin reservoir of pirimicarb, and a limited accumulation during the penetration process. This conclusion is based on the observation that penetration into the receptor chamber continues for at least 6 h after pirimicarb was removed from the donor chamber, the low flux during the last part of the experimental period, and the very low skin deposition at 48 h. These observations on skin deposition are also in agreement with the lower lipophilicity of pirimicarb when compared with methiocarb and the relatively low lag-time compared with the other pesticides.

The influence of molecular size was studied on three pesticides (methiocarb, paclobutrazol and prochloraz) with comparable solubilities, but different molecular weights. The flux and consequently the calculated Kp values for methiocarb and paclobutrazol were almost identical irrespective of whether the exposure period was 6 or 24 h, whereas flux as well as Kp for the pesticide with the highest molecular weight, prochloraz, was lower (significantly different for the 24 h experiment only) (Tables 1 and 2). The amount of pesticide penetration during the 48 h experimental period decreased with increasing molecular weight irrespective of exposure periods of 6 or 24 h (Tables 3 and 4). Likewise, a significantly (P < 0.01) higher fraction of the administered dose was removed from the donor chamber in experiments with methiocarb (Mw = 225 g/mol) when compared with paclobutrazol (Mw = 293 g/mol) (Tables 3 and 4). The significantly higher removal of pesticide from the donor chamber was also reflected through a higher skin deposition in experiments with methiocarb when compared with experiments with paclobutrazol (Tables 3 and 4). Prochloraz, on the other hand, had the lowest total penetration but a distribution between donor and skin compartments that was more like methiocarb than paclobutrazol, although prochloraz had the largest molecular weight (Tables 3 and 4). Despite equal solubilities in water, the log Pow value of prochloraz was a full unit higher than log Pow for methiocarb and paclobutrazol, which could indicate a higher preference for prochloraz to remain in the skin compartment when compared with the more hydrophilic donor and receptor solutions. The apparent preference of prochloraz for the skin compartment is most significantly reflected through the distribution in the experiment with 24 h exposure, where more than 90% of the administered dose was estimated to remain in the skin compartment when the experiment was stopped at 48 h (Table 4).

Based on the data from the five pesticides, we conclude that solubility characteristics significantly affect penetration rates as well as skin deposition, and that a too high as well as a too low lipophilicity may limit the rate and degree of skin penetration. These observations pertain to studies on full-thickness skin, in which a significantly larger potential for skin deposition (reservoir) exists. Several mathematical models have been developed to describe the relationship between dermal penetration and the underlying physical parameters such as log Pow and molecular weight (McKone and Howd, 1992Go; Guy and Potts 1993Go; Wilschut et al., 1995Go). Equal for these theoretical models is their unidirectional dependence on molecular weight and log Pow as well as their lack of dependence upon experimental conditions related to skin thickness and choice of receptor fluids. Our observation that a too high as well as a too low lipophilicity limits dermal penetration is thus at variance with those models. A possible explanation might be that these models do not sufficiently consider the penetration as a process involving penetration from a hydrophilic donor to a more lipophilic membrane as well as from the lipophilic membrane and into a more hydrophilic receptor. In the first process, penetration of hydrophilic compounds (like dimethoate) will be limited, and in the second process, more lipophilic compounds (like prochloraz) will prefer to remain in the lipophilic skin compartment.

The present study included an exposure time of 6 h to mimic an occupational exposure situation. The use of infinite dosing is at variance with most occupational exposures, but is not important for the study of temporary deposition in the skin and subsequent penetration to the receptor. As the lag-times for most pesticides tested were more than 6 h, it was not surprising that the maximal fluxes for these pesticides obtained in the 6 h-exposure experiments were generally lower than in the 24 h-exposure experiments, whereas pirimicarb with a short lag-time had comparable Kp-values in experiments with 6 h as well as 24 h. An important observation was that most of the applied dose of the two most hydrophilic pesticides (pirimicarb and dimethoate) was recovered in the donor wash after 6 h, which implies that preventive measures in the form of hand wash will significantly reduce the skin penetration of these pesticides following occupational exposures. However, an equally important observation was that dermal penetration continues long after exposure has ended due to absorption of temporarily deposited pesticide present in the skin compartment at the end of exposure.

A previous study on penetration of methiocarb, paclobutrazol and pirimicarb reported lag-times between 10 and 18 h (Nielsen and Nielsen, 2000Go). The present study found lag-times significantly deviating from these earlier observations. The receptor fluid did, however, differ between the two studies. Thus, a more physiological receptor (5% BSA in a 0.9% NaCl aqueous solution) was used in the present study when compared with the use of 50% ethanol in the earlier study. These differences make direct comparisons difficult as ethanol is known to significantly affect the barrier characteristics of the skin membrane (Nielsen, 2000Go). In general, these observations are in agreement with other studies demonstrating the influence of the vehicle on dermal penetration characteristics (Singer and Tjeerdema, 1993Go; Baynes et al., 2002Go; Brand and Mueller, 2002Go).

As illustrated in Fig. 1, different combinations of flux and lag-time may cause identical amounts of chemical to cross the dermal barrier at a certain point in time. Thus, the total amount of a chemical penetrating during a certain time period is not very informative on its own. This is especially relevant in relation to short-term exposures such as most occupational exposures, where exposure time will often be less than or close to the experimental lag-time. Under these circumstances, an exposure assessment based on measurement of blood concentration at the end of a work shift will ignore pesticide deposited in the skin and demonstrate zero absorption and thus underestimate the actual exposure. Although literature indicates that the systemic concentration of a number of pesticides tested did not increase after exposure was ended (Zendzian, 2003Go), the continued presence of pesticide in the blood compartment indicates continued absorption from the dermal reservoir and thus a continued rise in systemic dose. As a prolonged presence of a lower concentration of a toxicant may be equally important as a short exposure causing a high blood concentration, we suggest that data used for regulatory agencies should include steady-state flux (or Kp) and lag-time as well as an estimation of the potential importance of the skin reservoir.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
REFERENCES
 
The authors are grateful to F. Lind for technical assistance. This research was a part of the project ‘Evaluation and predictions of dermal absorption of toxic chemicals’ (QLRT-2000-00196), which was supported by the Fifth Framework Programme of the European Commission.

Received March 11, 2004; in final form June 14, 2004


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 

Archibald BA, Solomon KR, Stephenson GR. (1994) Estimating pirimicarb exposure to greenhouse workers using video imaging. Arch Environ Contam Toxicol; 27: 126–9.[Web of Science][Medline]

Baynes RE, Brooks JD, Mumtaz M et al. (2002) Effect of chemical interactions in pentachlorphenol mixtures on skin and membrane transport. Toxicol Sci; 69: 295–305.[Abstract/Free Full Text]

Benford DJ, Cocker J, Sartorelli P et al. (1999) Dermal route in systemic exposure. Scand J Work Environ Health; 25: 511–20.[Web of Science][Medline]

Brand RM, Mueller C. (2002) Transdermal penetration of atrazine, alachlor, and trifluralin: Effect of formulation. Toxicol Sci; 68: 18–23.[Abstract/Free Full Text]

Bronaugh RL, Stewart RF, Simon M. (1986) Methods for in vitro percutaneous absorption studies. VII: Use of excised human skin. J Pharmacol Sci; 75: 1094–7.[CrossRef][Web of Science][Medline]

Guy RH, Potts RO. (1993) Penetration of industrial chemicals across the skin: A predictive model. Am J Ind Med; 23: 711–9.[Medline]

McKone TE, Howd RA. (1992) Estimating dermal uptake of non-ionic organic chemicals from water and soil: Unified fugacity-based models for risk assessment. Risk Anal; 12: 543–57.[Medline]

Nielsen JB. (2000) Effects of four detergents in the in-vitro barrier function of human skin. Int J Occup Environ Health; 6: 143–7.[Medline]

Nielsen JB, Nielsen F. (2000) Dermal in vitro penetration of methiocarb, paclobutrazol and pirimicarb. Occup Environ Med; 57: 734–7.[Abstract/Free Full Text]

Nielsen GD, Nielsen JB, Andersen KE et al. (2000) Effects of industrial detergents on the barrier function of human skin. Int J Occup Environ Health; 6: 138–42.[Medline]

OECD-428. (2000) Guidance document for the conduct of skin absorption studies. OECD environmental health and safety publications; series on testing and assessment #28. Paris: Organisation for Economic Cooperation and Development.

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