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Protocol no. 53
AUTOMATED IN VITRO DERMAL ABSORPTION (AIDA) PROCEDURE

The AIDA system can be used, under precisely controlled environmental conditions, to predict the dermal absorption of the test substance in vivo.

CONTACT

Dr. R.P. Moody
Department of Health and Welfare Canada Room B-35
Environmental Health Centre Tunney's Pasture
Ottawa Ontario, K1A 0L2 Canada
Tel: Canada - (613) 954-6921 Fax: Canada - (613) 941-4545

RATIONALE

Percutaneous absorption of chemicals such as pesticides can be a major route of occupational exposure. A determination of the degree of dermal absorption that is likely to occur should therefore be integrated into the toxicity testing of these substances. The AIDA system provides a means for the in vitro testing of dermal absorption, by measuring the amount of radiolabelled test substance that passes through a sample of skin mounted in a chamber. The time course of the absorption may be determined by maintaining a constant flow-through of receiver solution, which is collected in aliquots. The passage of a flow of air at a controlled velocity over the skin permits wind conditions to be reproduced, while a UV lamp can be used to simulate the effects of solar radiation, thus providing an approximation to exposure in the field.

BASIC PROCEDURE

A skin specimen is clamped between two washers in the specially-constructed AIDA chamber. Radiolabelled test compound, dissolved in acetone, is applied to the corneal side of the skin, and the acetone is evaporated off under an air current. The AIDA chamber is then mounted, with the exposed side of the sample facing downwards, in a gas chromatograph oven at 37°C and, if required, illuminated by a UV lamp. Water-saturated air is passed over the exposed side of the skin and is then extracted by passage through a 50% aqueous methanol bubbler trap connected in series with an XAD polymer column. Receiver solution is passed through the upper part of the chamber, to come into contact with the unexposed side of the skin sample, and is harvested at 2 hour intervals in a fraction collector. After 24 hours, the skin is washed with soapy water and following study termination at 48 hours of exposure, the skin is rinsed with methanol, and then the tissue is digested. The percentage distribution of radioactivity in the receiver fluid samples, washes, tissue digest and air traps (methanol and XAD) is determined by scintillation counting.

CRITICAL ASSESSMENT

Comparison with in vivo testing of dermal absorption A number of systems have already been developed for the in vitro modelling of absorption of chemicals through human skin (reviewed in Bronaugh & Maibach, 1985), thus underlining the requirement for reliable alternatives to standard in vivo techniques. In vitro methods offer a more elegant and precise means to assay the dermal absorption process. By avoiding the need for testing in the living organism, in vitro methods avoid the problem of species difference that arises when animals are used for direct testing, and they also avoid the risk of potentially adverse consequences in human volunteers. There are a number of other advantages to using in vitro tests in preference to in vivo studies of dermal absorption. In vivo tests require that a correction be made for incomplete renal excretion, as exemplified in a human study by Feldmann & Maibach (1970). In order to do this, it is necessary to determine the pharmacokinetics of the test compound following intravenous injection of a labelled tracer. Where this is considered not acceptable in human volunteers, the pharmacokinetics must be determined in animals, which again allows for errors due to extrapolating from another species. The dermal absorption study itself may require a high degree of cooperation if carried out in human volunteers; for example, Feldmann & Maibach (1970) considered it necessary to collect all urine from the volunteers for 5 days. The pharmacokinetic data will not account for any test compound that remains bound to the skin. In the case of dermal absorption by rats of the herbicide fenoxaprop-ethyl (FPE), Moody & Ritter (1992) were able to show, using the AIDA system, that up to 25% of the chemical persisted as a dermal reservoir. Another disparity that was noticed between in vivo and in vitro kinetics during the FPE study, was the fact that the maximum % urinary excretion was obtained at about 48 hours, whereas the maximum skin penetration occurred at about 10 hours. Moody and Ritter, (1992) consider this an indication of the potential fallacy of trying to model dermatopharmacokinetics from urinary excretion data alone, and suggest that blood plasma clearance data would be required for a direct comparison of in vitro/in vivo half-lives, which would necessitate the use of more invasive procedures in human volunteers. In their study of FPE absorption, Moody and Ritter (1992) made a direct comparison between in vivo dermal absorption following topical application of the chemical to the shaved skin of the back, and in vitro dermal absorption in the AIDA system using skin samples taken from another part of the backs of the same rats. They found no significant difference between the mean % permeation in vitro and in vivo, and concluded that the AIDA procedure provides an accurate model of the in vivo percutaneous absorption process. Comparison with other in vitro systems The AIDA system offers a number of advantages over other cell chamber systems for the measurement of dermal absorption. It permits simultaneous multireplicate runs of the assay under precise environmental control. The temperature control that is possible with the gas chromatographic oven is superior to that obtained using a water jacket. The accurate control of dermal illumination provided by the UV lamp provides a more realistic model of field conditions, where solar radiation can damage the corneal layer of the skin and alter dermal barrier properties, and provides the means to test for potential photolytic effects on the test substance. Horstman et al., (1989) reported difficulties in their attempts to obtain large enough skin samples (1cm2) for use in the small Franz cell. The AIDA system requires a smaller exposure area (0.2cm2). Not only are smaller samples more easily obtained, but there is also less likelihood of strain-related ruptures of the epidermis that can occur when larger exposure areas are used. Even smaller skin samples (0.049cm2) can be used in the chamber developed by Akhter et al., (1984), but this chamber does not have flow-through chambers which permit air velocity to be regulated. AIDA, which does use a regulated air-flow, is therefore better able to mimic the effects of wind on evaporation from the skin. Spencer et al., (1979) have emphasized the importance of allowing for evaporation effects in penetration studies, in view of the volatility of many of the compounds that are applied to the skin. For example, they found that an average of about 10% of diethyltoluamide applied to the forearm was lost by evaporation. A similar amount was lost by evaporation in an in vitro system using frozen cadaver abdominal skin. A further refinement in the AIDA system, compared with that of Akhter et al., is the inverted design that eliminates the possibility of air becoming trapped at the skin-receiver interface. The finding of no significant difference between the mean % permeation of fenoxaprop-ethyl through rat skin in vivo and in vitro in the AIDA system is considered by Moody and Ritter (1992) to be further evidence of the superiority of AIDA to other in vitro systems. Fenoxaprop-ethyl is relatively lipophilic, and other authors (Hawkins & Reifenrath, 1986), using other systems, have reported problems in obtaining permeation comparable to that seen in vivo with compounds of even lower lipophilicity. The reason that other in vitro systems encounter problems with testing lipophilic compounds is that they are not flow-through systems and/or do not use a 24 hour soap wash followed by skin surface wash and tissue digest analysis with helical flow mixing. The capacity to reliably test lipophilic compounds is important, as many environmental contaminants of concern are highly lipophilic. One disadvantage of the AIDA system is the need to use radiolabelled test substances, which not only raises safety considerations, but also requires the use of expensive radiotracers. Currently the AIDA procedure is being developed in-house to permit the use of non-labelled test compounds by interfacing the receiver and donor (air) eluent lines with HPLC and GLC analytical equipment. Skin samples The manner in which skin samples are obtained may influence the results of the absorption assay. It is standard in many in vitro procedures to rely on heat separation in order to isolate the outermost layer of skin with the stratum corneum. This involves immersing the skin in water at 60°C for 1 minute and then peeling off the blister that forms. It is quite likely that the heat treatment inhibits cellular metabolism, however, the underlying assumption is that dermal absorption is a passive process, and that skin viability is therefore not of any relevance. A further consequence of this assumption is the frequent use of frozen human cadaver tissue for in vitro studies. Bronaugh et al., (1986) found that freezing at -20°C for up to one year had no significant effect on the water permeability of human skin. Similarly, Moody & Ritter (1992) showed that freezing in liquid nitrogen did not affect the barrier properties of the stratum corneum of rat skin to fenoxaprop-ethyl as assessed using the AIDA system. Carver et al., (1989), however, on the basis of studies in perfused porcine skin flaps, concluded that it is invalid to assume that skin viability does not influence dermal absorption. Kao et al., (1985) presented in vitro evidence for the influence of dermal metabolic viability on the penetration of benzo[a]pyrene through the skin of various mammalian species, including man. Although Hawkins & Reifenrath (1986) reported that neither freezing nor metabolic inactivation with ethylene oxide had any significant effect on the permeation of diethyltoluamide through pig skin, Moody & Martineau (1990) found that permeation of the same chemical in the AIDA system was much greater through fresh human skin than through frozen tissue. They also found permeation through heat-separated tissue to be faster than through fresh split-thickness tissue, although this could be due to the different thickness of the samples (0.2mm and 3mm, respectively). Skin samples can be obtained with a dermatome. These tissue sections may be up to several mm thick and will therefore contain viable tissue layers in addition to the external, non-living corneal layer. Replicate runs using the AIDA system and a dialysis membrane control sample gave a total diethyltoluamide permeation of 47.2 ± 3.16% (Moody & Martineau, 1990). The consistency of the replicate results with dialysis membrane indicates that AIDA offers a high level of precision. Total permeation through fresh human breast skin was very close to this value, but showed more variability (48.0 ± 10.18%). This was due to inherent differences in the samples, for example, relating to skin thickness and the presence of hair follicles and sebaceous glands. These structural differences may also be responsible for the different rates of permeation that have been reported at various anatomical sites. For example, the 48% permeation found for diethyltoluamide through fresh breast tissue in the AIDA system is much greater than the 17% absorption reported for the same chemical applied to the forearm of human volunteers (Feldmann & Maibach, 1970), and lower than the 68% reported following application of diethyltoluamide to the monkey ventral forepaw (Moody et al., 1989). It is necessary to investigate more fully the effects of factors such as sample processing and the site from which the sample is taken before developing a fully standardised AIDA procedure which can be subjected to a formal validation. Conclusions AIDA offers a simple procedure for the testing of skin permeation under precisely controlled physiological and environmental conditions, which can be approximated to those expected to arise during normal use of the test substance. The system has advantages over other in vitro systems that have been proposed, and has been found to give results comparable to those obtained in in vivo studies. In addition to testing the dermal permeability of chemicals, AIDA can also be used with other non-skin samples in order to test the efficacy of materials intended for use in protective clothing. Furthermore, the washing and rinsing step that forms part of the procedure offers the possibility of testing the efficacy of skin washing agents against various compounds.

TEST STATUS

In-house development

REFERENCES

  1. Akhter, S.A., Bennet, S.L., Waller, I.L. & Barry, B.W. (1984) An automated diffusion apparatus for studying skin penetration. Int. J. Pharm. 21, 17-26.
  2. Bronaugh, R.L. & Maibach, H.I. (1985) In vitro models for human percutaneous absorption. In: Models in dermatology (eds. H.I. Maibach and N.J. Lowe) Karger; New York, pp. 178-188.
  3. Bronaugh, R.L., Stewart, R.F. & Simon, M. (1986) Methods for in vitro percutaneous absorption studies. VII. Use of excised human skin. J. Pharm. Sci. 75, 1094-1097.
  4. Carver, M.P., Williams, P.L. & Riviere, J.E. (1989) The isolated perfused porcine skin flap. III. Percutaneous absorption pharmacokinetics of organophosphates, steroids, benzoic acid and caffeine. Toxicol. Appl. Pharmacol. 97, 324-337.
  5. Feldmann, R.J. & Maibach, H.I. (1970) Absorption of some organic compounds through the skin in man. J. Invest. Dermatol. 54, 399-404. Hawkins, G.S. & Reifenrath, W.G. (1986) Influence of skin source, penetration cell fluid, and partition coefficient on in vitro skin penetration. J. Pharm. Sci. 75, 378-381.
  6. Horstman, S.W., Rossner, A., Kalman, D.A. & Morgan, M.S. (1989) Penetration of pentachlorophenol and tetrachlorophenol through human skin. J. Environ. Sci. Health A24, 229-242.
  7. Kao, J., Patterson, F.K. & Hall, J. (1985) Skin penetration and metabolism of topically applied chemicals in six mammalian species, including man: an in vitro study with benzo[a]pyrene and testosterone. Toxicol. Appl. Pharmacol. 81, 502-516.
  8. Moody, R.P. & Martineau, P.A. (1990) An automatic in vitro dermal absorption procedure: I. Permeation of 14C-labelled N,N-diethyl-m-toluamide through human skin and effects of short-wave ultraviolet radiation on permeation. Toxicology in Vitro 4, 193-199.
  9. Moody, R.P. & Ritter, L. (1992) An automatic in vitro dermal absorption procedure: II. Comparative in vivo and in vitro dermal absorption of the herbicide, fenoxaprop-ethyl (HOE-33171) in rats. Toxicology in Vitro 6, 53-59.
  10. Moody, R.P., Benoit, F.M. & Ritter, L. (1989) Dermal absorption of the insect repellent DEET (N,N-diethyl-m-toluamide) in rats and monkeys: effect of anatomical site and multiple exposure. J. Toxicol. Environ. Health 26, 137-147.
  11. Spencer, T.S., Hill, J.A., Robert, B.S., Feldmann, R.J. & Maibach, H.I. (1979) Evaporation of diethyltoluamide from human skin in vivo and in vitro. J. Invest. Dermatol. 72, 317-319. IP-53 © July 1992

IP-53 © July 1992