Dermal penetration and metabolism of five glycidyl ethers in human,rat and mouse skin

1. Glycidyl ethers (GE), an important class of industrial chemicals, are considered to be potentially mutagenic in vivo because some GE have been shown to be direct mutagens in short-term in vitro tests. 2. The percutaneous penetration and metabolism of representatives of different classes of GE was studied in the fresh, full-thickness C3H mouse, and dermatomed human and Fisher 344 rat skin to determine the apparent permeability constants, lag times and metabolic profiles. 3. Five different GE, the diglycidyl ethers of bisphenol A (BADGE), 4,4'-dihydroxy3,3',5,5'-tetramethylbiphenyl (Epikote YX4000) and 1,6-hexanediol (HDDGE) and the GE of 1-dodecanol (C₁₂GE) and o-cresol (o-CGE), were synthesized by reaction of their alcohols with epichlorohydrin. Their radiolabelled analogues were synthesized with a ¹⁴Clabel using [U-¹⁴C]-epichlorohydrin. 4. There was a large variation (four orders of magnitude) in percutaneous penetration between the five GE. In general, penetration through full-thickness mouse skin was higher than through dermatomed rat skin, whereas dermatomed human skin was the least permeable. The permeability increased in the order YX4000<BADGE<C₁₂GE<o CGE<HDDGE. 5. The relative skin permeability of the five GE could be explained for a significant part by the lipophilicity, expressed as log Po/w, in combination with the molecular weight of the compounds. 6. During skin penetration, all GE were extensively metabolized to their corresponding (bis-)diols. Virtually no YX4000, and only very small amounts of C₁₂GE and BADGE, penetrated the skin unchanged, but significant amounts of HDDGE and o-CGE penetrated the skin unchanged. For o-CGE, but none of the other GE, the percentage of the applied dose that penetrated the skin unchanged increased over time. 7. The large variation in response observed with the five selected GE indicates that GE should not be considered as a single class of compounds but rather on the basis of their individual properties.     

Dermal penetration potential of perfluorooctanoic acid (PFOA) in human and mouse skin

Recent data, using a murine model, have indicated that dermal exposure to perfluorooctanoic acid (PFOA) induces immune modulation, suggesting that this may be an important route of PFOA exposure. To investigate the dermal penetration potential of PFOA, serum concentrations were analyzed in mice following topical application. Statistically significant and dose-responsive increases in serum PFOA concentrations were identified. In vitro dermal penetration studies also demonstrated that PFOA permeates both mouse and human skin. Investigation into the mechanisms mediating PFOA penetration demonstrated that dermal absorption was strongly dependent upon the ionization status of PFOA. In addition, PFOA solid, but not 1% PFOA/acetone solution, was identified as corrosive using a cultured epidermis in vitro model. Despite its corrosive potential, expression of inflammatory cytokines in the skin of topically exposed mice was not altered. These data suggest that PFOA is dermally absorbed and that under certain conditions the skin may be a significant route of exposure.     

Percutaneous penetration and metabolism of 2-nitro-p-phenylenediamine in human and fuzzy rat skin

2-Nitro-p-phenylenediamine (2NPPD) is a dye used in semipermanent and permanent (tinting color) hair dye formulations. National Toxicology Program toxicology and carcinogenesis testing of 2NPPD has raised concerns about its safety. Therefore, we initiated in vitro studies to measure absorption and metabolism of 2NPPD in human and fuzzy rat skin and rat jejunal tissue. Intestinal tissue metabolism of 2NPPD was compared to skin metabolism since toxicology data from oral 2NPPD studies will be used for future safety assessment purposes. Absorption was measured over 24 h by using flow-through diffusion cells with a receptor fluid consisting of Hepes-buffered Hank's balanced salt solution. Dosing vehicles were applied to skin and intestine in the diffusion cells for 30 min. 2NPPD metabolites were determined by high-performance liquid chromatography methodology. In human skin, the percentages of total applied dose absorbed (receptor fluid + skin) over 24 h were 9.2 +/- 5.7 (mean +/- SD) and 9.5 +/- 3.2 for the ethanol and semipermanent vehicles, respectively, with approximately 3% remaining in skin. In rat skin, the percentages of total applied dose absorbed over 24 h were 9.3 +/- 1.2 (mean +/- SE), 6.9 +/- 1.2, and 4.2 +/- 0.1 for the ethanol, semipermanent, and permanent formulation vehicles, respectively, with approximately 3% remaining in skin. In rat intestinal tissue, the percentage of total applied dose absorbed over 24 h was 10.9 +/- 1.2, with approximately 5% remaining in the tissue. In human and rat skin, 2NPPD was metabolized to triaminobenzene and N4-acetyl-2NPPD. 2NPPD was also metabolized to a sulfated 2NPPD metabolite in rat skin, but not in human skin. 2NPPD was extensively metabolized in both human and rat skin with ethanol application; metabolism was not as extensive with a semipermanent formulation application. In rat intestinal tissue, 62% of 2NPPD was metabolized upon absorption to triaminobenzene and N4-acetyl-2NPPD. Differences in the metabolic profiles (proportion of each metabolite formed) were found between the skin and intestinal tissue. These results suggest that 2NPPD is rapidly absorbed and extensively metabolized in both skin and intestinal tissue. The extent of metabolism and the metabolic profile were found to be species-, tissue-, and dosing vehicle-dependent. Metabolism information will be useful in predicting the extent of 2NPPD and/or 2NPPD metabolite systemic absorption relative to a dermal exposure, which will improve the health hazard assessment of 2NPPD.     

Dermal penetration of bisphenol A in human skin contributes marginally to total exposure

Bisphenol A (BPA) is ubiquitous and many exposure scenarios have been described during the last decades. While oral uptake is considered as the major route of exposure, the contribution of skin penetration has been recently discussed. In the present study, the dermal penetration rate of BPA has been determined in human skin in an in vitro test method according to the OECD Test Guideline 428. This analysis resulted in penetration of 8.6% and a total amount of bio-available BPA of 9.3% of the dose applied after 24h incubation under realistic exposure conditions. This confirms that the systemic exposure to BPA via the skin contributes in a negligible way to total systemic BPA exposure.     

Metabolic inactivation of five glycidyl ethers in lung and liver of humans, rats and mice in vitro

1. Some glycidyl ethers (GE) have been shown to be direct mutagens in short-term in vitro tests and consequently GE are considered to be potentially mutagenic in vivo. However, GE may be metabolically inactivated in the body by two different enzymatic routes: conjugation of the epoxide moiety with the endogenous tripeptide glutathione (GSH) catalysed by glutathione S-transferase (GST) or hydrolysis of the epoxide moiety catalysed by epoxide hydrolase (EH). 2. The metabolic inactivation of five different GE, the diglycidyl ethers of bisphenol A (BADGE), 4,4'-dihydroxy-3,3',5,5'-tetramethylbiphenyl (Epikote YX4000) and 1,6-hexanediol (HDDGE) and the GE of 1-dodecanol (C12GE) and o-cresol (o-CGE), has been studied in subcellular fractions of human, C3H mouse and F344 rat liver and lung. 3. All GE were chemically very stable and resistant to aqueous hydrolysis, but were rapidly hydrolysed by EH in cytosolic and microsomal fractions of liver and lung. The aromatic GE were very good substrates for EH. In general, microsomal EH is more efficient than cytosolic EH in hydrolysis of GE, and human microsomes are more efficient than rodent microsomes. 4. The more water-soluble GE, o-CGE and HDDGE, were good substrates for GST whereas the more lipophilic GE, YX4000 and C12GE, were poor substrates for GST. In general, rodents are more efficient in GSH conjugation of GE than humans. 5. In general, the epoxide groups of YX4000 are the most and those of HDDGE the least efficiently inactivated of the five GE under study. For the other three GE no general trend was observed: the relative efficiency of inactivation varied with organ and species. 6. The large variation in metabolism observed with five representative GE indicate that GE have variable individual properties and should not be considered as a single, homogenous class of compounds.     

Metabolic inactivation of five glycidyl ethers in lung and liver of humans,rats and mice in vitro

1. Some glycidyl ethers (GE) have been shown to be direct mutagens in short-term in vitro tests and consequently GE are considered to be potentially mutagenic in vivo. However, GE may be metabolically inactivated in the body by two different enzymatic routes: conjugation of the epoxide moiety with the endogenous tripeptide glutathione (GSH) catalysed by glutathione S-transferase (GST) or hydrolysis of the epoxide moiety catalysed by epoxide hydrolase (EH). 2. The metabolic inactivation of five different GE, the diglycidyl ethers of bis phenol A (BADGE), 4,4'-dihydroxy-3,3',5,5'-tetramethylbiphenyl (Epikote YX4000) and 1,6- hexanediol (HDDGE)and the GE of 1-dodecanol (C₁₂GE)and o-cresol (o-CGE), has been studied in subcellular fractions of human, C3H mouse and F344 rat liver and lung. 3. All GE were chemically very stable and resistant to aqueous hydrolysis, but were rapidly hydrolysed by EH in cytosolic and microsomal fractions of liver and lung. The aromatic GE were very good substrates for EH. In general, microsomal EH is more efficient than cytosolic EH in hydrolysis of GE, and human microsomes are more efficient than rodent microsomes. 4. The more water-soluble GE, o-CGE and HDDGE, were good substrates for GST whereas the more lipophilic GE, YX4000 and C₁₂GE, were poor substrates for GST. In general, rodents are more efficient in GSH conjugation of GE than humans. 5. In general, the epoxide groups of YX4000 are the most and those of HDDGE the least efficiently inactivated of the five GE under study. For the other three GE no general trend was observed: the relative efficiency of inactivation varied with organ and species. 6. The large variation in metabolism observed with five representative GE indicate that GE have variable individual properties and should not be considered as a single, homogenous class of compounds.     

In vitro skin penetration of griseofulvin in rat and human skin from an ointment dosage form

Penetration and permeation of griseofulvin into and across the rat skin after application of three ointment formulations containing either dimethylacetamide (DMAC) or diethylene glycol monoethylether (DGME) or the ointment base alone (control--without DMAC or DGME) were studied, in vitro. Penetration and permeation of griseofulvin into and across the human skin after application of DGME ointment was also studied. Permeation of griseofulvin across the rat skin was highest for the DMAC ointment, followed by the DGME ointment and lowest for the control. Concentration of griseofulvin in the upper layers of the skin (i.e., surface to 100 micron depth) was also highest for DMAC and lowest for the control. However, the skin levels from the DGME ointment could not be distinguished (statistically) from the other two formulations. In comparison to rat skin, human skin is much less permeable. Amount of griseofulvin (from DGME ointment) that permeated through the rat skin was 14 times the amount that permeated through the human skin. Concentration of griseofulvin in the upper layers of the rat skin were 4 times the concentration of the drug in the upper layers of the human skin. The concentration of griseofulvin in the various layers of the human skin after one topical application were far greater than those reported after prolonged peroral administration.     

Percutaneous penetration and absorption of parathion using human and pig skin models in vitro and human skin grafted onto nude mouse skin model in vivo

This study determined and compared the percutaneous penetration and absorption of an organophosphorus (OP) pesticide, parathion (PA), using three experimental skin models: namely the human abdominal- and pig-ear skin in vitro models and the Human Skin grafted onto a nude mouse (HuSki) in vivo model. The percentage of topically applied dose absorbed and the doses present in the stratum corneum and skin were systematically determined at 24 h under similar experimental conditions. The three experimental skin models were first compared. Then, the advantages of the HuSki model for in vivo PA skin absorption studies were evaluated compared with the pig in vivo model previously used by others. Lastly, the relevance of each skin model to predict the permeability of human skin to PA in vivo was assessed by comparing our results with previously published in vivo human volunteer values. It was demonstrated that (a) pig-ear skin is relevant for predicting the in vitro human abdominal skin absorption taking into account a 2-3 times higher skin permeability to PA, (b) using ethanol as the vehicle, the absorption of PA was 4-5 times higher in the HuSki model than in the pig model but supports the usefulness of the HuSki model to easy mass balance studies, (c) both human in vitro and HuSki models closely predict the in vivo human volunteer absorption at 24 h when acetone is used as a vehicle but the HuSki model overcomes the known limitations of in vitro models for studying the fate of PA in the different skin layers after topical application.     

Percutaneous penetration and genotoxicity of 4,4'-methylenedianiline through rat and human skin in vitro

4,4'-Methylenedianiline (MDA) is a primary aromatic amine used in the plastics industry and is classified by the International Agency for Research on Cancer as an animal carcinogen and possible human carcinogen. In order to estimate human exposure it is useful to determine percutaneous penetration. Previous studies have suggested that both rat and human skin were permeable to MDA, with greater penetration being seen through human skin. In this study no significant difference was seen between the percutaneous penetration of MDA through human or rat skin for three different treatment levels: 0.01, 0.1 and 1mg per skin membrane (0.32 cm(2)). The apparent dermal flux was calculated as 0.7 +/- 0.3 and 10.1 +/- 2.0 microg/cm(2)/h for the 0.01 and 0.1mg treatments, respectively. The permeability constant K(p) was estimated at 1.8 x 10(-3) cm/h and the lag time at 3.5 +/- 0.5 h. MDA absorbed into the skin was found to be bioavailable. Experiments also showed that after application of 0.1mg MDA, 4% penetrated through latex and nitrile gloves, respectively. The potential genotoxicity of MDA in human skin was assessed by DNA (32)P-postlabelling; levels of DNA adducts were detected, following the treatment and penetration of 1mg MDA.     

Comparative metabolism of honokiol in mouse,rat, dog,monkey, and human hepatocytes

Honokiol has antitumor, antioxidative, anti-inflammatory, and antithrombotic effects. Here we aimed to identify the metabolic profile of honokiol in mouse, rat, dog, monkey, and human hepatocytes and to characterize the enzymes responsible for the glucuronidation and sulfation of honokiol. Honokiol had a high hepatic extraction ratio in all five species, indicating that it was extensively metabolized. A total of 32 metabolites, including 17 common and 15 different metabolites, produced via glucuronidation, sulfation, and oxidation of honokiol allyl groups were tentatively identified using liquid chromatography–high resolution quadrupole Orbitrap mass spectrometry. Glucuronidation of honokiol to M8 (honokiol-4-glucuronide) and M9 (honokiol-2′-glucuronide) was the predominant metabolic pathway in hepatocytes of all five species; however, interspecies differences between 4- and 2′-glucuronidation of honokiol were observed. UGT1A1, 1A8, 1A9, 2B15, and 2B17 played major roles in M8 formation, whereas UGT1A7 and 1A9 played major roles in M9 formation. Human cDNA-expressed SULT1C4 played a major role in M10 formation (honokiol-2′-sulfate), whereas SULT1A1*1, 1A1*2, and 1A2 played major roles in M11 formation (honokiol-4-sulfate). In conclusion, honokiol metabolism showed interspecies differences.     

Xenobiotic-metabolizing enzymes in the skin of rat,mouse, pig,guinea pig,man, and in human skin models

The exposure of the skin to medical drugs, skin care products, cosmetics, and other chemicals renders information on xenobiotic-metabolizing enzymes (XME) in the skin highly interesting. Since the use of freshly excised human skin for experimental investigations meets with ethical and practical limitations, information on XME in models comes in the focus including non-human mammalian species and in vitro skin models. This review attempts to summarize the information available in the open scientific literature on XME in the skin of human, rat, mouse, guinea pig, and pig as well as human primary skin cells, human cell lines, and reconstructed human skin models. The most salient outcome is that much more research on cutaneous XME is needed for solid metabolism-dependent efficacy and safety predictions, and the cutaneous metabolism comparisons have to be viewed with caution. Keeping this fully in mind at least with respect to some cutaneous XME, some models may tentatively be considered to approximate reasonable closeness to human skin. For dermal absorption and for skin irritation among many contributing XME, esterase activity is of special importance, which in pig skin, some human cell lines, and reconstructed skin models appears reasonably close to human skin. With respect to genotoxicity and sensitization, activating XME are not yet judgeable, but reactive metabolite-reducing XME in primary human keratinocytes and several reconstructed human skin models appear reasonably close to human skin. For a more detailed delineation and discussion of the severe limitations see the “Overview and Conclusions” section in the end of this review.     

The metabolism of n-butyl glycidyl ether in the rat and rabbit

[1-14C]Butyl glycidyl ether administered orally to male rats and rabbits (20 mg/kg) is rapidly absorbed and metabolized. Most of the administered compound, 87% (rat) and 78% (rabbit), is eliminated in the 0-24 urine. Major metabolites in the rat include 3-butoxy-2-hydroxypropionic acid (9%), 3-butoxy-2-acetylaminopropionic acid (23%) and butoxyacetic acid (10%). 3-Butoxy-2-hydroxypropionic acid (35%) and butoxyacetic acid (5%) are also major metabolites in the rabbit. Biotransformations of the glycidyl ether and their likely biochemical mechanisms are discussed.     

In vitro kinetics of styrene and styrene oxide metabolism in rat,mouse, and human

Styrene oxide (SO), a labile metabolite of styrene, is generally accepted as being responsible for any genotoxicity associated with styrene. To better define the hazard associated with styrene, the activity of the enzymes involved in the formation (monooxygenase) and destruction of SO (epoxide hydrolase and glutathione-S-transferase) were measured in the liver and lungs from naive and styrene-exposed male Sprague-Dawley rats and B6C3F1 mice (three daily 6-h inhalation exposures at up to 600 ppm styrene) and Fischer 344 rats (four daily 6-h inhalation exposures at up to 1000 ppm styrene), and in samples of human liver tissue. Additionally, the time course of styrene and SO in the blood was measured following oral administration of 500 mg styrene/kg body weight to naive Fischer rats and rats previously exposed to 1000 ppm styrene. The affinity of hepatic monooxygenase for styrene, as measured by the Michaelis constant (K _m), was similar in the rat, mouse, and human. Based on theV _max for monooxygenase activity and the relative liver and body size, the mouse had the greatest capacity and humans the lowest capacity to form SO from styrene. In contrast, human epoxide hydrolase had a greater affinity (i. e., lowerK _m) for SO than epoxide hydrolase from rats or mice while the apparent Vmax for epoxide hydrolase was similar in the rat, mouse, and human liver. However, the activity of epoxide hydrolase relative to monooxygenase activity was much greater in the human than in the rodent liver. Hepatic glutathione-S-transferase activity, as indicated by theV _max, was 6- to 33-fold higher than epoxide hydrolase activity. However, the significance of the high glutathione-S-transferase activity is unknown because hydrolysis, rather than conjugation, is the primary pathway for SO detoxification in vivo. Human hepatic glutathione-S-transferase activity was extremely variable between individual human livers and much lower than in rat or mouse liver. Prior exposure to styrene had no effect on monooxygenase activity or on blood styrene levels in rats given a large oral dose of styrene. In contrast, prior exposure to styrene increased hepatic epoxide hydrolase activity 1.6-fold and resulted in lower (0.1>P>0.05) blood SO levels in rats given a large oral dose of styrene. Qualitatively, these data indicate that the mouse has the greatest capacity and the human the lowest capacity to form SO. In addition, human liver should be more effective than rodent liver in hydrolyzing low levels of SO. Quantitative evaluation of the species differences in enzyme levels are being evaluated with the development of a physiologically based pharmacokinetic model for styrene that includes SO.     

Comparative metabolism of geranyl nitrile and citronellyl nitrile in mouse, rat, and human hepatocytes.

Geranyl nitrile (GN) and citronellyl nitrile (CN) are fragrance components used in consumer and personal care products. Differences in the clastogenicity of these two terpenes are postulated to result from differential biotransformation, presumably involving the conjugated nitrile moiety. The metabolic clearance and biotransformation of GN and CN were compared in primary hepatocytes from mice, rats, and humans. For determination of intrinsic clearance, GN and CN were incubated with hepatocytes in sealed vials, and the headspace was sampled periodically by solid-phase microextraction and analyzed by gas chromatography/mass spectrometry. For metabolite identification, GN and CN were incubated with hepatocytes from each species for 60 min, and reaction mixtures were extracted and analyzed by mass spectroscopy. Both GN and CN were rapidly metabolized in hepatocytes from all species (T1/2, 0.7-11.6 min). Within a species, intrinsic clearance was similar for both compounds and increased in the order human < rat < mouse. Major common pathways for biotransformation of GN and CN involved 1) epoxidation of the 6-alkenyl moiety followed by conjugation with glutathione, 2) hydroxylation of the terminal methyl group(s) followed by direct conjugation with glucuronic acid in rodents or further oxidation to the corresponding acid in human cells, and 3) hydroxylation of the allylic C5 position. No evidence for either phase I or phase II metabolism of the conjugated nitrile moiety was obtained. Thus, the presumed metabolic basis for differences in genotoxicity remains elusive.     

Enhanced penetration of mitomycin C through hairless mouse and rat skin by enhancers with terpene moieties

The effects of four new percutaneous absorption enhancers containing an azacyclo ring and terpene chain (1-geranylazacycloheptan-2-one (GAH), 1-farnesylazacycloheptan-2-one (FAH), 1-geranylazacyclopentan-2,5-dione (GAPD), and 1-farnesylazacyclopentan-2-one (FAP] and 1-dodecylazacycloheptan-2-one (Azone) on the percutaneous penetration of mitomycin C (MMC) through hairless mouse and rat skin in-vitro has been investigated. GAH, FAH, FAP and Azone enhanced MMC penetration by 20 to 60 times that of the control (ethanol). During the early part of the experiments, when the sink condition was maintained, FAH was the most effective for hairless mouse skin, whereas Azone showed the highest effect in the rat skin. The enhancing effect of GAPD was only about half that of the other enhancers, suggesting the importance of the polar group of the ring moiety in these compounds. The penetration of MMC through rat skin was also increased by pretreatment with these compounds, suggesting that the enhancers had a direct effect on the skin.     

In vitro metabolism of the analgesic agent, tramadol-N-oxide, in mouse, rat, and human.

Tramadol-N-oxide (TNO, RWJ-38705) is a new analgesic agent, which is believed to produce its analgesic effect following metabolic conversion to tramadol. In the present study, API ionspray-MS and MS/MS techniques were used to profile the in vitro metabolism of TNO in mouse, rat, and human hepatic S9 fractions in the presence of an NADPH generating system. Unchanged TNO represented 60, 24, and 26% of the sample in mouse, rat, and human, respectively. Tramadol, and seven other metabolites were profiled and tentatively identified on the basis of MS analysis and by comparison to synthetic reference samples. TNO metabolites were formed via four Phase I reactions: (1) N-oxide reduction, (2) O-demethylation, (3) N-demethylation, and (4) cyclohexylhydroxylation. TNO was found to be substantially metabolized in hepatic S9 from all three species. The metabolism of TNO to tramadol via N-oxide reduction was greater in rat and human than in mouse.     

In vitro metabolism of the analgesic bicifadine in the mouse, rat, monkey, and human.

The in vitro metabolism of [(14)C]bicifadine by hepatic microsomes and hepatocytes from mouse, rat, monkey, and human was compared using radiometric high-performance liquid chromatography and liquid chromatography/tandem mass spectrometry. Two main metabolic pathways were identified in all four species. One pathway was an NADPH-dependent pathway in which the methyl group was oxidized to form a hydroxymethyl metabolite (M2). Its formation was inhibited in human microsomes only by quinidine, a CYP2D6 inhibitor. In incubations with individual cDNA-expressed human cytochromes P450, M2 was formed only by CYP2D6 and CYP1A2, with CYP2D6 activity 6-fold greater than that of CYP1A2. M2 was oxidized further to the carboxylic acid metabolite (M3) by hepatocytes from all four species. The second major metabolic pathway was an NADPH-independent oxidation at the C2 position of the pyrrolidine ring, forming a lactam metabolite (M12). This reaction was almost completely inhibited in human hepatic microsomes and mitochondria by the monoamine oxidase (MAO)-B-specific inhibitor selegiline. Clorgyline, a specific inhibitor of MAO-A, was less effective in inhibiting M12 formation. Other metabolic pathways of variable significance among the four species included the formation of carbamoyl-O-glucuronide, hydroxymethyl lactam, and carboxyl lactam. Overall, the data indicate that the primary enzymes responsible for the primary metabolism of bicifadine in humans are MAO-B and CYP2D6.     

Influence of skin source, penetration cell fluid, and partition coefficient on in vitro skin penetration

Using split-thickness pig skin mounted on in vitro skin penetration-evaporation cells, standard conditions were developed to preserve the viability of the skin as judged by its ability to successfully graft to nude mice. The effects of variations from these conditions on the disposition of radioactivity of radiolabeled compounds were determined. No differences in percutaneous penetration were found for N,N-diethyl-m-toluamide, parathion, and progesterone when Tyrode's solution was used in place of tissue culture media. The percutaneous penetration of benzo(a)pyrene on human and pig skin was unaffected by the presence of sodium azide in the tissue culture media; however, with mouse skin, penetration was lower when sodium azide was present. The disposition of radioactivity following topical application of five radiolabeled compounds was similar on fresh skin compared with skin that had been frozen and exposed to ethylene oxide, although variability of the values was greater with the treated skin. The percutaneous penetration of several compounds was determined on skin with and without the epidermis. The penetration of compounds with a lower log P (log octanol-water partition coefficient) increased to a greater extent (e.g., benzoic acid, log P = 2, sixfold increase) than compounds with a higher log P (e.g., DDT, log P = 5, twofold increase). To further validate the use of pig skin, the percutaneous penetration of 11 compounds on pig skin were correlated (r = 0.79) with the values obtained for human skin under standardized in vitro conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dermal penetration and metabolism of p-aminophenol and p-phenylenediamine: Application of the EpiDerm™ human reconstructed epidermis model

To address the provision of the 7th Amendment to the EU Cosmetics Directive banning the use of in vivo genotoxicity assays for testing cosmetic ingredients in 2009, the 3D EpiDerm™ reconstructed human skin micronucleus assay has been developed. To further characterise the EpiDerm™ tissue for potential use in genotoxicity testing, we have evaluated the dermal penetration and metabolism of two hair dye ingredients, p-aminophenol (PAP) and p-phenylenediamine (PPD) in this reconstructed epidermis model. When EpiDerm™ tissue was topically exposed to PAP or PPD for 30 min (typical for a hair dye exposure), the majority (80–>90%) of PAP or PPD was excluded from skin tissue and removed by rinsing. After a 23.5 h recovery period, the PAP fraction that did penetrate was completely N-acetylated to acetaminophen (APAP). Similarly, 30 min topical application of PPD resulted in the formation of the N-mono- and N,N′-diacetylated metabolites of PPD. These results are consistent with published data on the dermal metabolism of these compounds from other in vitro systems as well as from in vivo studies. When tissue was exposed topically (PAP) or via the culture media (PPD) for 24 h, there was good batch-to-batch and donor-to-donor reproducibility in the penetration and metabolism of PAP and PPD. Overall, the results demonstrate that these two aromatic amines are biotransformed in 3D EpiDerm™ tissue via N-acetylation. Characterising the metabolic capability of EpiDerm™ tissue is important for the evaluation of this model for use in genotoxicity testing.