James N. McDougal

A physiological pharmacokinetic model for dermal absorption of vapors in the rat

Absorption of chemical vapors through the skin is a passive process that is not easily quantitated, but may be important in the assessment of health hazards in some occupational circumstances. Physiological modeling is a quantitative technique which may provide insight into the system being modeled and can be used for interspecies extrapolation. We developed a physiological model for the penetration of organic vapors through skin in vivo which allows the prediction of blood concentrations, after dermal vapor exposures in the rat, when chemical distribution coefficients, physiological and metabolic parameters, and skin permeability constants are known. We used the model in two distinct ways. First, permeability constants for dibromomethane (DBM), bromochloromethane (BCM), and methylene chloride (DCM) were calculated by using a physiologically based pharmacokinetic model for dihalomethanes to relate blood concentrations during dermal vapor exposures to the total amount of chemical which was absorbed through the skin. Second, a skin compartment was added to the model which had input based on the permeability-area-concentration product. This predictive model adequately described blood concentrations after DBM, BCM, and DCM dermal vapor exposures over a wide range of concentrations. This model could easily be modified for use with other organic vapors, and could be used to extrapolate to human vapor exposure conditions by substituting human physiological parameters for the animal values, providing permeability constants are known or can be determined.

Skin absorption and human risk assessment.

A common practice is to assume that percutaneous absorption does not significantly contribute to total bioavailability and therefore, absorption through other routes is more important to human risk assessment. The skin can represent a significant barrier to absorption, but some substances are absorbed to a significant extent. Since there is a potential for percutaneous penetration that is not consistent between species or substances, the assessment of the potential contribution of total body burden from dermal exposures should be considered. This review briefly discusses some theories, practices, and factors that affect percutaneous absorption with an emphasis on how percutaneous absorption evaluations apply to human risk assessment.

Methods for Assessing Risks of Dermal Exposures in the Workplace

The skin as a route of entry for toxic chemicals has caused increasing concern over the last decade. The assessment of systemic hazards from dermal exposures has evolved over time, often limited by the amount of experimental data available. The result is that there are many methods being used to assess safety of chemicals in the workplace. The process of assessing hazards of skin contact includes estimating the amount of substance that may end up on the skin and estimating the amount that might reach internal organs. Most times, toxicology studies by the dermal route are not available and extrapolations from other exposure routes are necessary. The hazards of particular chemicals can be expressed as “skin notations”, actual exposure levels, or safe exposure times. Characterizing the risk of a specific procedure in the workplace involves determining the ratio of exposure standards to an expected exposure. The purpose of this review is to address each of the steps in the process and describe the assumptions that are part of the process. Methods are compared by describing their strengths and weaknesses. Recommendations for research in this area are also included.

In vitro dermal absorption rate testing of certain chemicals of interest to the Occupational Safety and Health Administration: Summary and evaluation of USEPA’s mandated testing

In 2004, the United States Environmental Protection Agency (USEPA) published a final test rule in the US Federal Register requiring in vitro dermal penetration rate testing for selected industrial chemicals. The test rule described procedures for determining a permeability coefficient (Kp) and two short-term dermal absorption rates at 10 and 60min using human cadaver skin mounted in an in vitro diffusion cell model. According to the USEPA announcement, the selected chemicals were to be spiked with their radiolabeled form and tested in either water, isopropyl myristate (IPM) or neat depending on their physical character at room temperature, their aqueous solubility, their potential to damage the skin and their ability to achieve the study endpoints as prescribed. Overall, and for the majority of chemicals, the short-term absorption rates were higher at 10min than at 60min and the portion of applied dose remaining in the skin at the end of the exposure period was greater than the portion of dose that had penetrated through the skin and was detected in the receptor solution. In contrast to this, the amount of chemical in the receptor solution at study termination for the Kp (steady-state) experiments was greater than the amount remaining in the skin. For the Kp experiments, which lasted from 2 to 48h, a majority of skins exposed to neat chemical exhibited a reduced barrier function. However, integrity was mostly unaltered for skins from the short-term experiments and Kp experiments using chemicals applied either in water or IPM. Quantitative structure activity relationship (QSAR) model-predicted Kp values were in fair agreement with experimental data for those chemicals that were applied in a water vehicle when the integrity of the skin was not compromised. However, QSAR-derived Kp values were not predictive for those chemicals when applied in IPM vehicle or neat. Absorption predictions, based on the measured Kp and steady-state flux data for chemicals applied in water or neat, respectively, were comparable to measured values at both 10 and 60min. Kp data for chemicals applied in water and the flux values for neat chemicals will be useful for making estimates of skin absorption in occupational settings. Kp measurements for chemicals applied in IPM vehicle are not envisioned to provide useful data for estimating the risk from dermal exposure to chemicals in the workplace. When available, in vitro dermal flux measurements should be combined with toxicity information in order to improve the utility of chemical skin notations.

The effect of m-xylene on cytotoxicity and cellular antioxidant status in rat dermal equivalents.

Exposure of the skin to volatile organic chemicals (VOCs) can lead to irritation, inflammation and cytotoxicity. Since VOCs are used in industrial, commercial and military applications, concern is mounting with respect to VOC safe exposure limits. Although traditional toxicological assessment of VOCs has utilized animal models, the use of alternative in vitro models is becoming more widespread. We have previously developed a sealed exposure system that prevents chemical loss through evaporation and enables calculation of target cell chemical dose. The present study utilized this in vitro exposure method to assess m-xylene-induced cytotoxicity and antioxidant status in dermal equivalents (dermal fibroblasts in a collagen matrix). At the end of a 1- or 4-h exposure, cytotoxicity was measured using the MTT assay and the EC50 values determined were 1481 +/- 88 and 930 +/- 33, respectively. Decreases in cellular thiols and catalase activity were observed, which occurred in a time and dose-dependent manner. Treatment of dermal equivalents with the antioxidants N-acetylcysteine (NAC) and catalase provided some protection against m-xylene-induced cytotoxicity. When compared to m-xylene exposures, treatment with either 1.0 or 5.0 mM NAC led to increases in the EC50 values at 1 and 4 h. Increases in these EC50 values ranged from 1.22- to 1.32-fold at 1 h and 1.27- to 1.54-fold at 4 h. Although treatment with catalase (1000 U/ml) led to a 1.35-fold increase in cell viability at 1 h, no significant differences were observed at either 1 or 4 h when compared to dermal equivalents exposed to m-xylene alone. These results suggest that exposure to m-xylene leads to a time- and dose-dependent decrease in cellular antioxidants and that cellular thiols may provide protection against the cytotoxic properties of m-xylene.

The Effects of Perchlorate on Thyroidal Gene Expression are Different from the Effects of Iodide Deficiency

Perchlorate (ClO4 −), which is a ubiquitous and persistent ion, competitively interferes with iodide (I) accumulation in the thyroid, producing I deficiency (ID), which may result in reduced thyroid hormone synthesis and secretion. Human studies suggest that ClO4 − presents little risk in healthy individuals; however, the precautionary principle demands that the sensitive populations of ID adults and mothers require extra consideration. In an attempt to determine whether the effects on gene expression were similar, the thyroidal effects of ClO4 − (10 mg/kg) treatment for 14 d in drinking water were compared with those produced by 8 wk of ID in rats. The thyroids were collected (n = 3 each group) and total mRNA was analyzed using the Affymetrix Rat Genome 230 2.0 GeneChip. Changes in gene expression were compared with appropriate control groups. The twofold gene changes due to ID were compared with alterations due to ClO4 − treatment. One hundred and eighty-nine transcripts were changed by the ID diet and 722 transcripts were altered by the ClO4 − treatment. Thirty-four percent of the transcripts changed by the I-deficient diet were also altered by ClO4 − and generally in the same direction. Three specific transporter genes, AQP1, NIS, and SLC22A3, were changed by both treatments, indicating that the membrane-specific changes were similar. Iodide deficiency primarily produced alterations in retinol and calcium signaling pathways and ClO4 − primarily produced changes related to the accumulation of extracellular matrix proteins. This study provides evidence that ClO4 −, at least at this dose level, changes more genes and alters different genes compared to ID.

Improved method for in vitro assessment of dermal toxicity for volatile organic chemicals

Cell culture methods are being developed to assess the dermal toxicity (irritancy and corrosion) of chemicals. These in vitro methods are being validated to categorize chemicals as irritating or non-irritating to humans. Currently, these cell culture tests are useful to assist in the ranking of chemicals for irritancy, but they are not useful for quantitative risk assessment for two reasons. First, for volatile chemicals the amount of chemical in the media that the cells are exposed to may decrease with exposure time. Also, effective concentrations such as EC(50) and IC(50) are reported as the concentrations in the media not the skin tissue/cells. We have developed an in vitro approach for dermal toxicity testing of volatile chemicals that avoids these problems. Using sealed vials lacking a headspace, dermal equivalents (dermal fibroblasts in a collagen matrix) were exposed to culture medium containing a test chemical (m-xylene) and compared to a traditional open well culture system. We found that about 90% of the m-xylene was lost from the open well plates and the viability was 4-6 times greater than in the closed system. Partition coefficients were measured and used to estimate the m-xylene concentration in the fibroblasts. The EC(50) for m-xylene in the dermal equivalents was 833.13+/-35.33 microg m-xylene per gram of fibroblasts. This method will provide an effective approach to relate target cell chemical concentration to cellular responses. Based on this method, a biologically-based mathematical model could be used to determine an equivalent external dose for a specific toxic end point.

Transcriptional changes in porcine skin at 7 days following sulfur mustard and thermal burn injury.

Severe cutaneous injuries continue to result from exposure to sulfur mustard [bis(2-chloroethyl)sulfide; HD] and thermal burns. Microarray analysis was utilized in this study to evaluate transcriptional changes in porcine skin assessing the underlying repair mechanisms of HD and thermal injury involved in wound healing. Four ventral abdominal sites on each of 4 weanling swine were exposed to 400 μL undiluted HD or a heated brass rod (70°C) for 8 minutes and 45–60 seconds, respectively. At 7 days postexposure, skin samples were excised and total RNA was isolated, labeled, and hybridized to Affymetrix GeneChip (Santa Clara, CA, USA) Porcine Genome Arrays (containing 20,201 genes). Based on the gene expression patterns in HD- and thermal-exposed skin at 7 days, the transcriptional profiles do not differ greatly. HD and thermal exposures promoted similar changes in transcription, where 270 and 283 transcripts were increased with HD and thermal exposures, respectively. Both exposures promoted decreases in 317 and 414 transcripts, respectively. Of the significantly increased transcripts, at least 77% were commonly expressed in both HD- and thermal-exposed skin, whereas at least 67% of decreased transcripts were common between both exposure types. Six of the top 10 biological functions were common to HD and thermal injury in which 9 canonical pathways were shared. The present study illustrates the similarities found between HD and thermal injury with respect to transcriptional response and wound healing and identifies specific genes (CXCL2, CXCR4, FGFR2, HMOX1, IGF1, PF4, PLAU, PLAUR, S100A8, SPP1, and TNC) that may be useful as potential therapeutic targets to promote improved wound healing.

Transcriptional responses associated with sulfur mustard and thermal burns in porcine skin

In military and civilian environments, serious cutaneous damage can result from thermal burns or exposure to the blistering agent sulfur mustard [bis (2-chloroethyl) sulfide; HD]. Similar therapies have historically been used to treat cutaneous thermal and HD injuries; however, the underlying molecular mechanisms of tissue damage and wound healing may differ between the types of burns. Using microarray analysis, this study assessed the transcriptional responses to cutaneous HD and thermal injury at 48 hours post-exposure to identify molecular networks and genes associated with each type of skin injury. Ventral abdominal sites on each of 4 weanling swine were exposed to 400 μl of undiluted HD or a heated brass rod (70°C) for 8 minutes and 45–60 seconds, respectively. At 48 hours post-exposure, total RNA was isolated from excised skin samples and hybridized to Affymetrix GeneChip Porcine Genome Arrays (containing 20,201 genes). Both HD and thermal exposure promoted significant transcriptional changes where 290 and 267 transcripts were increased and 197 and 707 transcripts were decreased with HD and thermal exposure, respectively. HD- and thermal-injured skin expressed 149 increased and 148 decreased common transcripts. Comparison of the 10 most significantly changed biological functions for HD and thermal exposures identified 7 overlapping functional groups. Canonical pathways analysis revealed 15 separate signaling pathways containing transcripts associated with both HD and thermal exposure. Within these pathways, 5 transcripts (CXCR4, FGFR2, HMOX1, IL1R1, and TLR4) were identified as known targets for existing phase II/III clinical trial or Food and Drug Administration (FDA)-approved drugs. This study is the first to directly assess transcriptional changes in porcine skin subjected to HD or thermal injury over the same time period.

Skin Penetration And Lag Times Of Neat And Aqueous Diethyl Phthalate, 1,2-Dichloroethane And Naphthalene

Cutaneous exposures to occupational chemicals may cause toxic effects. For any chemical, the potential for systemic toxicity from dermal exposure depends on its ability to penetrate the skin. Most laboratory studies measure chemical penetration from an aqueous solution through isolated human or laboratory animal skin, although most exposures are not from pure aqueous solutions. The US EPA Interagency Testing Committee (ITC) mandated by the Toxic Substances Control Act, has required industry to measure the in vitro penetration of 34 chemicals in their pure or neat form (if liquid). The goal of the present study was to measure skin permeability and lag time for three neat chemicals of industrial importance, representing the general types of chemicals to be studied by the ITC (non-volatile liquids, volatile liquids, and solids), and to examine interlaboratory variation from these studies. Steady state fluxes and lag times of diethyl phthalate (DEP, slightly volatile), 1,2-dichloroethane (DCE, highly volatile), and naphthalene (NAP, solid) were studied in two different laboratories using different analytical methods. One lab also measured fluxes and lag times from saturated aqueous vehicle. Static diffusion cells, dermatomed hairless guinea pig skin, and gas chromatography were used to measure skin penetration. In the two laboratories, the steady state fluxes (mean±SD; µg cm−2hour−1) of DEP applied neat were: 11.8±4.1 and 23.9±7.0; fluxes of DCE (neat) were 6280±1380 and 3842±712; fluxes of NAP from powder were 30.4±2.0 and 7.5±4.7. Compared with neat fluxes measured in the same laboratory, flux from saturated aqueous solution was higher with DEP (1.9 ×) but lower with DCE (0.17 ×) and NAP (0.45 ×). The three chemicals studied including a dry powder, demonstrate the potential for significant dermal penetration.