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A Toxicity Index of Skin and Wound Cleansers Used on In Vitro Fibroblasts and Keratinocytes

Wilson, Judy R. PhD; Mills, John G. DO; Prather, Irvine D. DO; Dimitrijevich, S. Dan PhD

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Quality of care is a critical requirement for wound healing. Strategies that optimize the tissue repair process have evolved with advances in understanding of the wound healing process.1 "Good" care of chronic wounds has been synonymous with topical prevention and management of microbial contamination2 because of the deleterious effects of infection on wound repair.3 Ideal topical therapy includes periodic reduction of bacterial contamination and removal of soluble debris without adversely impacting cellular activities vital to the wound healing process.4,5 One group of circulating cells involved in the tissue repair process includes polymorphonuclear leukocytes (PMNs), mast cells, macrophages, and inflammatory cells.1 Another major cellular group is composed of fibroblasts, keratinocytes, and vascular endothelial and smooth muscle cells.

In 2001, the Food and Drug Administration (FDA) released industry guidelines for development of products to treat chronic wounds.6 The FDA defined 2 broad wound care product categories by which claims may be classified: (1) those products contributing to improved healing, and (2) those products contributing to improvements in aspects of wound care other than healing. The manufacturer is responsible for providing evidence of efficacy. Demonstration of product biocompatibility, safety, and efficacy assists in regulation and assessment of risk.

Although certain skin and wound cleansers are designed as topical solutions with varying degrees of antimicrobial activity, wound cleansers may also be antimitotic and adversely affect normal tissue repair. The first step in a comprehensive strategy for defining wound care products, therefore, should be in vitro assays of antimitotic activity on each cellular component of injured target tissue. Thereafter, the effects of these products should be established on other wound healing events, including cell migration and angiogenesis, synthesis of extracellular matrix macromolecules, and wound closure.

Initially, studies should be performed on monolayer cultures of individual cell types. Later studies should include multicellular, 3-dimensional models of living tissue, such as a skin equivalent.7 In vitro models of the initial phase of this strategy have included monolayer cultures of human fibroblasts,5,8-10 mouse fibroblasts,11 keratinocytes,5,12 and PMNs.13 The ideal evaluation strategy for wound care products includes in vivo animal models of wound healing and correlates the findings with clinical experience with human subjects, when data are available.

The interest in finding alternatives to animal models for irritation and toxicity testing12 has led to further development of in vitro approaches for evaluating biomedical products. However, no one standard in vitro assay adequately simulates in vivo use of the wound care product. To understand the complete spectrum of effects on the wound environment, a strategy is needed that utilizes multiple cell types and various models for analysis of cytotoxicity and other cell functions.11,14

Frequently used antimicrobial products that fall within FDA guidelines on human skin cells have been investigated for cytotoxic effects.2,5,8-10,15,16 Little is known, however, about the biologic properties of nonantiseptic cleansing solutions not subject to FDA regulations. These agents are not primarily considered antiseptics, and it is often assumed that they do not affect the wound healing process.

Wright and Orr17 and Burkey et al11 evaluated the cytotoxicity of skin and wound cleansers on human cells, including the effect of pH on red and white blood cell integrity (benzidine method) and methylene blue toxicity on fibroblasts.17 Burkey et al11 used agarose overlay neutral red diffusion and fluorescein diacetate assays, red blood cell hemolysis assays, and direct contact assays (fluorescein diacetate) to evaluate fibroblast function (primarily cell viability).

Foresman et al13 used PMNs as a cellular model for evaluating wound cleanser cytotoxicity. Although PMNs are important in the wound healing process, fibroblasts are the principal cells involved in tissue repair and remodeling. To close the wound, fibroblasts migrate into the wound bed, populate the provisional fibrin-based matrix, divide, synthesize collagen and glucosaminoglycan, and initiate the tissue contraction process. In skin wounds, keratinocytes proliferate and differentiate to reestablish the epidermal barrier. Concurrently playing an important supporting role, endothelial cells initiate angiogenesis, which restores the microcirculation and remedies transient hypoxia. Resident and migratory macrophages supply the continuing source of cytokines necessary to stimulate and maintain epithelialization, fibroplasia, and angiogenesis.

The present study demonstrated that in vitro monolayers of normal human skin cells, fibroblasts, and keratinocytes are reliable models for screening wound and skin cleansers. The MTS assay [3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]18 determined cell viability and developed a toxicity index. This approach allows comparison and an informed choice of the numerous wound care products developed to meet the growing needs and changing strategies in chronic wound management.


Twenty commercial skin, wound, and skin/wound cleansers were evaluated, including saline and liquid bath soaps. Materials were obtained from manufacturers or distributors; bath soaps were purchased locally Table 1. For the initial test dilution, materials were used in their original concentrations.

Table 1
Table 1:

Human infant keratinocytes (IKs) were obtained by incubating human foreskin in dispase (5 mL, 20 U/mL; BD Biosciences, Bedford, MA) for 2 days at 4°C. The dermis and epidermis were separated, placed into centrifuge tubes (15 mL), and vigorously pipetted in trypsin-EDTA (1X; 0.05% trypsin with EDTA 4 Na; GIBCO; Invitrogen, Carlsbad, CA), giving a single cell suspension and undigested stratum corneum. Stratum corneum pieces were removed, and the remaining cell suspension was pelletted by centrifugation. The supernatant was aspirated, and the cells were rinsed twice with trypsin inhibitor (2 × 5 mL Dulbecco's Modified Eagle Medium [DMEM] GIBCO; Ivitrogen) containing fetal bovine serum (FBS; 10%). The final cell pellet was suspended in keratinocyte growth medium (KGM, 5 mL; Clonetics, Cambrex Corporation, East Rutherford, NJ) and plated into 75-cm2 tissue culture flasks (T75) precoated with FNC Coating Mix (FNC; BRFF, Ijamsville, MD). After adding an additional 15 mL of KGM to the flask, the cells were allowed to attach overnight. The medium was then changed and replaced every second day until the flask was 80% confluent.

Infant dermal fibroblasts (IDFs) were obtained as outgrowths from explanted tissue. Dermal tissue obtained from deepidermalization (approximately 3-mm squares) was placed in a flask (T75); after attachment, the medium (DMEM, containing 10% FBS) was added and changed every 48 hours. IKs and IDFs were cultured to 80% confluence (T150 flasks) and passaged into multiple flasks, providing the needed number of cells; only early-passage IDFs and IKs (4-7) were used for this study.

For exposure to the cleansing agents, IDFs were seeded into 96-well plates at a density of 20,000 cells/well, allowed to adhere in DMEM (containing 10% FBS for 24 hours), and cultured under the same conditions until ready for use. IKs were also seeded into 96-well plates (20,000 cells/well) that had been precoated with FNC and allowed to adhere for 48 hours in KGM. After 24 and 48 hours, DMEM and KGM were removed from IDFs and IKs, respectively, by aspiration. After the cells were rinsed with phosphate-buffered saline (PBS) and pelletted by centrifugation, the supernatant was removed by aspiration. The IDFs and IKs were then exposed to the various cleansing agents for 30 minutes at 37°C and assayed for viability.

Cell viability was determined using the MTS assay (Promega; Madison, WI). The cleansers (serial 1:10 dilutions) were tested until the results of the cells exposed to the test solutions were similar to control cells (in complete DMEM or KGM). Testing at each dilution was performed in triplicate.

The MTS assay (reagents purchased from Sigma-Aldrich, St. Louis, MO, and Promega, Madison, WI) is based on conversion of a tetrazolium salt into a colored, soluble formazan product by mitochondrial cell activity viable at 37°C. Formazan is produced by an amount of dehydrogenase enzymes directly proportional to the number of living cells in culture as measured at 492 nm.18 Preliminary hourly experiments determined that 3 hours provided the best measure of sensitivity (better than 1, 2, or 4 hours).


The toxicity index for each product was based on MTS cell proliferation/viability assay results and defined as the dilution required to generate experimental cell viability to be 85% of controls (cells exposed only to media DMEM or KGM). Toxicity indexes ranged from 10 to 100,000; therefore, a nontoxic dilution of 1/1000 corresponded to a toxicity index of 1000. Foresman et al13 proposed a similar toxicity index derivation: as the toxicity index becomes higher, the presence of the material becomes more detrimental to cell division. Cell death clearly has an indirect effect on other cell functions (eg, collagen biosynthesis and wound closure).


Reducing and eliminating the toxicity of cleansers tested on infant dermal fibroblasts required a wide range of dilutions, resulting in a broad toxicity index range Table 2. Shur-Clens was found to be the least toxic to fibroblasts, requiring no dilution to maintain viable cells, with SAF-Clens and saline close behind. Several wound cleansers (acetic acid, Biolex, boric acid, Cara-Klenz, and Puriclens) had a toxicity index of 10, and Restore Wound Cleanser had a toxicity index of 100. Dermal Wound Cleanser, povidone (10%), Techni-Care, and hydrogen peroxide were found to be as toxic as povidone-iodine (Betadine Surgical Scrub). Dove Moisturizing Body Wash, Hibiclens, and Hollister Skin Cleanser had a toxicity index of 10,000. Surprisingly, ordinary bath soaps (eg, Dial, Ivory) were still toxic to IDFs at 10−5 dilution Table 2.

Table 2
Table 2:


Keratinocytes proliferate and differentiate early in the tissue repair process, reestablishing the skin barrier by covering the wound. Also used as monolayers, keratinocytes are expected to be more sensitive (vulnerable) to external agents than completely stratified and cornified epidermal barriers. Table 3 indicates the difference between materials toxic to keratinocytes and those toxic to fibroblasts. Shur-Clens, Biolex, and Techni-Care were nontoxic and needed no dilution. Acetic acid, boric acid, and Cara-Klenz retained a toxicity index of 10; SAF-Clens and saline were slightly more toxic (toxicity index, 10) to keratinocytes than to fibroblasts (Table 2). The toxicity index increased for Puriclens, but decreased for Dermal Wound Cleanser from 1000 for IDFs to 100 for IKs. Liquid bath soaps were less toxic to IKs (toxicity index, 1000). Hibiclens and Hollister Skin Cleanser remained toxic to both IDFs and IKs (toxicity index, 10,000). Compounds historically toxic to intact skin were also toxic to IKs; povidone-iodine, hydrogen peroxide, modified Dakin's solution (0.025%), and povidone (10%) had a toxicity index of 100,000.

Table 3
Table 3:


Because the use of controls (untreated patients) is rarely feasible ethically, clinical evaluation of wound healing and wound management is difficult in humans. Consequently, most initial studies utilize animal models or cellular in vitro models. The most widely used small-animal models (eg, guinea pigs, mice, rabbits, and rats) are firmly established as appropriate for wound healing studies. In recent years, however, there has been a movement away from animal models14 and an increased interest in cellular models, particularly those using normal human cells. Cellular models are almost exclusively appropriate for studies of specific phases or events within a particular phase of tissue repair. More complex questions can be addressed using in vitro models based on co-cultures of 2 different cell types possibly encountered in vivo. The most relevant in vitro models are tissue equivalents (cultured tissue substitutes) that are 3-dimensional models of normal human tissue, usually comprised of at least 2 different cell types.14 These complex models of living tissue (eg, skin equivalent) only address aspects of tissue biology dependent on systemic dynamics of the organisms in vivo, when they are grafted to nude mice. Thus, integrated biologic responses more directly relevant to human subjects are best derived from in vivo experiments using animal models.

The ideal comprehensive evaluation strategy of wound care products in the present study would involve all the models discussed above, followed by analyzing clinical use outcomes. Studying cells relevant for a particular injury is the first step in the process of increasingly complex in vitro models and, eventually, in vivo animal studies. The events are studied in a controlled manner, addressing the complexities of the progressive in vivo process and increasing the likelihood of clarifying various involved mechanisms. Because of difficulty establishing controls and the effect of variations inherent in wound care protocols on outcome measures, evaluation is most difficult at the clinical level.

Skin cleansers in the present study were most toxic to fibroblasts, showing toxicity indexes between 10,000 and 100,000 (Dove Moisturizing Body Wash, Hibiclens, Hollister Skin Cleanser, Dial Antibacterial Soap, and Ivory Liqui-Gel). This was not surprising because fibroblasts (connective tissue cells) are not exposed to chemical insults in their normal environment. A similar argument may be applied to other cellular internal tissue components (eg, endothelial cells); circulating cells (eg, red blood cells), which are afforded additional protection within the circulatory system, should be even more vulnerable.

In contrast, keratinocyte monolayers, representing the in vivo basal layer of the epidermis that epithelializes the wound surface after injury, are more sensitive to wound cleansers such as hydrogen peroxide, modified Dakin's solution (0.025%), and povidone (10%). The different responses of these cell types to wound cleansers are due, in part, to varying abilities to respond to inflammatory events. Direct exposure to inflammatory events may increase hydrogen peroxide production, which fibroblasts are better equipped to handle; keratinocytes are able to handle other inflammatory mechanisms. This hypothesis of progressive cellular vulnerability or susceptibility to skin/wound cleansers could be used as a predictive guide.

The agarose overlay neutral red diffusion assay,11 an American Standard Test Methods cytotoxicity method,19 direct contact, and dilution direct contact assays represent a progressive increase in relevance to open wounds. However, using neutral red or fluorescein diacetate20 as end-point markers provides only semiquantitative results. Nevertheless, an empirical comparison of 13 skin cleansers yielded results from direct contact that could be helpful in the development of a toxicity profile. Cell viability was determined by the dilution direct contact approach and MTS. MTS, accepted as a considerably less hazardous but fully quantitative alternative to isotopic labeling of cell proliferation, is commercially available in kit format. After the present study was completed, a new reagent, sulforhodamine B (SRB) was incorporated into a cytotoxicity assay,21,22 which is somewhat more convenient because the final pigment (SRB) absorbed by the target cells is stable.

In vitro data of this type are difficult to translate into definitive guidelines and clinically meaningful recommendations for wound care specialists. The environment of a real wound is a complex combination of regulatory signals generated by living, injured, and necrosing cells and damaged extracellular matrix, which is compounded by varying degrees of vascularization and innervation that link the wound to the rest of the organism in an integrated biologic manner. In vitro models cannot simulate this. The opportunity to modify the cleanser through continuous changes in protein profile and microbial flora in a chronic wound, therefore, far exceeds most in vitro models. The "dwell time" of the cleanser within the wound may be shorter than those employed in the present study. The phase of wound healing that prevails at the time of application of the agent, be it invasion of the provisional matrix by fibroblasts or epithelialization, would also dictate the appropriate cleanser. However, repeated application of evaluated products may overwhelm the systemic dispersal and contribute to accumulation of significant concentration in the open wound. This scenario may contribute to a delay in the tissue repair phases that involve proliferation and functions of viable cells. Although there may not be issues with efficacy of cleansing action, the direct benefits to tissue repair should be carefully weighed.

It is difficult to establish direct correlation of in vitro findings with in vivo results. The findings of the present study, however, are supported by the in vivo work of Menton and Brown,23 in which application of SAF-Clens and Shur-Clens into a full-thickness guinea pig dorsum skin wound resulted in a healing process that did not differ from healing in wounds in which saline was applied. Povidone-iodine resulted in significantly slower dermal and epidermal healing. Other studies24-28 targeted different materials and employed significantly different methods and outcome measures, all of which make extrapolation less meaningful. When correlations are established, however, in vitro methodology could then accelerate the evaluation of any newly introduced wound cleanser.


Wound management strategies address the delicate balance between cytotoxicity and cellular activities. Irritation of intact healthy tissue could seriously impact the rate and quality of tissue repair. An extensive matrix of in vitro and in vivo tests is needed so that accurate predictive conclusions can be derived for the clinical application of products not closely monitored by the FDA.

Using a quantitative cell viability methodology and normal human skin cells, this study determined toxicity indexes for 13 wound cleansers, 2 skin and wound cleansers, and 5 skin cleansers. Results can be rationalized with the function and sensitivity of fibroblasts or keratinocytes in vivo, whether applying the products to intact skin or an open wound in the early stages of repair.

Although toxicity indexes do not represent a complete profile, they provide a relatively easy primary screening methodology essential in a strategy of progressively comprehensive in vitro models that should culminate in in vivo animal testing and derivation of human clinical outcomes.


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