Timothy B. Hellewell, BS
David A. Major, BS
Pamela A. Foresman, BA
George T. Rodeheaver, PhD

WOUNDS: A Compendium of Clinical Research and Practice
Volume 9, Number 1, January / February 1997

From Plastic Surgery Research, University of Virginia
Health Sciences Center, Charlottesville, VA

Address correspondence to:
George T. Rodeheaver, PhD University of Virginia
Health Sciences Center, Box 443, Charlottesville, VA 22908


ABSTRACT

Ten commercial wound cleansers were tested for their toxicity to polymorphonuclear leukocytes (PMNs). PMNs were attached to glass coverslips and exposed to the products, serial 1:10 dilutions of the products, or a physiologically normal solution for 30 minutes at 37°C. After these exposures, the PMNs were evaluated for viability with the trypan blue exclusion test and for function by measuring their ability to phagocytize yeast cells. The PMNs were exposed to the serial 1 : 10 dilutions until the resulting cellular viability and function were similar to PMNs exposed to a physiologically normal solution. A relative toxicity index was then defined as the reciprocal of the dilution that eliminated toxicity. The resulting toxicities for the ten cleansers ranged from 10, i.e. a 1 : 10 dilution, to 10,000, i.e. a 1 10,000 dilution. The non-antimicrobial wound cleansers had toxicity indexes of 10 to 1,000, while the toxicity indexes for the antimicrobial cleansers were 10,000. The implications of these data for wound management are discussed.

WOUNDS 1997;9(1):1′20

Wound cleansing is indispensable to any effective wound care protocol insofar as it removes inflammatory stimulants such as foreign matter, necrotic debris, inflammatory exudate, and bacteria from the damaged tissues. Until the wound is clean, inflammation persists and optimum healing cannot occur. With such a broad definition, wound cleansing could be taken to encompass everything from debridement to the use of topical antibiotics. However, as the term is more commonly understood, wound cleansing involves the gentle use of fluids to remove loosely adherent debris and necrotic tissue from the wound’s surface.(1)

Wound cleansing must be performed without inflicting undue mechanical or chemical trauma to tissues that have already been compromised. Therefore, the benefits of facilitating wound healing in a clean environment must be balanced against the damage that inevitably results from cleansing. Such damage is not always obvious because it can occur on a cellular level without gross manifestations. Cytotoxicity from topical agents is an example of chemical trauma that may outweigh the anticipated benefit. Since wound cleansers are used repeatedly in wound care it is important to use those of low toxicity.

In an earlier publication, we measured the relative cytotoxicities of sixteen wound and skin cleansers with a simple assay, based on the viability and functionality of polymorphonuclear leukocytes (PMNs).(2) Our motivation for that study was to differentiate the toxicity of skin cleansers from wound cleansers. Since the publication of our earlier study, a number of new wound cleansers have appeared on the market. The present study assessed the cytotoxicity of ten such products.


Table 1
List of Non-Antimicrobial and Antimicrobial Wound Cleansers
Evaluated for Their Relative Cytotoxicity

Non-Antimicrobial Wound Cleansers
Product Manufacturer
Clinswound™ Sage Laboratories, Inc., Shreveport, LA
Curaklense™ Wound Cleanser Kendall Healthcare Products Co., Mansfield, MA
Curasol™ Healthpoint Medical, Fort Worth, TX
Dermagran® Derma Sciences, Old Forge, PA
Gentell Wound Cleanser™ Gentell, Huntingdon Valley, PA
Sea-Clens® Wound Cleanser Coloplast Sween Corp., N. Mankato, MN
Antimicrobial Wound Cleansers
Micro-Klenz™ Antimicrobial Wound Cleanser Carrington Laboratories Inc., Irving, TX
Restore™ Hollister Incorporated, Libertyville, IL
Royl-Derm™ Acme United Corporation, Farifield, CT
Septicare™ Antimicrobial Wound Cleanser Sage Laboratories, Inc., Shrevport, LA

METHODS

Antimicrobial and non-antimicrobial wound cleansers. The four antimicrobial and six non-antimicrobial preparations evaluated during this study are listed in Table 1. All of the agents were obtained from their manufacturers or their distributors and used as received. Testing bias was eliminated by transferring all of the agents to separate sterile containers that were identified by code numbers. The codes were broken only at the end of the study.

Experimental design. PMNs were isolated from fresh rabbit blood onto glass coverslips. These PMNs were exposed to the cleansing solutions for 30 minutes and then assayed for viability and functionality. Viability was assessed by the trypan blue dye exclusion test, and functionality by the ability of the exposed cells to phagocytize yeast cells. Serial 1: 10 dilutions of a given cleanser were tested until the results for the cells exposed to the diluted cleansing solution were similar to those of cells exposed to Hank’s balanced salt solution (HBSS).

Testing of each cleanser involved blood from three different rabbits. The test agent and each of its 1: 10 dilutions were evaluated on eight cover- slips from blood from each rabbit for a total of 24 coverslips per concentration – twelve each for the viability and functionality tests. During each experiment, 24 coverslips (eight from each rabbit’s blood sample) were used as control samples and were exposed only to HBSS. Viability and functionality of agent-exposed PMNs were com- pared to controls for each animal and the results expressed as a percentage.

Preparation of cells. This procedure was modified from that of Patselas, et al.(3) and used in the earlier study of skin and wound cleansers.(1) Unheparinized blood was obtained by means of intracardiac puncture of adult female New Zealand white rabbits that had been anesthetized with halothane. Aliquots of blood (0.8 ml) were immediately placed onto clean #2 coverslips and incubated for 30 minutes at 37°C in humidified chambers. During this incubation, the red blood cells formed a clot that was separated from the glass coverslip by a layer of serum. PMNs and monocytes adhered to the coverslip and remained attached, while the blood clot and serum were removed by carefully rinsing the coverslip in HBSS. PMNs comprised 90 percent of the adherent cells and were the only cells counted during this evaluation. Each coverslip with its adherent PMNs was placed cell-side up into its own container of HBSS with 10 percent added autologous serum. The coverslips were stored in this solution at room temperature until utilized. The time between PMN isolation and usage did not exceed 4 hours.

Exposure to test agent. The coverslips were carefully removed from their container, held vertically, and drained of their excess HBSS by placing gauze to their lower edge. Each coverslip was then placed cell-side up in a humidified chamber and covered with 0.3 ml of a test agent. The latter was either a cleanser, one of its 1 : 10 dilutions, or HBSS control solution. The coverslips were incubated for 30 minutes at 37°C with the PMNs in contact with the test agent. After the incubation, the test agents were drained from the coverslips, which were then swirled gently in a 37°C HBSS bath to remove any excess test agents before the viability and functionality tests were performed.

Viability assay-trypan blue dye exclusion. Following a 30-minute exposure to a test agent and rinsing, each coverslip was placed cell-side down onto a slide containing a drop of 0.125 percent trypan blue dye (wt/vol in sterile isotonic saline). Using gentle pressure with gauze at the coverslip edges only, the excess dye solution was removed, and the coverslip edges sealed with melted paraffin. The slides were viewed within ten minutes with a light microscope at 40X and 50 to 100 cells counted. Viable cells remained unstained, while the nuclei of non-viable cells were stained blue. The ratio of viable cells to the total number of cells counted was recorded as a percent viability for each coverslip. The mean percent viability for the twelve coverslips exposed to each concentration of cleanser was determined. The concentration of cleanser that resulted in a mean viability of 85 percent or more was considered not significantly different from samples exposed to HBSS, and thus was considered non-toxic.

Functionality assay-phagocytic efficiency. Following exposure to a test agent for 30 minutes and rinsing, the cells on each cover slip were exposed to 0.5 ml of saline containing 10,000 yeast cells (see Yeast preparation below). The PMNs were incubated with the yeast cells for 30 minutes at 37° C in humidified chambers before the yeast solution was rinsed away. The coverslips were then fixed for one minute in absolute methanol, stained for fifteen minutes in Giemsa (0.325 mg/ml in methanol), rinsed in distilled water, and air dried. The coverslips were then placed cell-side down onto microscope slides and the edges sealed with melted paraffin.

The slides were examined by means of 100X oil immersion microscopy. The number of yeast cells ingested per PMN was recorded for 50 to 100 PMNs. The mean number of yeast cells ingested per PMN was then calculated for each coverslip. These results were compared to corresponding HBSS’treated controls for that rabbit, and the test cell results expressed as a percentage of control cell function. For each concentration of a cleanser, the percent function for all twelve separate coverslips was used to calculate the mean phagocytic efficiency for that dilution of cleanser. When the mean phagocytic efficiency of a dilution of test cleanser was 85 percent or more of that of cells exposed only to HBSS, then that dilution of cleanser was considered to be not significantly different than the controls, and thus was considered non-toxic.


Table 2
Relative Toxicity Indexes of Non-Antimicrobial and Antimicrobial Wound Cleansers
(Combination of Present Findings and Those of Previous Study (2))

Non-Antimicrobial Wound Cleansers
Product Manufacturer Toxicity
Index
Dermagran® Derma Sciences, Inc. 10
Shur-Clens® Wound Cleanser ConvaTec® 10
Biolex™ Bard Medical Division, C.R. Bard, Inc. 100
Cara-Klenz™ Wound & Skin Cleanser Carrington Laboratories, Inc. 100
Saf’Clens® Chronic Wound Cleanser ConvaTec® 100
Clinswound™ Sage Laboratories, Inc. 1,000
Constant-Clens™ Dermal Wound Cleanse Sherwood Medical – Davis & Geck 1,000
Curaklense™ Wound Cleanser Kendall Healthcare Products Company 1,000
Curasol™ Healthpoint Medical 1,000
Gentell Wound Cleanser™ Gentell 1,000
Sea-Clens® Wound Cleanser Coloplast Sween Corp. 1,000
UItra-Klenz™ Wound Cleanser Carrington Laboratories, Inc 1,000

Antimicrobial Wound Cleansers
Product Manufacturer Toxicity
Index
Clinical Care® Dermal Wound Cleanser Care-Tech® Laboratories, Inc 1,000
Dermal Wound Cleanser Smith & Nephew United, Inc. 10,000
MicroKlenz™ Antimicrobial Wound Cleanser Carrington Laboratories, Inc. 10,000
Puri-Clens™ Wound Deoderizer and Cleanser Coloplast Sween Corp. 10,000
Restore™ Hollister Incorporated 10,000
Royl-Derm™ Acme United Corporation 10,000
SeptiCare™ Antimicrobial Wound Cleanser Sage Laboratories, Inc. 10,000

Yeast preparation.

One colony forming unit of a clinical isolate of Candida albicans (B311– University of Virginia) was incubated for 24 hours in 30 ml of trypticase soy broth. The yeast were then collected by centrifugation and the broth decanted. The pellet was washed twice with sterile saline and then resuspended in 2 ml of saline. The yeast was diluted in sterile saline, and the final solution was made in HBSS with 10 percent autologous serum. The final concentration was 10,000 yeast/0.5 ml. Toxicity index. This was assigned to the cleanser based upon the results from the viability and phagocytic efficiency assays. A dilution of the cleanser was identified in which both the viability and phagocytic efficiency of treated cells were similar to HBSS-treated control cells. The toxicity index was the denominator of the dilution; thus, if the non-toxic dilution was 1 : 1,000, the toxicity index would be 1,000.

RESULTS

The relative cytotoxicity indexes for the six non-antimicrobial wound cleansers spanned two 1 : 10 dilutions from 10 to 1,000. Dermagran® (Derma Sciences, Inc., Old Forge, PA) had a toxicity index of 10; the other five non-antimicrobial wound cleansers had a toxicity index of 1,000.  The relative cytotoxicity index for all four wound cleansers containing an antimicrobial agent was 10,000.

DISCUSSION

The results obtained in this study were similar to those obtained in the previous study.(2) In that study, nine wound cleansers were shown to have toxicity indexes ranging from 10 to 10,000. when the results for those nine wound cleansers are combined with the results of the present study involving ten wound cleansers, a composite list of most of the commercial wound cleansers can be generated (Table 2).

A review of Table 2 indicates a marked difference between non-antimicrobial wound cleansers and antimicrobial wound cleansers. Without the presence of an antiseptic agent, the toxicity indexes ranged from 10 to 1,000. When an antiseptic was added, the toxicity index, with the exception of Clinical Care® Dermal Wound Cleanser (Care-Tech® Laboratories, Inc., St. Louis, MO.), increased to 10,000. Antiseptic agents are known to be toxic to cells(1) and their presence in these wound cleansers at concentrations of 0.1 percent result in a toxicity index value of 10,000.

Addition of antiseptics to wound cleansers has not been shown to be of any benefit. So why add these toxic agents? Antiseptics are added to satisfy the continued requests of wound care professionals who mistakenly believe that antiseptics are needed in wound cleansing to reduce bacterial contamination. This belief is based on tradition, not science. Yet the FDA in its monograph on over-the-counter (OTC) first aid antiseptic drug products defined a wound cleanser as a formulation that contains an antiseptic agent.(4) This definition has to be viewed in the context of the mono- graph, which is for first aid products that must contain an antimicrobial agent. In this context, a wound is an acute injury that results in a minor cut, scrape or burn. This monograph and these definitions do not pertain to acute or chronic wounds that require intervention by healthcare professionals, nor do they pertain to wound cleansers that do not contain an antimicrobial agent.

The belief that antiseptics kill high levels of bacteria is based on tests using suspensions of bacteria in aqueous solutions of antiseptics. However, the inclusion of blood, wound exudate, or necrotic tissue in these suspensions attenuates or even eliminates the antiseptic’s efficacy. Antiseptics also bind actively to other organic substrates that are present in wound beds.(5-7) It is probable, therefore, that clinically suitable concentrations of antiseptics do not reach bacteria in lethal concentrations. This supposition is not new. It was first advanced in 1919 by Fleming(6) and has not been repudiated since. The benefits that have been ascribed to antiseptics during wound healing studies probably arise from other features of the protocols, such as debridement; most of a wound’s bacterial burden arises from bacteria sequestered in necrotic matter. When this debris is removed, so is the bulk of a wound’s bacterial flora. Unless a patient is severely immunocompromised, these bacteria can be controlled with standard, physiologically sound wound management. As a rule, bacteria will not survive in clean, healthy tissue.

The foregoing arguments imply that debridement, coupled with the maintenance of a normal wound environment, will stimulate the body’s innate capacity for healing. To this end, it is logical to use the least cytotoxic wound cleansers available. Although the FDA provides monographs that define wound cleansers, it does not regulate them and so it is up to each practitioner to select the least cytotoxic cleansers that are compatible with the demands of an open wound.

Although water or saline are adequate in many cases for cleansing the wound surface, there are instances in which more active formulations are needed. These typically feature surfactants, which reduce the adherence of contaminants and debris to the wound surface and thus enhance cleansing efficacy. In addition, surfactants significantly reduce the friction between wound tissue and the scrubbing device because of their lubricity.(8) Unfortunately the greater the surfactant activity the greater the cytotoxicity. Therefore, cleansing efficacy has to be balanced against wound trauma.

REFERENCES

  1. Rodeheaver GT. Wound cleansing, wound irrigation, wound disinfection. In: Krasner D, Kane D. Chronic Wound Care, Second Edition. Wayne, PA, Health Management Publications, Inc., pp 97-108.

  2. Foresman PA, Payne DS, Becker D, et al. A relative toxicity index for wound cleansers. WOUNDS 1993;5:226-231.

  3. Patselas TN, Sullivan CW, Mandell CL. Effect of Trimethoprim/Sulfamethoxazol (™P/SMX) on human neutrophil function and survival of mice infected with candida. In: Cillissen C, Operkuch W, Peters C, Pulverev (eds). The Influence of Antibiotics on the Host-Parasite Relationship III. Berlin, Springer-Verlag, 1989, pp 174-183.

  4. Topical antimicrobial drug products for over-the-counter human use; tentative final monograph for first aid antiseptic drug products; proposed rule. Federal Register 1991;56(140):33644-33680.

  5. Zamora JL, Price MF, Chuang P, Gentry LO. Inhibition of povidone-iodine’s bactericidal activity by common organic substances: an experimental study. Surgery 1985;98(1):25-9.

  6. Fleming A. The action of chemical and physiological antiseptics in a septic wound. Br J Surg 1919;7:99-129.

  7. Lacey RW. Antibacterial activity of povidone towards non-sporing bacteria. J Applied Bacteriology 1979;46:443-9.

  8. Rodeheaver CT, Smith SL, Thacker JC, Edgerton MT, Edlich RF. Mechanical cleansing of contaminated wounds with a surfactant. Am J Surg 1975;129(3):241-5.