Polyhexamethylene Biguanide
Polyhexanide (polyhexamethylene biguanide, PHMB) is a polymer used as a disinfectant and antiseptic. In dermatological use,[2] it is spelled polihexanide (INN) and sold under names such as Lavasept, Serasept, Prontosan and Omnicide.[3] Polyhexamethylene biguanide has been shown to be effective against Pseudomonas aeruginosa, Staphylococcus aureus (also the methicillin-resistant type, MRSA), Escherichia coli, Candida albicans (yeast), Aspergillus brasiliensis (mold), vancomycin-resistant enterococci, and Klebsiella pneumoniae (carbapenem-resistant enterobacteriaceae).[4]
Some products containing Polyhexamethylene biguanide are used for inter-operative irrigation, pre- and post-surgery skin and mucous membrane disinfection, post-operative dressings, surgical and non-surgical wound dressings, surgical bath/hydrotherapy, chronic wounds like diabetic foot ulcer and burn wound management, routine antisepsis during minor incisions, catheterization, scopy, first aid, surface disinfection, and linen disinfection.[5] Polyhexamethylene biguanide eye drops have been used as a treatment for eyes affected by Acanthamoeba keratitis.[6]
Branded as Baquacil, it also has an application as a swimming-pool and spa water sanitizer in place of chlorine- or bromine-based products. It is available as Baqua-Spa 3 sanitize, as Revacil Spa 3 sanitizer, and in the Leisure Time Free system.
Polyhexamethylene biguanide is also used as an ingredient in some contact lens cleaning products, cosmetics, personal deodorants and some veterinary products. It is also used to treat clothing (Purista), purportedly to prevent the development of unpleasant odors.
The Polyhexamethylene biguanide hydrochloride salt (solution) is used in the majority of formulations.
Safety
In 2011, Polyhexamethylenbiguanide (Polyhexamethylene biguanide, Polyhexanide) has been classified as carcinogenic category 2 by the European Chemical Agency (ECHA). Products containing concentrations of 1% Polyhexamethylene biguanide and more have to be declared as «suspected of causing cancer» and concentrations of 0.1% or above have to be noted in the safety datasheet. Polyhexamethylene biguanide is allowed as a part of cosmetic products (max. 0.1%) if exposure by inhalation is impossible.
On the 20th of April 2018, the european commission decided to ban preservative uses of Polyhexamethylene biguanide PT9 (Fibre, leather, rubber and polymerised materials preservatives). It’s still allowed for uses as disinfectants PT2 (Disinfectants and algaecides not intended for direct application to humans or animals). Furthermore, Polyhexamethylene biguanide has been declared as a candidate for substitution by the ECHA.
Studies suggest that iodine’s mechanism of action is through destabilization of the bacterial cell wall and disruption of the membrane that results in leakage of the intracellular components.25
Polyhexamethylene biguanide (PHMB). Polyhexamethylene biguanide (PHMB), also known as polyhexanide and polyaminopropyl biguanide, is a commonly used antiseptic. It is used in a variety of products including wound care dressings, contact lens cleaning solutions, perioperative cleansing products, and swimming pool cleaners.
Wound care products containing Polyhexamethylene biguanide include Kerlix AMD™, Excilon AMD™, and Telfa AMD™ (all from Tyco HealthCare Group, Mansfield, Mass) and XCell® Cellulose Wound Dressing Antimicrobial (Xylos Corp, Langhorne, Pa).
A review of the literature demonstrates in-vivo and in-vitro safety and effectiveness of Polyhexamethylene biguanide for a number of applications. For wound dressings, Wright and colleagues26 compared the effectiveness of a silver dressing to a dry gauze dressing containing Polyhexamethylene biguanide (Kerlix AMD). Results demonstrated reduction in bioburden with both dressings when tested in an in-vitro bactericidal assay. Using a Kirby-Bauer zone of inhibition study, the gauze was not as effective. This was believed to be due to a tight bond between the dressing and Polyhexamethylene biguanide, which was not released and therefore did not result in killing beyond the edge of the dressing.26 Alternatively, Motta and associates6 demonstrated a good response using Kerlix AMD compared to gauze without Polyhexamethylene biguanide in wounds where packing the dressing into the wound was required. Results suggested that the Polyhexamethylene biguanide in the gauze resulted in a decrease in the number of organisms present in the wound.
The majority of literature describes effectiveness of Polyhexamethylene biguanide on various microorganisms associated with contact lens disinfecting solutions. Antimicrobial effectiveness has been demonstrated on Acanthamoeba polyphaga, A castellanii, and A hatchetti.25,27,28 Additional effectiveness was demonstrated for Polyhexamethylene biguanide use in water treatment. Barker and colleagues29 tested the effect of Polyhexamethylene biguanide on Legionella pneumophila. This bacterium causes Legionnaire’s disease and can be found in water systems, air conditioning machinery, and cooling towers.
Gilbert and colleagues30,31 have performed numerous studies on bacteria, especially those that form biofilms, such as Klebsiella pneumoniae. In studying biofilms produced from E coli and S epidermidis, they noted that those compounds with higher activity against planktonic bacteria, including Polyhexamethylene biguanide, were also the most effective agents against sessile bacteria found within biofilms. They suggested that the differences in effects of concentration of Polyhexamethylene biguanide on planktonic versus sessile bacteria was due to either the mechanism of action or the number or disposition of cationic binding sites.30–32 Kramer et al33 have studied the effects of various antiseptics including Polyhexamethylene biguanide on fibroblast proliferation and cytotoxicity. They noted that while octenidine-based products retarded wound healing, Polyhexamethylene biguanide promoted contraction and aided wound closure significantly more than octenidine and placebo.
The mechanism of action of Polyhexamethylene biguanide has been described in a number of articles. Broxton et al34,35 demonstrated that maximal activity of the Polyhexamethylene biguanide occurs at between pH 5–6 and that initially the biocide interacts with the surface of the bacteria and then is transferred to the cytoplasm and cytoplasmic membrane. Ikeda and colleagues36 showed that the cationic Polyhexamethylene biguanide had little effect on neutral phospholipids in the bacterial membrane—its effect was mainly on the acidic negatively charged species where it induced aggregation leading to increased fluidity and permeability. This results in the release of lipopolysaccharides from the outer membrane, potassium ion efflux, and eventual organism death.37
Clinically, Polyhexamethylene biguanide has been used as a perioperative cleansing agent,38 in mouth wash,39 in ophthalmology,38,40 and as a topical wash.18 Hohaus et al19 reported on the oral use of Polyhexamethylene biguanide (Lavasept 1%, Fresenius-Kabi, Bad Homburg, Germany). A combination of oral terbinafine and topical ciclopirox and Polyhexamethylene biguanide were used to successfully treat a deep fungal infection (Trichophyton mentagrophytes) of the throat. Petrou-Binder40 describes the germicidal effects of Polyhexamethylene biguanide (Lavasept 0.02%) as eye drops prior to cataract surgery. It was well tolerated with low tissue response and minimal patient discomfort.
While there is no peer-reviewed clinical literature of Polyhexamethylene biguanide used on wounds, industry literature describes the effectiveness of AMD Gauze (Kerlix) as a bacterial barrier against Staphylococcus epidermidis (penicillin resistant) on volunteers. Results suggest that clinically, this dressing was an effective barrier against bacterial colonization.41 The studies suggested that AMD gauze did not elicit any skin reactions.42
Biosynthesized Cellulose Wound Dressing—
Antimicrobial (BWD-Polyhexamethylene biguanide)
Biosynthesized cellulose wound dressings (XCell Cellulose Wound Dressing and XCell Cellulose Wound Dressing Antimicrobial) were developed to maintain a moist wound environment without causing maceration, reduce pain, and enable autolytic debridement. This is possible because the dressings effectively absorb exudate and hydrate dry areas of a wound different from other dressings that have only a single function.43
A 49-patient, multicenter, controlled, randomized clinical study was conducted to demonstrate effectiveness of BWD compared to standard of care on venous leg ulcers. Significantly more autolytic debridement, significantly reduced pain, and cleaner wound margins were demonstrated after the 12-week study period.44,45 Improved rate of wound closure, as demonstrated by increased epithelialization and granulation tissue, was also noted.43
The antimicrobial version of BWD (BWD-Polyhexamethylene biguanide) contains cellulose, water, and 0.3% polyhexamethylene biguanide (PHMB). BWD-Polyhexamethylene biguanide is indicated for use on partial- and full-thickness wounds. It is designed to cover a wound or burn, absorb areas of wound exudate, and provide a moist wound environment that supports autolytic debridement of nonviable tissue. The dressing may be used on moderately exuding, nonexuding, and dry wounds. It also protects against abrasion, desiccation, and external contamination. The moist environment has a cooling effect that has demonstrated a significant reduction of pain.45
Preclinical efficacy testing. BWD-Polyhexamethylene biguanide demonstrates it effectiveness against a variety of organisms. Following a modified American Association of Textile Chemists and Colorists (AATCC) Method 100, samples were incubated with approximately 106 CFU/mL of the various challenge organisms. After 24 hours, a second count was made to determine the reduction in the number of organisms present. Results indicated 99.9% reduction of MRSA, Escherichia coli, Enterococcus faecalis, Bacillus subtilis, and Candida albicans within the 24-hour period.
Release of Polyhexamethylene biguanide from BWD-Polyhexamethylene biguanide. A study was performed to demonstrate the release of Polyhexamethylene biguanide from BWD-Polyhexamethylene biguanide. Five sterile 3.5-in x 3.5-in samples were used. One quarter of the dressing was used to determine the initial Polyhexamethylene biguanide concentration in each dressing using UV-Vis (Ultraviolet-Visible) Spectroscopy (Genesys™ 10 UV, Thermo Spectronic, Rochester, NY) at a wavelength of 234 nm. The remainder of the sample was weighed and placed into 20 times its weight in filtered water. At various times, including 0.5, 1, 2, 3, 4, 5, 6, and 24 h, the solution was assayed for Polyhexamethylene biguanide concentration. At the 24-h time the dressing was removed from the tray, weighed, and an extract was taken and assayed for Polyhexamethylene biguanide concentration.
Figure 1 illustrates the concentration of Polyhexamethylene biguanide over time. Equilibrium was reached after about 3 hours with the concentration (in ppm) in the dressing equaling the concentration in the solution. This demonstrates that the Polyhexamethylene biguanide is not bound to the cellulose and therefore can be released into surrounding fluid along a concentration gradient.
Clinical case series. BWD-Polyhexamethylene biguanide was evaluated in an open enrollment, noncontrolled clinical trial. Standard procedures for wound care were followed and samples of wound fluid were tested for type and level of microbial colonization at initial administration and 1–7 days after BWD-Polyhexamethylene biguanide placement.
Materials and Methods
BWD-Polyhexamethylene biguanide pads (XCell Cellulose Wound Dressing–Antimicrobial) measuring 3.5-in x 3.5-in were provided to 2 clinical sites and used as the primary dressing. Secondary dressings, including compression wraps (where indicated), were the standard of care for the facilities. Patients were chosen on an “as needed” basis and neither randomized nor controlled.
The 2 sites evaluated a total of 12 patients with 26 wounds of various etiologies including venous stasis ulcers (12), diabetic (4), traumatic (8), vasculitic (1), and necrobiosis diabetica lipoidica (1). Eleven of the 12 patients were unresponsive to a silver impregnated or an iodine containing dressing in the 3–4 weeks prior to use of the BWD-Polyhexamethylene biguanide dressing. In these cases the wound had either increased in size or failed to progress. One patient was treated directly with BWD-Polyhexamethylene biguanide.
Swabs of the wound were taken to determine if bacterial colonization was the reason for the lack of response to previous dressings. Organisms were identified in the wounds of 8 patients prior to and after BWD-Polyhexamethylene biguanide application. Systemic antibiotics were not given in conjunction with the use of BWD-Polyhexamethylene biguanide to ensure bacterial reductions were solely due to the Polyhexamethylene biguanide.
The organisms identified included methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus aureus, Pseudomonas aeruginosa, Proteus mirabilis, Diphtheroid gram-positive rods, beta hemolytic Streptococcus B, Enterobacter aerogenes, mixed skin flora, and Enterococcus sp. The most common was Staphylococcus (including MRSA) and Pseudomonas. Semiquantitative scores ranged from 0 to 4+ (0 represents no bacterial growth and 4+ represents the largest amount of bacterial growth on the culture). The various bacteria found in the wounds of all 8 patients and the relative abundance prior to and after application of the BWD-Polyhexamethylene biguanide dressing are shown in Table 1.
Results
Four patients (5 wounds) from 1 site were used strictly for the economic analysis below. Of the remaining 8, 1 patient (3 wounds) was lost to follow-up after 1 week of BWD-Polyhexamethylene biguanide treatment. The remaining patients had BWD-Polyhexamethylene biguanide applied over periods of 1 to 7 weeks. Results of the 8 patients demonstrated a decrease in wound size on average from 6.79 cm2 to 4.57 cm2 (42% reduction) in an average of 25 days (Table 2). Two of the wounds completely healed during the study, 13 improved, and 2 showed a slight increase in size.
Case Reports
Case 1. A 58-year old woman presented with a full-thickness draining wound over the dorsal foot secondary to an incision (Figure 2). The patient's wound extended to the level of tendon and was recalcitrant to topical gels, ointments, foam dressings, silver dressings, and moist saline gauze. Past medical history was significant for Hodgkin’s disease, heart valve replacement, pacemaker, hemolytic anemia, and chemo and radiation therapy for breast cancer, which was on-going at the time of presentation. After 3 weeks of treatment with a papain-urea ointment (Panafil®, Healthpoint, Fort Worth, Tex), the majority of fibrotic tissue was removed although the wound did not decrease in size. The patient was then placed exclusively on BWD-Polyhexamethylene biguanide for approximately 4 weeks with the dressing being changed once a week. The wound rapidly improved and progressed to complete closure during this time period.
Case 2. A 78-year-old woman presented with a large wound secondary to a hematoma occurring after trauma (Figure 3). The patient was not on anticoagulants and had a medical history significant for hypertension. The wound had been present for 1 week prior to presentation. Following extensive debridement, the patient was started exclusively on BWD-Polyhexamethylene biguanide dressing changes every 4 days. The wound closed completely in approximately 2 months. The patient had a history of similar lesions that required up to 6 months of treatment.
Case 3. An 89-year-old woman with diabetes presented with venous disease and psoriasis (Figure 4). She had 2 wounds, one each on her right and left lower extremities (RLE and LLE) that were treated separately over a period of 209 days.
Upon presentation, the RLE wound was 17.5 cm x 7.0 cm x 0.3 cm. It was treated for 167 days using various products including Acticoat™ (46 applications, [Smith & Nephew, Largo, Fla]), Santyl® (7 applications, [Healthpoint, Fort Worth, Tex]), Apligraf® (6 applications, [Organogenesis, Canton, Mass]), and Xeroform™ (7 applications, [Tyco-Kendall HealthCare Group, Mansfield, Mass]). After these treatments the wound measured 9.0 cm x 4.4 cm x 0.1 cm. Following an initial decrease in size, the wound became unresponsive to these treatments. At that time, BWD-Polyhexamethylene biguanide was substituted as the exclusive primary dressing. Over the next 42 days, a total of 10 BWD-Polyhexamethylene biguanide dressings were applied. The patient subsequently went on to heal 1 week after her final treatment (49 days total) using this protocol.
Upon presentation, the LLE wound was 1.0 cm x 0.9 cm x 0.3 cm. It was treated for 156 days using various products including Acticoat (2 applications), XCell (2 applications), Santyl/Panafil (70 applications), Apligraf (4 applications), Sulfamylon (26 applications), Aquacel® (3 applications, [ConvaTec, Skillman, NJ]), OpSite™ (8 applications, [Smith & Nephew, Largo, Fla]), and Xeroform (7 applications). The wound remained unhealed after these treatments. The wound was recalcitrant to care; therefore, BWD-Polyhexamethylene biguanide was substituted as the exclusive primary dressing. Over the next 53 days, a total of 12 BWD-Polyhexamethylene biguanide dressings were applied as the exclusive treatment. The wound healed at approximately 60 days.
Case 4. A 79-year-old woman presented with venous leg ulcer on her lower extremity (Figure 5). She was treated over a period of 104 days. The wound was 15.0 cm x 9.0 cm x 0.1 cm. The wound was initially treated for 34 days using Panafil (13 applications) and Iodosorb (22 applications). After these treatments the wound measured 10.0 cm x 9.0 cm x 0.3 cm. The wound was determined to be recalcitrant after an initial decrease in size (15.0 cm x 9.0 cm to 10.0 cm x 9.0 cm, [≈ 35%]) and BWD-Polyhexamethylene biguanide was substituted as the exclusive primary dressing. Over the next 70 days, a total of 10 BWD-Polyhexamethylene biguanide dressings were applied.
Effect on wound bioburden and pain. By evaluating the bacterial load pre- and post-BWD-Polyhexamethylene biguanide, it was demonstrated that the dressing resulted in elimination of Pseudomonas aeruginosa, Diptheroid gram-positive rods, beta hemolytic streptococcus, and Enterobacter aerogenes in some patients. In other patients, decreased levels of Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus mirabilis were observed.
A reduction in pain has been noted with BWD44 as was observed in the present study.
Economics of BWD-Polyhexamethylene biguanide. The estimated cost for the treatment of chronic wounds including services and associated products is close to $40,000 or in some cases even more.45 Any delay to heal a wound can increase that cost. Mulder46 described an economic model for determining the cost of 2 different treatments for removing necrotic tissue. The analysis demonstrated that a hydrogel/polyurethane combination was slightly more expensive than wet-to-dry gauze but was more cost effective when time to reach ≥ 50% debridement was included.
The cost of BWD-Polyhexamethylene biguanide is similar to other advanced wound dressings. An economic analysis was performed in this study to determine the cost of BWD-Polyhexamethylene biguanide use over time. An economic analysis of the use of BWD-Polyhexamethylene biguanide dressings demonstrates the low cost of using BWD-Polyhexamethylene biguanide on recalcitrant wounds. The average cost of material was calculated to be $5.99 to $9.01 per day with the wounds demonstrating improvement or healing. No attempt was made to quantify the remaining cost of treatment (clinic visit, staff time, etc.).
Data were gathered retrospectively for 2 patients that presented at the UCSD Healthcare System in San Diego, Calif. These patients had a total of 3 wounds that were initially treated with an array of advanced wound care products prior to exclusive use of a BWD-Polyhexamethylene biguanide dressing. The costs associated with the products used in Cases 3 and 4 appear in Tables 3 and 4, respectively. Table 5 illustrates the cost of the use of BWD-Polyhexamethylene biguanide including the use of saline and gauze to clean the wound.
Conclusion
A greater understanding of the role bacteria plays in the wound matrix repair process is resulting in an increasingly important role for antimicrobial dressings and products used in chronic wound care. The differences between various antimicrobial components and dressings require that clinicians have a basic understanding of different antimicrobial agents and their role in tissue repair before selecting the most appropriate dressing for a wound. The introduction of noncytotoxic levels of antimicrobial agents, including silver and Polyhexamethylene biguanide, provides a means to potentially decrease levels of bacterial colonization that may impede closure while providing dressings that may assist with the development of a wound environment conducive to tissue repair, and ultimately, successful wound closure. Currently, Polyhexamethylene biguanide does not have a history of resistance or cytotoxicity, has demonstrated promotion of healing,33 and may play a new and important role as an antimicrobial agent in dressings. The need for decreased frequency of dressing changes, dressing tolerance, and ease-of-use are factors, which are equally important when selecting an appropriate antimicrobial dressing.
The limited amount of information on the ability of antimicrobial dressings to significantly affect the healing process and wound closure supports the need for well designed and adequately powered clinical trials to determine the true role of these devices in the treatment of chronic wounds. Current information and publications indicate a potential benefit regarding the use of these products in wounds where bacterial burden may be delaying or impeding wound closure.
Polyhexamethylene biguanide (PHMB) is an antiseptic with antiviral and antibacterial properties used in a variety of products including wound care dressings, contact lens cleaning solutions, perioperative cleansing products, and swimming pool cleaners. There are regulatory concerns with regard to its safety in humans for water treatment. We decided to assess the safety of this chemical in Sprague-Dawley rats. Polyhexamethylene biguanide was administered in a single dose by gavage via a stomach tube as per the manufacturer's instruction within a dose range of 2 mg/kg to 40 mg/kg. Subchronic toxicity studies were also conducted at doses of 2 mg/kg, 8 mg/kg and 32 mg/kg body weight and hematological, biochemical and histopathological findings of the major organs were assessed. Administration of a dose of 25.6 mg/kg, i.e. 1.6 mL of 0.4% Polyhexamethylene biguanide solution (equivalent to 6.4x103 mg/L of 0.1% solution) resulted in 50% mortality. Histopathological analysis in the acute toxicity studies showed that no histopathological lesions were observed in the heart and kidney samples but 30% of the animals had mild hydropic changes in zone 1 of their liver samples, while at a dosage of 32 mg/kg in the subchronic toxicity studies, 50% of the animals showed either mild hepatocyte cytolysis with or without lymphocyte infiltration and feathery degeneration. Lymphocyte infiltration was, for the first time, observed in one heart sample, whereas one kidney sample showed mild tubular damage. The acute studies showed that the median lethal dose (LD50) is 25.6 mg/kg (LC50 of 1.6 mL of 0.4% Polyhexamethylene biguanide. Subchronic toxicological studies also revealed few deleterious effects on the internal organs examined, as seen from the results of the biochemical parameters evaluated. These results have implications for the use of Polyhexamethylene biguanide to make water potable.
Keywords: polyhexamethylene biguanide, toxicity, biochemical hematology, histopathology, LD50, therapeutic index
Introduction
Polyhexamethylene biguanide (PHMB) is an antiseptic with antiviral and antibacterial properties used in several ways including wound care dressings, contact lens cleaning solutions, perioperative cleansing products, and swimming pool cleaners. It is also known as polyhexanide and polyaminopropyl biguanide, polymeric biguanide hydrochloride; polyhexanide biguanide. It is a commonly applied antiseptic, often used as a preservative in cosmetics and personal care products (Schnuch et al., 2007).
The antimicrobial efficacy has been demonstrated on Acanthamoeba polyphaga, A castellanii, and A hatchetti (Hughes et al., 2003; Wright et al., 2003; Burgers et al., 1994; Hiti et al., 2002). In vivo studies have also demonstrated that a miltefosine–polyhexamethylene biguanide combination is highly effective for the treatment of Acanthamoeba keratitis (Polat et al., 2013).
As a biocide, additional pharmacological effects have been demonstrated against Legionella pneumophila, against gram positive and gram negative bacteria. It is a broad spectrum virucide and has amebicidal activities (Gilbert et al., 1990; Kramer et al., 2004; Broxton et al., 1984; Lee et al., 2007). Polyhexamethylene biguanide retains its activity in hard water and does not cause surface streaks or tackiness (Broxton et al., 1984b; Ikeda et al., 1984). Consistent with previous studies, a Polyhexamethylene biguanide mouthrinse was shown to inhibit plaque re-growth and reduced oral bacterial counts, indicating that Polyhexamethylene biguanide could be an alternative to established mouth rinses in preventive applications (Welk et al., 2005). Recreational water maintained and sanitized with Polyhexamethylene biguanide is however assumed to serve as a medium for transmission of ocular adenovirus infections, mainly because at a concentration of 50 ppm, Polyhexamethylene biguanide was not virucidal against adenovirus at temperatures consistent with swimming pools or hot tubs (Romanowski et al., 2013).
Previous studies have shown increased frequency of sensitization to 0.5% and 0.4% Polyhexamethylene biguanide in unselected dermatitis patients (Schnuch et al., 2007). Polyhexamethylene biguanide proved also toxic to keratocytes (Lee et al., 2007) and was shown to have acute toxic effects in human cells where it caused severe inflammation, atherogenesis, and aging. Moreover, Polyhexamethylene biguanide produced embryo toxicity and heart failure in zebrafish (Kim et al., 2013).
Though, officially not used in the treatment of drinking water, there have been instances where toxic effects were experienced in certain individuals. For example in the period from August 2006 to May 2007, more than 12,500 patients were admitted to hospital with a history of drinking illegal cheap “vodka” in 44 different regions in Russia, of whom 9.4% died. In reality, the “vodka” was an antiseptic liquid composed of ethanol (≈93%), diethyl phthalate, and 0.1-0.14% polyhexamethylene guanidine (PHMG) (“Extrasept-1”) (Ostapenko et al., 2011). Previous studies have also shown that another biocide – polyhexamethylene guanidine hydrochloride – has an LD50 of 600 mg/kg in rats (Asiedu-Gyekye et al., 2014). There have been various regulatory concerns with regard to the use of these biocides in water treatment. We therefore evaluated the safety of Polyhexamethylene biguanide when used in treating water to make it potable and also in the case of survivors of drowning events, concentrating especially on its effect on the major organs.
Materials and methods
Polyhexamethylene biguanide concentrate was purchased from AGRIMAT-Ghana as an aqueous solution. A stock solution of 0.1% concentration of Polyhexamethylene biguanide was prepared using deionized water. This was equivalent to 1.0 mg/mL of Polyhexamethylene biguanide. Further dilutions were made using deionized water.
Animal husbandry and groupings
Eight-week-old Sprague-Dawley rats (250 g body weight) of both sexes were acquired from Noguchi Memorial Institute for Medical Research, University of Ghana, Legon and housed in rooms with regulated room temperature of 26°C and humidity of 40 to 60%. The animals were exposed to 12 h light and 12 h darkness. The females were nulliparous and non-pregnant. The animals were randomly assigned to 4 groups of 10 animals each for the acute toxicity test. A similar provision was made for the subchronic study. Animal feed (Kosher Feed Mills Ltd, Osu, Accra) and water were given ad libitum. To ensure effective absorption from the gastrointestinal tract after oral administration, feed was withdrawn 8 h prior to treatment and further withheld for an extra 30 min after administration of Polyhexamethylene biguanide before being reintroduced. Equal numbers of rats were randomized and each marked in their individual cages for 7 days prior to Polyhexamethylene biguanide administration. Equal numbers of animals of both sexes were used at each dose level of Polyhexamethylene biguanide.
Acute toxicity
Polyhexamethylene biguanide was administered as a single dose by gavage in view of the potential mode of ingestion. The animals received doses of 2 mg/kg (500 mg/L), 4 mg/kg (2000 mg/L), 32 mg/kg (8000 mg/L) and 40 mg/kg (10000 mg/L of 0.1% Polyhexamethylene biguanide solution). Since the maximum volume of liquid that could be administered was 1 mL/100 g of body weight, an appropriate adjustment was made in preparing the concentrations so as to avoid exceeding the recommended volume of not more than 2 mL for oral administration (Lee, 1985). Thus 5 different concentrations were prepared. Control animals received only deionized water. The animals were observed every 30 min for the first 4 h, and every 8 h for the next 24 h. The number of animals that died within the 24 h period was recorded for each treatment. The rest of the animals were observed daily for 14 days and any clinical signs were recorded. Clinical signs monitored included respiratory distress, frequency of urination, swellings, abnormal gait, etc.
Discussion
This study aimed to assess the safety level of Polyhexamethylene biguanide when used to sanitize water to make it potable. The LD50 calculated from the study was found to be 25.6 mg/kg (equivalent to 6.4x103 mg/L of 0.1% Polyhexamethylene biguanide solution). Blood chemistry studies also indicated little or no adverse reaction on cellular components of the blood. All the indices examined were comparable to those of controls, suggesting that the chemical may not have any adverse effects on cellular components of the blood at doses below 25.6 mg/kg, the dose which elicited LD50.
Potassium concentrations detected were very high compared to controls, p<0.0057, suggesting that at LD50 level most of the rats might have experienced abnormal heart beats, but this was not confirmed by the histopathological study on the heart as there were no cellular lesions detected. This finding may suggest that in spite of the high dose tested, which possibly might have caused abnormal heart beat in some animals, the integrity of the architecture of the vital organs was not compromised. This observation is similar to that made for PHMGH (Asiedu-Gyekye et al., 2014). Sodium concentrations, on the other hand, did not show any change in either
Polyhexamethylene biguanide-treated or control groups (p<0.08), thus most of the clinical manifestations such as lethargy, weakness, etc, usually associated with sodium imbalance were not observed in the study. It is worth noting that most of the animals that died exhibited various nervous manifestations such as abnormal gait and tonic-clonic convulsions. These observations were not supported by the electrolyte profile obtained from blood chemistry analysis. A chronic toxicity study, which is beyond the goal of this study, is recommended to be carried out to further investigate this nervous phenomenon. It should be emphasized that mortality only occurred at very high doses.
