Antimicrobial agents help maintain cleanroom integrity
The key is to select the right products for the right jobs
by Elaine M. Kopis, Steris Corp.
There are a number of choices available in antimicrobial agents and formulated one-step disinfectants. The key to establishing an effective microbial control program lies in recognizing the differences in the abilities of the key antimicrobial products, and in selecting the right products for the right jobs. In most cases, more than one product will be necessary in order to meet all of the goals of a facility. For example, a phenolic disinfectant may be used for routine cleaning due to its effectiveness and relative compatibility with cleanroom substrates, whereas a strong sporicidal agent would be used periodically (thus minimizing substrate damage) to complement the phenolic agent by providing spore control. Additionally, while not sporicidal or always a particularly good cleaner, 70-percent alcohol solutions are indispensable for glove sanitizing and residue removal.
Controlling forces
For over 200 years we have been trying to control microbial growth. Prior to these efforts, we tried to control diseases without having a complete understanding of their microbial origin. Over the centuries several methodologies have been evaluated and ultimately adopted for these purposes including, filtration, steam sterilization, irradiation, and chemical treatment with gases and liquids. Although these methods are effective, they differ in their effects on substrates and products. In some cases, while the technology is quite effective at reducing microbial levels, resulting damage to product and substrates is unacceptable. These methods also differ in terms of the mechanism by which the organisms are destroyed, inactivated or removed from products and surfaces. For example, thermal applications and radiation kill microorganisms, but do not remove them from surfaces. Filtration removes microorganisms, but may not be effective in killing them. Cleaning with the proper chemical agent results in removal and kill of the microorganisms. When conducted properly, chemical cleaning may be a reliable, cost effective, and reproducible means of achieving microbial control for cleanrooms.
Establishing an effective microbial control program requires an understanding of product chemistry and application issues. There are a variety of products available for cleaning hard surfaces in cleanrooms; however, they are not all created equally in terms of their antimicrobial abilities, formulations and support packages. An understanding of these issues is critical to establishing the optimum microbial control program for a facility.
Sanitizers are the most basic of the antimicrobial products available for cleanroom users. These formulations are designed to provide a significant microbial reduction (99.9 to 99.999 percent) which is highly dependent upon the level of soil present. Sporicidal ability is typically where the line is drawn between a disinfectant and a sterilant. A sterilant provides 100 percent kill, including bacterial spores. A disinfectant will provide 100 percent kill of vegetative bacteria, selected fungi and selected viruses, but is typically ineffective against bacterial spores. Although some products may fall into both the disinfectant and the sterilant category, the key differentiation between the two will be in terms of concentration, contact time and the necessity of precleaning the surface prior to application (sterilants always require pre-cleaning of the surface prior to use). An example of this is chlorine dioxide which may be an intermediate level disinfectant at 10 minutes and a sterilant at 10 hours.
One-step disinfection
Sanitizers and sterilants are typically fairly simple in formulation due to the fact that they are not designed to provide 100 percent reduction in the presence of soil. However, one-step disinfectants (also known as germicidal cleaners) are required to function in the presence of soils, and typically their formulations are more complex than those for either sterilants or sanitizers. In addition to the antimicrobial agent, these formulations often include the following chemicals: surfactants (to increase wetting and cleaning ability); chelants (to bind calcium, magnesium and iron present in potable water which may interfere with the disinfectant); pH buffers (to boost the efficacy of the antimicrobial agent); and builders (to boost the cleaning ability of the solution). Disinfectants are also formulated to be safe to use on most cleanroom substrates on a daily basis. Sterilants, which are typically strong oxidizing agents, may cause damage to surfaces, even stainless steel, if used too frequently or at elevated concentrations. The most commonly used one-step disinfectants are formulated with either phenolic agents (phenols) or quaternary ammonium chloride compounds (quats).
Phenolic chemicals have been studied for nearly two centuries. One of the earliest uses of these types of chemicals (in the form of carbolic acid) was as wound sanitizers, an application introduced by Lister. Phenolic compounds are still found in antimicrobial handwashes, although due to their cytotoxic effects they are no longer routinely used for wound sanitizing, having been replaced by more gentle formulations and better wound management techniques. Over the years it was discovered that by altering the original phenol molecule (carbolic acid) by adding alkyl groups and halides, a much more potent molecule could be formed. Presently, ortho phenyl phenol, and para tertiary amyl phenol are the most commonly used phenolic agents for hard surface applications.
Phenolics work well against gram positive and gram negative organisms, certain fungi, and lipophilic viruses. However, they are not effective against bacterial endospores. The antimicrobial action of phenolics includes penetration and disruption of the cell wall, precipitation of proteins, and inactivation of enzyme systems. Phenolic disinfectants are available in both acidic and alkaline formulations, which are useful from a rotational perspective as studies have shown that the use of an alkaline and an acidic phenolic product in rotation has helped to prevent selection for resistant organisms.
Quaternary ammonium chloride compounds are cationic (carrying a positive charge) surfactants. It is this characteristic which is the basis for their antimicrobial action, as the positively charged molecule is attracted to the slightly negatively charged cell surface where adsorption and diffusion through the cell wall occurs. Binding to the cytoplasmic membrane is followed by disruption, and release of potassium ions and other cell constituents. Cell death follows shortly thereafter.
Quats are good broad spectrum disinfectants which are available in EPA-registered formulations. Although they are effective against both gram negative (and possibly to a larger extent) gram positive organisms, they are not typically TB effective. They tend not to be reliably virucidal against hydrophilic viruses and perform somewhat better in an alkaline medium.
Sporicidal concerns
Although both quats and phenols may be formulated to provide effective cleaning and disinfection for hard surface applications in cleanrooms, neither of these types of products are effective sporicidal agents. Therefore, the use of a sporicidal agent on a routine basis is highly recommended in order to ensure that these resistant organisms are adequately controlled. The most commonly used sporicidal agents include: sodium hypochlorite (bleach), chlorine dioxide, and hydrogen peroxide/peracetic acid blends.
Sodium hypochlorite has been used as a disinfectant for over 100 years. The first documented uses were in water treatment to control a typhoid epidemic. Apparently it worked well because sodium hypochlorite has been used in water treatment and other disinfectant applications since then. Later, hypochlorite solutions were used in controlling transmission of puerperal (child bed) fever, which was a common cause of death among women in the early 1800s. In order to control the spread of disease-causing organisms from one patient to another via the hospital staff, hands were washed with calcium hypochlorite solutions. Their uses as wound and hand sanitizers were limited due to their cytotoxic and corrosive effects. The primary antimicrobial action from bleach is oxidation, whereby sensitive chemical bonds, sulfhydryl, carbon, etc., are broken by the chemical action of the hypochlorous acid species of the solution. The destruction of these bonds leads to precipitation of essential enzymes, DNA inactivation, and a host of other effects which lead to cell death. Bleach solutions demonstrate good broad spectrum activity including sporicidal ability, and unlike some of the other agents available, they are not affected by hard water.
Although bleach is a good broad spectrum disinfectant and also a sporicidal agent, it does not hold EPA registrations in these areas. Therefore the burden of proof of efficacy is much greater for the consumer with this material. Even though the efficacy is not affected by hard water, it is affected by many other parameters, including pH, organic soil load, UV and heat exposure. Additionally, bleach, although it may have terrific bleaching qualities on certain materials, contains no surfactants; therefore it is not really a good cleaner. Probably the most commonly cited limitation of bleach is the fact that it is very corrosive, even to stainless steel. Additionally, in the presence of heavy organic material (as may be the case with cell culture operations), bleach generates several trihalomethane chemicals, including chloroform, a suspect carcinogen.
Chlorine dioxide is also an oxidizing chemical and is available in EPA-registered formulations as a disinfectant or a sterilant. It provides fast activity with sterilization time of six to 10 hours.
Chlorine dioxide is formed from the reaction of sodium chlorite with an organic acid (e.g., lactic). This reaction must take place in water in order to disperse the chlorine dioxide for application purposes and in order to absorb the heat of this exothermic reaction. The sodium chlorite, lactic acid and water must be blended at a defined ratio in order to achieve full efficacy.
Although chlorine dioxide is a powerful antimicrobial agent, there are several limitations to practical applications. As previously mentioned, the product requires exact activation which may lead to errors in preparation and consequently problems with efficacy. The use-life after dilution is typically 48 hours. Most disinfectants may be used for up to seven days with no loss in efficacy. Additionally, the product is corrosive to many substrates, including stainless steel. Perhaps the most serious issue with chlorine dioxide is regarding employee safety. The product produces offensive and noxious fumes and if used in a poorly ventilated area, a respirator should be worn. Sensitization has been associated with the use of this material.
Hydrogen peroxide is an oxidizing chemical that has been referred to as the natural sterilant. In nature it is found in small amounts in honey, where it acts as a preservative. It is also naturally occurring in saliva, where it helps to keep the mouth clean, and in the blood stream where it aids the phagocytes in eliminating foreign materials. It has been used for a number of years as a skin and wound sanitizer and as a sterilant for soft contact lenses.
Hydrogen peroxide is quite effective as a chemical sterilant or a high level disinfectant providing broad spectrum ability including spore control when used at concentrations of 6 to 25 percent. One of the chief advantages of this material is that it decomposes to water and oxygen; therefore minimizing the risk of surface and product contamination or residue build-up. However, it is quickly inactivated in the presence of organic soil.
Peracetic acid is the strongest of the oxidizing antimicrobial chemicals discussed here. It has been most commonly used as a broad spectrum disinfectant or sterilant in fogging applications for isolators. It has also been used at lower concentrations ( 2 percent) as a liquid. One of the chief advantages of peracetic acid is its fast rate of kill. Another advantage is that the material is strong enough to be effective without precleaning, in some cases. Although the decomposition products, acetic acid, oxygen and water are not hazardous, they do not leave surfaces completely residue free.
There are several reasons why peracetic acid has not been widely adopted for hard surface disinfection. Some find the odor objectional, and it can be an irritant at higher concentrations. Additionally, the material must be temperature controlled in order to prevent rapid decomposition. Typically the material should not be stored above 30 degrees Celsius. Finally, peracetic acid is corrosive to soft metals.
Product innovations
Manufacturers have recently blended the speed and efficacy of peracetic acid with the relative mildness of hydrogen peroxide to introduce a new line of EPA-registered cold sterilants which are a blend of these two oxidizing chemicals. Peracetic acid/hydrogen peroxide blends typically have a much higher ratio of hydrogen peroxide than peracetic acid (100:1). This minimizes the odor and the corrosivity of the blend, making it safer to handle, while maintaining the speed and efficacy as a sterilant. Some of these products have sterilization times of as little as 5.5 hours. As they do not require activation, they may be considered truly “self-sterilizing,” obviating aseptic filtration prior to entry into the sterile core.
Although the peracetic acid/hydrogen peroxide blends are milder than peracetic acid alone, they do have some limitations. They are still somewhat corrosive to soft metals, although milder than other types of sporicidal agents, including chlorine dioxide. Precleaning is required prior to use of this material, which requires controlled temperature storage at less than 30 degrees Celsius. Although minimal when compared to peracetic acid, some find the acetic acid (vinegar) odor objectionable even though inhalation toxicity is much less of a concern with this type of material than with chlorine dioxide, for example.
An old standby
The primary sanitizing agent utilized in pharmaceutical cleanrooms is alcohol. Alcohol holds no EPA registration as a sanitizer; however, it has been used for decontamination of gloves, equipment and other hard surfaces for years. According to a recent survey conducted by the Parenteral Drug Association, 70-percent isopropyl alcohol is used in approximately 90 percent of the parenteral cleanroom facilities. Although 70-percent isopropyl alcohol is more commonly used, 70-percent ethanol is also used for decontamination. Both chemicals provide good broad spectrum activity, including viruses; however, it is widely held that ethanol is somewhat more virucidal than isopropanol and that isopropanol is somewhat more bactericidal then ethanol due to molecular weight differences. Both products work on the basis of protein denaturation. The chief advantages of using these antimicrobial agents is that they evaporate readily, leaving behind no evidence of residue. In those cases where a denaturant other than methanol is used, low-level brucine sulfate residue should be considered.
Alcohol solutions have limitations that preclude their use as a substitute for formulated germicidal cleaners. Alcohol solutions do not contain surfactants, and consequently may not be effective cleaners, unless a particular soil is alcohol soluble. They also become quickly overwhelmed in the presence of heavy soil. Alcohol solutions are not sporicidal, and if not prepared properly, they can contribute to the spread of contamination in cleanrooms. Most facilities prepare their own 70-percent alcohol solutions with water for injection (WFI) or USP purified water, which is sterile filtered into sterile containers. This is an acceptable practice; although, depending upon the volume generated, the volatility (VOC) and flammability of alcohol may preclude this practice at certain facilities.
Elaine Kopis is the manager of the technical services department for the scientific division of Steris Corp. Her focus area is microbial control in cleanrooms and other critical environments. Additionally, she is on the editorial advisory board of CleanRooms magazine and is a faculty member of the PDA Training and Research Institute.
References:
1. Seymour S. Block, Disinfection, Sterilization, and Preservation, 4th ed. (Philadelphia, London: Lea & Febiger, 1991).
2. V.F. Denny and F.J. Marsik, “Current Practices in the Use of Disinfectants Within the Pharmaceutical Industry,” PDA Journal of Science and Technology, 51. 227-228 (1997).
3. D.E. Conner and M.K. Eckman, “Rotation of Phenolic Disinfectants,” Pharmaceutical Technology, 148-162 (September 1992).
4. D.E. Conner and M.K. Eckman, “Rotation of Phenolic Disinfectants Enhances Efficacy Against Adherent Pseudomonas aeruginosa,” Pharmaceutical Technology, 94-104 (October 1993).
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An understanding of product chemistry and application issues is key to an effective microbial control program.