REVIEW OF LITERATURE

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INTRODUCTION

Requirements for aseptic processing areas include readily cleanable floors, walls, and ceilings that have smooth, non-porous surfaces; particulate, temperature, and humidity controls; and cleaning and disinfecting procedures to produce and maintain aseptic conditions. These conditions combined with careful and thorough evaluation of the chemical agents used for the cleaning and disinfection program should lead to achieving the specified cleanliness standards and control of microbial contamination of products. In recent years, the use of disinfectants in pharmaceutical, biotechnology, medical device facilities, and associated controlled environments has been the subject of scrutiny by regulatory agencies. An effective cleaning and disinfection program in aseptic processing areas of a Good Manufacturing Practice (GMP) facility is critical to assure the quality of the products. Manufacturers are being held to a high standard when it comes to product sterility and regulatory agencies are increasingly asking for validation data to support sanitization and disinfection procedures. Regulatory authorities now expect evidence of the efficacy of disinfection agents against environmental isolates. The FDA Guideline for Aseptic Processing states, “The suitability, efficacy, and limitations of disinfecting agents and procedures should be assessed. The effectiveness of these disinfectants and procedures should be measured by their ability to ensure that potential contaminants are adequately removed from surfaces”. Basic knowledge regarding the effectiveness of different chemical agents against vegetative bacteria, fungi, and spores will aid in selecting chemical agents. A good understanding of test methods used to assess disinfectant effectiveness is important. Most methods are adaptable allowing the user to customize the methods to their specific requirements.

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H I S T O RY O F D I S I N F E C TA N T S JOSEPH LISTER & ANTISEPTIC SURGERY

Introduction: By the middle of the nineteenth century, post-operative sepsis infection accounted for the death of almost half of the patients undergoing major surgery. In 1839 the chemist Justin von Liebig had asserted that sepsis was a kind of combustion caused by exposing moist body tissue to oxygen. It was therefore considered that the best prevention was to keep air away from wounds by means of plasters, collodion or resins. Joseph Lister (1827-1912) Joseph Lister, a British surgeon, doubted this explanation. For many years he had explored the inflammation of wounds, at the Glasgow infirmary. These observations had led him to consider that infection was not due to bad air alone, and that 'wound sepsis' was a form of decomposition. When the Regius Professorship of Surgery at Glasgow University fell vacant in 1859 Lister was selected from seven candidates. In August 1861 he was appointed surgeon at the Glasgow Royal Infirmary and put in charge of its new surgical building. The hope was that the new building would decrease the number of deaths caused by what was then called hospital disease (now known as operative sepsis). This proved a vain hope when Lister reported that between 45 and 50 percent of his amputation cases died from sepsis between 1861 and 1865 in his Male Accident Ward. It was in this ward that Lister began his experimental work with antisepsis. Having tried methods to encourage clean healing, with little or no success, Lister began to form theories to account for the prevalence of sepsis. He discarded the popular concept of direct infection by bad air and postulated that sepsis might be caused by a 'pollen-like dust'. Although, there is no evidence GERM CONNECTION WITH WOUND SEPSIS When, in 1865, Louis Pasteur suggested that decay was caused by living organisms in the air, which on entering matter caused it to ferment, Lister made the connection with wound sepsis. A meticulous researcher and surgeon, Lister recognized the relationship between Pasteur's research and his own. He considered that microbes in the air were likely causing the putrefaction and had to be destroyed before they entered the wound.

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In the previous year Lister had heard that 'carbolic acid' was being used to treat sewage in Carlise, and that fields treated with the affluent were freed of a parasite causing disease in cattle.

CARBOLIC ACID
Lister now began to clean wounds and dress them using a solution of carbolic acid. He was able to announce at a British Medical Association meeting, in 1867, that his wards at the Glasgow Royal Infirmary had remained clear of sepsis for nine months. Operations using Lister’s carbolic spray invented in 1869 although his methods initially met with indifference and hostility, doctors gradually began to support his antiseptic techniques. In 1870, Lister’s antiseptic methods were used, by Germany, during the Fransco-Prussian war saving many Prussian soldiers’ lives. In Germany, Robert Koch (1878) demonstrating the usefulness of steam for sterilizing surgical instrument and dressings.

German surgeons were beginning to practice antiseptic surgery, which involved keeping wounds free from micro-organisms by the use of sterilized instruments and materials. Lister became aware of a paper published (in French) by the French chemist Louis Pasteur which showed that rotting and fermentation could occur without any oxygen if microorganisms were present. Lister confirmed this with his own experiments. If micro-organisms were causing gangrene, the problem was how to get rid of them. Pasteur suggested three methods: filter, heat, or expose them to chemical solutions. The first two were inappropriate in a human wound, so Lister experimented with the third. Carbolic acid (phenol) had been in use as a means of deodorizing sewage, so Lister tested the results of spraying instruments, the surgical incisions, and dressings with a solution of it. Lister found that carbolic acid solution swabbed on wounds markedly reduced the incidence of gangrene and subsequently published a series of articles on the Antiseptic Principle of the Practice of Surgery describing this procedure in Volume 90, Issue 2299 of The Lancet published on 21 September 1867. He also made surgeons wear clean gloves and wash their hands before and after operations with 5% carbolic acid solutions. Instruments were also washed in the same solution and assistants sprayed the solution in the operating theatre. One of his conclusions was to stop using porous natural materials in manufacturing the handles of medical instruments. As the germ theory of disease became more widely accepted, it was realized that infection could be better avoided by preventing bacteria from getting into wounds in the first place. This led to the rise of sterile surgery. Some consider Lister "the father of modern antisepsis." In 1879 Listerine mouthwash was named after him for his work in antisepsis. Also named in his honor is the bacterial genus Listeria, typified by the food-borne pathogen Listeria monocytogenes.
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The first major development towards modifying basic antimicrobial agents to make them more specialized, more effective, was in 1906 when Bechhold and Ehrlich synthesized the bis-phenols. They found that by linking two phenol rings directly or by mean of an alkaline group, the bactericidal and bacteriostatic effects of the phenols were greatly enhanced. Further work was done on the bis-phenol (During 1931) leading eventually to the synthesis of hexachlorophene (Gump 1941; Smylie et al, 1959). Iodine has also been modified for use as an antiseptic. The Iodine molecules can be added to other substances which have the ability to increase the antimicrobial qualities of the elements, whilst reducing the irritancy of iodine. The more recent modification of iodine has been the production of iodine-alcohol tinctures and polyvinyl-pyrrolidon-iodine complexes (Gershinfield 1957). Alcohols, whilst proven to be excellent antimicrobial agents, have hardly been modified since the 1880. It has been shown that, for instance, an increase in the chain length of amyl alcohols will increase their bactericidal activity (Wirgins 1904). Alcohols can also be incorporated into other antiseptics to boost the antimicrobial activity. Some compounds which have been developed as antiseptics include the surface active cationic quaternary ammonium compounds (Jacobs, 1916), although these compounds did not have their germicidal activity realized until 1935 by Domagk. During the latter part of 19th century, many test procedures were made available for examining the efficacy of skin antiseptic (Sykes 1985).
a) Skin infection test- which measure the antiseptic efficacy of germicides in contact

with artificially contaminated skin wounds
b) Skin penetration test- which asses the retention of activity of an antiseptic by the

skin over a period of time
c) Skin disinfection test- which evaluate antiseptic properties on intact skin.

While the scientific application of disinfectants, sterilants, and preservatives is limited to, at most, the past 150 years, empirical practices that were more or less effective go back to ancient times. Bible is filled with injunctions on cleanliness, strict dietary rules, regulations regarding lepers, care regarding wastes, these matters were almost an obsession.

The man who deserves the laurel wreath for having first recorded adequate experiment proof as well as interpretation of the role of a microorganism in the causation of disease is IssacBenedict Prevost of Montauban, France. He also used chemical disinfectants and showed in field tests that they were effective in preventing infection. In prevention of human disease, the first half of the 19th century was a period of awakening. The association between foul odors and disease was the basis for selection prior to the establishment of the germ throng of human disease. Kronig and Paul in their classical research published in 1897 laid the foundation of scientific knowledge of chemical disinfection. They observed that disinfectants can be accurately compared only when tested under controlled conditions. The number of bacteria
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must be constant and brought in to the contact with the disinfectant without the interference of other organic matter such as the nutritive medium. The action of the disinfectant must be promptly arrested after a stated period of time and the bacteria transferred to the most favorable medium at optimum temperature. The results of test are determined by accurate count of survivors in plate cultures. Cleaning methods today in hospitals have largely been developed from studies of dust control carried out in the early 1960s. These studies examined the efficacy of different cleaning methods, by comparing not only various techniques for removing dust, but also numerous disinfectants used for the chemicals treatment of environmental contamination. From ‘cleaning’ point of view, the pharmaceutical units may be regarded as consisting of two distinct types of environment; ‘sterile’ production areas where injections, vaccine’s and other products are prepared from living microorganisms and ‘non-sterile’ production areas where medicines for internal and external use are made in bulk. Difference between the two types environment have become more marked in recent years, largely as a result of the introduction of clean room technology to sterile production area, microorganism may be introduced to the environment via the raw materials, air and water supplies, packing components, processing or cleaning equipment; or by the staff who work in the unit. Aerobic spore bearers are generally accepted as being inevitable contaminants in many supplies, and hence are often found in pharmaceutical units. Likewise, unless protective clothing is worn by the staff, large numbers of Gram positive cocci may be shed from the skin into the manufacturing environment. It is known that contaminated medicines may serve as a vehicle for the spread of cross-infection when used in the wards. In particular, certain disinfectants are susceptible to contamination by Pseudomonas aeruginosa, when incorrectly made or used and ironically made or used and ironically these may contributed to, rather than reduce the microbial flora of the area to be cleaned. Studies in pharmaceutical units have shown that floors and other horizontal surfaces are often heavily contaminated with microorganisms. Washing these surfaces with hot water and soap alone removes a large proportion of contaminants; washing with disinfectants can result in a significant improvement. Surfaces such as walls and ceilings generally have a much smaller microbial population if kept in good repair and hence requires cleaning much less often than floors. Several studies have suggested that surfaces cleaned too vigorously and frequently may become thereby encouraging environmental contamination. The number of range of microorganisms that are resistant to one or more antibiotic is a continual and increasing problem, especially in hospitals. With the emergency of pathogens such as methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin resistant enterococci (VRF) and gram-negative bacteria, for example, Pseudomonas aeruginosa and Acinetobacter species which are resistant to multiple antibiotics, there is an increasing need for effective disinfection. This is especially true in clean rooms where pharmaceuticals, dressing and instrument are prepared and sterilized. In general, there were no disinfection policies in place for the use and concentration of the disinfectants available since last many years. The situation has changed markedly and now many pharmaceutical industries have such policies, but implements are still frequently unsatisfactory.
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Clearly, there is a need for pharmaceuticals unit to understand the role and uses of disinfectants, the factor that influences their activity, the possibility that microbial resistance can arise and the relevance, if any of rotation of disinfectants. Disinfectant: an agent that frees from infection; usually, a chemical agent which destroys disease germs or other harmful microorganisms; commonly used as substances applied to inanimate objects. While this definition has been in general use for many years, it is sometimes misused, as in “skin disinfectant”; “skin antiseptic” is more appropriate in describing a germicide for use on the body or inanimate surfaces. One another definition employed for disinfectant is- an agent that frees from infection; usually a chemical agent which destroys disease germs or other microorganisms or inactivates viruses. The official definition of the word “Disinfection” adopted by the American public health Association (1950) is as follows: “Disinfection – killing of pathogenic agents by chemical or physical means directly applied”. A sound cleaning and sanitization program is needed for controlled environment used in the manufacture of pharmacopeial articles to prevent the microbial contamination of these products. Sterile drug products may be contaminated via their pharmaceutical ingredients, process water, packaging components, manufacturing environment processing equipment and manufacturing operators. Current good manufacturing practices (cGMP) emphasize the size, design, construction and location of buildings and construction materials and the appropriate material flow to facilitate cleaning, maintenance and proper operations for the manufacture of drug products. When disinfectants are used in a manufacturing environment, care should be taken to prevent the drug product from becoming contaminated form chemical disinfectant as a result of the inherent toxicity of the disinfectants. The requirement for aseptic processing include readily cleanable floors, walls and ceilings that have smooth and non porous surfaces; particulate, temperature and humidity controls; cleaning and disinfecting procedures to produce and maintain aseptic conditions. The cleaning and sanitization program should achieve cleanliness standards, control microbial contamination of products, and be designed to prevent chemical contamination of pharmaceutical ingredients; products contact surfaces or equipments, packaging materials and ultimately the drug products. These principles also apply to non sterile dosage forms where the microbial contamination is controlled by the selection of appropriate pharmaceutical ingredients, utilities, manufacturing environments, sound equipments cleaning procedures, products especially formulated to control water activity, inclusion of suitable preservatives, and products packaging design. In addition to disinfectants, antiseptics are used to decontaminate human skin and exposed tissues and may be used by personnel prior to entering the manufacturing area. Chemical sterilants mat be used to decontaminate surfaces in manufacturing and sterility testing areas. Furthermore, sterilants may be used for the sterilization of pharmacopeial articles, and UV irradiation may be used as a surface sanitizer.

This general introduction will discuss the selection of suitable chemical disinfectants and antiseptics:
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The demonstration of their bactericidal, fungicidal and sporicidal efficacy; The application of disinfectant in the sterile pharmaceutical manufacturing area; Regulations and safety consideration.

Definitions:Antiseptics: An agent that inhibits or destroys microorganisms on living tissue including skin, oral cavities and open wounds. Chemical disinfectants: A chemical agent used on inanimate surfaces and objects to destroy infectious fungi, viruses and bacteria but not necessarily their spores. Sporicidal and antiviral agents may be considered a special class of disinfectants. Disinfectants are often categorized as high level, intermediate level, and low level by medically oriented groups based upon their efficacy against various microorganisms. Decontamination: The removal of microorganisms by disinfection or sterilization. Disinfectants: A chemical or physical agent that destroys or removes vegetative forms of harmful microorganisms when applied to a surface. Sanitizing agents: An agent which reduce microbes on inanimate surfaces, the number of all forms of microbial life including fungi, viruses and bacteria. Sporicidal agents: An agent that destroys bacterial and fungal spores when used in sufficient concentration for a specified contact time. It is expected to kill all vegetative microorganisms. Sterilants: An agent that destroys all forms of microbial life including fungi, viruses and all forms of bacteria and their spores. Sterilants are liquid or vapor-phase agents.

Microorganisms differ greatly in their resistance to disinfecting agents. The order of resistance of clinically significant microorganisms to chemical disinfectants from most to least resistant.
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Table-1. The resistance of some clinically important microorganisms to chemical disinfectants (Listed in order of decreasing resistance). 1) Type of Microorganisms Examples

1) Bacterial spores 2) Mycobacteria 3) Non lipid-coated viruses 4) Fungal spores and vegetative molds 5) Vegetative bacteria

Bacillus subtilis and Clostridium sporogenes. Mycobacterium tuberculosis. Poliovirus and Rhinovirus. Trichophyton, Crytococcus and Candida sp.and yeasts. Pseudomonas.aeruginosa, Staphylococcus aureus and Salmonella etc.

6) Lipid-coated viruses

Herpes simplex virus, Hepatitis B virus, HIV

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C L A S S I F I C AT I O N O F D I S I N F E C TA N T S
Chemical disinfectant is classified by their chemical type. This includes Aldehydes, alcohol, halogens, and per-oxides, quaternary ammonium compounds and phenolic compounds. Table-2: General classification of Antiseptics, Disinfectants and Sporicidal Agents. Chemical Entity 1) Aldehydes 2) Alcohol Classification Sporicidal agents Examples 2% Gluteraldehyde

General purpose disinfectants, antiseptic, 70% Isopropyl antiviral agents. alcohol 70% Alcohol. 3) Chlorine and Sporicidal agents 0.5% Sod. Sod.hypochlorite hypochlorite 4) Phenolics General purpose disinfectant 500µg per gm chloroxylenol. 5) Ozone Sporicidal agent 8% gas by weight 6) H2O2 Vapor phase sterilant, liquid sporicidal 4µg per gm H2O2 agent, antiseptic vapor 10-25% solution, 3% solution. 7) Substituted Antiseptic agents 0.5% Chlorhexidine Diguanides gluconate. 8) Per-acetic acid Liquid sterilant, vapor phase sterilant 0.2% Per-acetic acid, 1µg per gm per-acetic acid. 9) Ethylene oxide Vapor-phase sterilnt 660µg per gm ethylene oxide. 10) Quaternary General purpose disinfectant, antiseptic 200µg per gm Amm. Benzalkonium compounds chloride 11) β- Propiolactonic Sporicidal agent 100µg per gm βPropiolactone.

Under the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA), the Environment protection agency (EPA) registers chemical disinfectant marketed in the United States and requires manufacturers to supply product information on the use dilution, and type of microorganisms killed and the necessary contact time. Certain liquid chemicals sterilizers intended for use on critical or semi critical medical device are defined and regulated by the U.S Food and Drug Administration (FDA).Selection of an antiseptic for hand and surgical site disinfection:

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SELECTION OF AN ANTISEPTIC FOR HAND AND SURGICAL SITE DISINFECTION: Hands and surgical sites are disinfected in a hospital setting to reduce the resident flora and to remove transient flora (e.g. – Streptococcus pyogenes and methicillin resistant Staphylococcus aureus and Pseudomonas aeruginosa) that have been implicated in hospital- associated infection. Use of antiseptic to disinfectant hands has been shown to be more effective than soap and water in reducing the counts of bacteria on the skin; repeated antiseptic use further reduces these counts. These principles may be applied to clean room operation in the pharmaceutical industry.

MECHANISM OF DISINFECTANT ACTIVITY Target
1) 2) 3) 4) Cell wall Cytoplasmic membrane, action on membrane potential Membrane enzymes, action on electron transport chain Action on ATP Action on enzymes with –SH group

Disinfectants
Formaldehyde, hypochlorite’s and mercurials Anilides and hexachlorophene Hexachlorophene

Chlorhexidine and ethylene oxide Ethylene-oxide, Gluteraldehyde, Hydrogen peroxide, hypochlorite’s, Iodine and mercurial’s 5) Action on general membrane Alcohols, Chlorhexidine and quaternary permeability ammonium compounds. 6) Cell contents, general coagulation Chlorhexidine, Aldehydes, Hexachlorphene and quaternary ammonium compounds 7) Ribosome’s Hydrogen peroxide and mercurial’s 8) Nucleic acids Hypochlorite’s 9) Thiol-groups Ethylene oxide, gluteraldehyde, hydrogen peroxide, hypochlorite’s and mercurial’s 10) Amino groups Ethylene oxide, gluteraldehyde and hypochlorite’s 11) General oxidation Ethylene oxide gluteraldehyde and hypochlorite’s

MICROBIAL RESISTANCE TO DISINFECTANTS:
The development of microbial resistance to antibiotics is a well described phenomenon. The development of microbial resistance to disinfectant is less likely, as disinfectants are more powerful biocidal against low population of microorganisms usually not growing actively, so the selective pressure for the development of resistance is less profound. However, the most frequently isolated microorganism from an environmental monitoring program may be periodically subjected to use dilution testing with the agents used in the disinfection program to confirm their susceptibility.
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DISINFECTANT CHALLENGE TESTING
The EPA requires companies that register public health antimicrobial pesticide products including Disinfectants, Sanitization agents, Sporicidal agents and sterilants to ensure the safety and effectiveness of their products before they are sold or distributed. Companies registering those products must address the chemical composition of their product include toxicology data to document that their product is safe? If used as directed on the label, include efficacy data to document their claims of effectiveness against specific organisms and to support the directions for use provided in the labeling and provide labeling that reflects the required elements for safe and effective use. While these directions provide valuable information, they may not be helpful in terms of the products use as disinfectants in a manufacturing environment. To demonstrate the efficacy of a disinfectant with a pharmaceutical manufacturing environment, it may be deemed necessary to conduct the following tests;
1) In Use-dilution method: - Screening disinfectant for their efficacy at various

concentration and contact times against a wide range of standard test organisms and environment isolates.

2) Surface

challenge test: - Using standard test micro-organisms and test microorganisms that are typical environmental isolates, applying disinfectant to surfaces at the selected used concentration with specified contact time, and determining the log reduction of the challenge microorganisms.

3) A statistical comparison of the frequency of isolation and numbers of microorganisms isolated prior to and after the implementation of a new disinfectant. This is considered necessary because critical process steps like disinfection of aseptic processing areas as required by EMP regulations need to be validated, and the EPA registration requirements do not address how disinfectants are used in the pharmaceutical, biotechnology, and medical device industries. For the surface challenge tests, the test organisms are enumerated using swabs, surface rinse, or contact plate methods. Neutralizers that inactivate the disinfectant should be included in either the diluents or microbiological media used for microbial enumeration or both. TABLE: - NEUTRALIZING AGENTS FOR COMMON DISINFECTANT Disinfectant 1) 2) 3) 4) 5) Alcohols Gluteraldhyde Sodium hypochlorite’s Chlorhexidine Mercuric chloride 6) Quaternaryammonium Neutralizing Agents Dilution or polysorbate 80 Glycine and Sodium bisulfate Sodium thiosulfate Poltsorbate 80 and lecithins Thioglycolic acid Polysorbate 80 and lecithins
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compounds Universal neutralizer’s broths may be formulated to contain a range of neutralizing agents. For example, Dey-Engley (D/E) broth contains 0.5% polysorbate 80, 0.7% lecithin, 0.1% Sodium thioglycolate, 0.6% Sodium thiosulfate, 0.25% Sodium biosulfate, 0.5% tryptone, 0.25% yeast extract, and 1.0% dextrose; letheen broth contains 0.5% polysorbate 80, 0.07 % lecithin, 1.0% peptamin, 0.5% beef extract, and 0.5% Sodium chloride; and TryptoneAzolecithin-Tween (TAT) broth base contains 4.0%(v/v) polysorbate 20, 0.5% lecithin, and 2.0% Tryptone. In practice, sufficient organisms need to be inoculated on a 2 inch× 2 inch square of the surface being decontaminated, i.e. a coupon, to demonstrate at least a 2 (bacterial spore) to 3 (for vegetative bacteria) log reduction during a predetermined contact time ( i.e. 10 minutes over and above the recovery observed with a control disinfectant application). The efficacy of the neutralizers and their ability to recover inoculated microorganisms from the material should be demonstrated during the use dilution or surface challenge studies. Points to be remember are that disinfectants are less effective against the higher numbers of microorganisms used in laboratory challenge tests than they are against the numbers that are found in clean rooms; that inoculate from the log growth phase that are typically employed in laboratory tests are more resistant, with the exception of spores formed during the static phase, than those from a static or dying culture or stressed organisms in the environment; and that microorganisms may be physically removed during actual disinfectant application in the manufacturing area. Although not all inclusive, typical challenge organisms that may be employed are listed in Table;

TYPICAL CHALLENGE ORGANISMS

AOAC Challenge Organisms Isolates Bactericide: Escherchia. coli Staphylococcus. aureus Pseudomonas aeruginosa Fungicide: Penicillium. Chrysogenum

Typical Environmental Bactericide: Micrococcus. luteus Streptococcus. Epidermis Coynebacteria.jeikeium Fungicide: C.albicans, Aspergillus niger Penicillium. Chrysogenum

Sporicide: Bacillus. Subtilis

Sporicide: Bacillus. Sphaericus Bacillus.thuriengiensis

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Because a wide range of different materials of construction are used in clean rooms and other controlled areas, each material needs to be evaluated separately to validate the efficacy of a given disinfectant. Table contains a list of common materials used in clean room construction. Table: - Typical surfaces to be decontaminated by disinfectants in a Pharmaceutical manufacturing Area. Materials 1) Stainless steel 2) Glass 3) Plastic, Vinyl 4) Plastic, Polycarbonate 5) Lexan(Plexiglas’s) 6) Epoxy-Coated gypsum 7) Fiber glass-Reinforced plastic 8) Terrazzo tiles Application Work surfaces, filling equipments and tanks. Windows and vessels. Curtains. Insulation coating. Shields. Walls and Ceilings. Wall paneling. Floors.

DISINFECTION PROGRAM

IN

CLEANING

AND

S A N I T I Z AT I O N

The selection of suitable disinfectants and the verification of their effectiveness in surface challenge testing are critical in the development of a cleaning and sanitization program. Issue associated with the successful implementation of such a program are the development of written procedure, staff training, decisions on disinfectant rotation, institution of application methods and contact times, environmental monitoring to demonstrate efficacy, and personnel safety. Staff involved in disinfection requires training in microbiology, industry practices for cleaning and sanitization, safe handling of concentrated disinfectants, the preparation and disposal of disinfectant, and appropriate application methods. It should be emphasized that the preparation of the correct dilution is critical because many disinfectant failures can be attributed to use of disinfectant solution that are too dilute. Typically disinfectants used in aseptic processing and filling areas are diluted with sterile purified water, and are prepared aseptically. Alternately, the disinfectant may be diluted with purified water, and then sterile filtered to eliminate microorganisms that may potentially persist in a disinfectant. Dilute disinfectants must have an assigned expiration dating justified by effectiveness studies. Because it is theoretically possible that the selective pressure of the continuous use of a single disinfectant could result in the presence of disinfectant could result in the presence of disinfectant- resistant microorganisms in a manufacturing area, in some quarters the rotation of disinfectant has been advocated. However, the literature supports the belief that the exposure of low numbers of microorganism on facility equipment surfaces within a clean room where they are not actively proliferating will not result in the selective pressure that may be seen with the antibiotics. It is prudent to augment the daily use of a bactericidal disinfectant with weekly (or monthly) use of a sporicidal agents is not generally favored because of their tendency to corrode equipment and because of the potential safety issues
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with chronic operator exposure. Other disinfection rotation schemes may be supported on the basis of a review of the historical environment monitoring data. Disinfectants applied on potential product contact surfaces are typically removed with 70% alcohol wipes. The removal of residual disinfectants should be monitored for effectiveness as a precaution against the possibility of product contamination The greatest safety concerns are in the handling of concentrated disinfectants and the mixing of incompatible disinfectants. For example, concentrated sodium hypochlorite solutions (at a concentration of more than 5%) are strong oxidants and will decompose on heating, on contact with acids, and under the influence of light, producing toxic and under the corrosive gases including chlorine. In contrast, dilute solutions (at a concentration of less than 0.5%) are not considered as hazardous. Under no circumstances should disinfectants of different concentration be mixed. Material safety data sheets for all the disinfectants used in a manufacturing area should be available to personnel handling the disinfectant preparation, and personnel must be trained in the proper use of this equipment. Safety showers and eye wash stations must be situated in the work area where disinfectant solutions are prepared. APPLICATIONS OF DISINFECTANTS Disinfectant Alcohols Uses Ethyl or isopropyl alcohol at 70-80% concentration is a good general purpose disinfectant; not effective against bacterial spores. Phenols Effective against vegetative bacteria, fungi and viruses containing liquids, unpleasant odor. Formaldehyde Concentration of 5-8% formalin is a good disinfectant against vegetative bacteria, spores and viruses; known carcinogen; irritating odor. Quaternary Ammonium Cationic detergents are strongly surfaced active; extremely Compounds effective against lipoviruses; ineffective against bacterial spores; may be neutralized by anionic detergents (i.e. soaps). Chlorine Low concentrations (50-500 ppm) are active against vegetative bacteria and most viruses; higher concentrations (2,500 ppm) are required for bacterial spores; corrosive to metal surfaces; must be prepared fresh; laundry bleach (5.25% chlorine) may be diluted and used as a disinfectant. Recommended for general use; effective against vegetative Iodine bacteria and viruses; less effective against bacterial spores; Wescodyne diluted 1 to 10 is a popular disinfectant for washing hands.

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FACTORS AFFECTING DISINFECTION

The following are six primary variables that influence the efficacy of disinfection: 1) Nature of the item to be disinfected: The rougher the surface, the longer the contact time required for disinfection.

2) Number of microorganisms present: The number of microorganism present will lengthen the time for effective disinfection to take place. In general, higher numbers of organisms require more time for disinfection.

3) Resistance of microorganisms: Some microorganisms are more resistant to disinfection than others. The generally accepted order from the most resistance to the least resistant is: bacterial spore, mycobacteria, hydropjillic viruses, fungi, vegetative bacteria, lipid viruses. Disinfecting a spill with a small concentration of bacterial spores will require longer disinfection time than a large concentration of lipid viruses.

4) Type and concentration of disinfectant used: Resistance of microorganisms depends on the type of disinfectant used. A particular microorganism may be more resistant to one type of disinfectant than another. For instance, alcohol (Isopropyl or ethyl) is effective against vegetative bacteria and most lipophilic viruses. Many disinfectants are broad spectrum; that is, effective against all or most forms of microbial life. Some non-broad spectrum disinfectants include phenolics and quaternary ammonium compounds. Some broad spectrum disinfectants include Gluteraldehydes, sodium hypochlorites, and hydrogen peroxides. Alcohols lie somewhere in between these two. The concentration of a particular disinfectant effects disinfection. In most cases, a higher concentration increases microbial killing power and decreases time necessary for disinfection.

However, some disinfectants are not as effective in higher concentrations. Iodophors must be diluted according to the directions on the label; over-diluting or under-diluting may substantially lower the microbial potency. Alcohols used in concentration above 90% are less effective because the water added to dilute the alcohol allows it to penetrate better and reach its target. Optimal concentration range is between 70-90%.
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5) Presence of organic material: The presences of organic soiling matter will compromises disinfection. Blood, blood product, bodily fluids and feces contain significant amounts of proteins, and protein will bind and inactivate some disinfectants or slow their action. Therefore, in the presence of large amounts of protein, a higher concentration of disinfectant and longer contact time will be necessary to achieve maximal disinfection.

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6) Duration of exposure and temperature: Duration of exposure and temperature influences the disinfection process. The longer the duration of exposure, the higher the degree of disinfection achieved. Some disinfectants require a longer contact time to achieve killing, and some microorganisms need longer exposures to be killed. Higher temperature increases the killing power of most disinfectants, where lower temperature may slow the killing power of most disinfectants. .

The following disinfectants were taken up for the Efficacy and Shelf life study: General Description of disinfectants used: 1) Incidur: Incidur is a broad spectrum; Microbiocidal action including HIV, Hepatitis B etc.It shows long residual effect with sustained release. It is manufactured by Henkel Germany. It is purposely used for surface disinfection and fumigation.

Active ingredients in 100 gms solutions:

• •

Glyoxal…………..8.8gm. Gluteral…………..4.5gm.

Range of application:   High risk areas like Operation theatres, Intensive care Unit (ICU), laboratories and drug manufacturing units. Commonly used for general purpose in laboratories and pharmaceuticals units.

Mode of Action:   It interferes with enzymes with –SH groups and Thiol group of proteins. It is also responsible for general oxidation of cellular components in microbial cells.

Recommended concentration and contact time (According to manufacturer): • According to manufacturer (Henkel Germany), Incidur is effective at 1% concentration for 1 hour exposure or contact time.

2) Levermed: Levermed is broad spectrum hand disinfectant. It acts as Bactericidal, Sporicidal, Fungicidal and Virucidal agent. It is manufactured by Universal Healthcare Silvassa.
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It is marketed by Johnson Diversy. It is purposely used for hand disinfection and laboratory practices. Levermed is ready to use prepared hand disinfectant.

Active Ingredients (w/w): • • • • n- popanol………………………50% Iso-propyl Alcohol...……………20% Benzalkonium chloride………...0.5% Emollents.

Range of Application: • Industrial Quality Control Laboratories, Research Institutes, Academics, Drug Manufacturing units.

Mode of Action: • It generally shows action towards membrane permeability. It coagulates cellular components.

Recommended concentration and contact time (According to manufacturer): • According to manufacturer (Universal Healthcare, Silvassa), Levermed is effective at 10 minutes exposure or contact time.

3) Oxivir: Oxivir is broad spectrum environmental cleaner & disinfectant. It is oxygen based Bactericidal, Sporicidal, Virucidal, Fungicidal, Yeasticidal. Oxivir is concentrated oxygen based liquid cleaner disinfectant for cleaning and disinfectant of all water resistant surfaces. It is manufactured by Johnson Diversy. It is effective against Bacteria, Fungi, Yeasts, Virus. It is purposely used for Fumigation, Cleaning and disinfecting equipment. It is compatible with metals and surfaces used in hospitals. However, material compatibility on a small and inconspicuous place is recommended before use. Composition: Hydrogen peroxide Alkyl Benzene sulphonic acid
19

Alcohol ethoxylate (C12-15) 3EO Hydroxyethene-1-1-diphosphonic acid 2- butoxyethanol Feature Advantage Benefits Broad spectrum Microbial kill

Accelarated hydrogen Higher grade disinfectant peroxide Aldehyde free product Cleaner disinfectant No negative hazards

occupational Eco-friendly and user friendly Time and cost effective

and Single shot product

Range of Application:  Oxivir is purposely used in Hospitals, laboratories, Institutes and Drug manufacturing units.

Recommended Concentration and contact time:  According to manufacturer (Johnson Diversy), Oxivir is effective at 2.5% for 5 minute contact time. Mode of Action:  Oxivir shows action on enzymes with –SH group.  Oxivir also cause general oxidation of cellular components. 4) Virex II 256: Virex II 256 is high level instrument & surface disinfectant Bactericidal, Sporicidal, Virucidal, Fungicidal, Yeasticidal. Virex II 256 is a high level disinfectant of surfaces and critical instruments (which come in contact with blood stream or normally sterile areas of the body, such as rigid and flexible surgical instruments) in healthcare application. . It is purposely used for Cleaning and disinfecting equipment. It is compatible with metals and surfaces used in hospitals. However, material compatibility on a small and inconspicuous place is recommended before use. Virex II 256 is blue colored liquid disinfectant manufactured by Johnson Diversy. It is effective against Bacteria, Fungi, Yeasts, Virus.

Composition:
20

    

Didecyl dimethyl ammonium chloride……………………..8.7% n-alkyl dimethyl benzyl ammonium chloride………………8.19% Lauryl amine amine oxide Ethylene diamine tetra acetic acid sodium salt Sodium biocarbonate

Feature

Advantage

Benefits

N-alkyl dimethyl benzyl Combination of most potent Broad spectrum kill ammonium chloride & Didecyl QAC’s dimethyl ammonium chloride Aldehyde free product Cleaner and disinfectant No negative occupational hazard Single shot product Eco friendly and user friendly Time and cost effective

Range of Application:  Virex II 256 is purposely used for instruments disinfection. It is also used for surface disinfection. It is also used for washing and other tiled surfaces. It is compatible with most metals and surfaces used in pharmaceuticals units, Laboratories and Research Institute. Recommended Concentration and contact time:  According to manufacturer (Johnson Diversy), Virex II 256 is effective at 0.4% for 10 minutes contact time or exposure. Mode of Action:  It causes general cell components coagulations.

21

AIMS AND OBJECTIVES
AIM: Assessment of efficacy and shelf life of various disinfectants used in Vaccine formulations Plant by In-Use Dilution Method.

OBJECTIVES

To check the efficacy of various disinfectants under laboratory conditions, stimulating their possible practical use.

 Levermed.  Incidur.  Oxivir.  Virex II 256.

22

M AT E R I A L S A N D M E T H O D O LO GY

23

TESTING THE EFFICACY OF DISINFECTANTS

Materials and Reagents:1) Disinfectants.
2) Microbial Cultures. a) Staphylococcus aureus NCIM 2079. b) Pseudomonas aeruginosa NCIM 2200. c) Bacillus subtilis NCIM 2063. d) Escherchia coli NCIM 2065. e) Candida albicans NCIM 3471. f) Salmonella abony NCIM 2257. g) Environmental Isolates of E. coli. h) Environmental Isolates of Bacillus. i) Environmental Isolates of Staphylococcus aureus.

Glassware and other materials:1) Sterile bottles. 2) Sterile test tubes. 3) Sterile Petri plates. 4) Stopwatch. 5) Test tube stand.
24

6) Inoculating loop. 7) Glass beads.
8) Micro pipette (1000µL). 9) Micro pipette (200µL). 10)Micro pipette tips (1000µL). 11)Micro pipette tips (200µL).

12) 10 mL sterile syringe.
13)0.2µm filters.

14) Anhydrous calcium chloride. 15) Anhydrous magnesium chloride. 16) 10 % dextrose solution. 17) Nutrient broth.
18)Soybean Casein Digest Agar. 19)Dey- Engley neutralizing Broth.

Preparation of solutions:

Preparation of sterile hard water: Dissolve 0.304gm Calcium chloride and 0.065gm anhydrous magnesium chloride in purified water. Make it up to 1 liter. Dispense into glass container and sterilize by autoclaving at 121°c for 30 minutes.

• • •

3) Preparation of 10 % dextrose solution:•

Took 10gm of dextrose in a volumetric flask Add distill water to make up to 100 mL shake well to dissolve completely.

25

Transfer dextrose solution in a clean glass bottle and sterilize by autoclaving at 121ºc for 30 minutes.

3) Preparation of Nutrient broth solution:4) Dissolve 13 gm nutrient broth in 1000 ml water 5) Sterilize by autoclaving at 121ºc for 30 minutes. 6) To each liter of nutrient broth solution add 10 ml sterile dextrose solution for the growth of the organism.

4) Preparation of Disinfectant solution:•

Prepare disinfectant solution as recommendation by manufacturer. After dilution filter the disinfectant solution from 0.2µm filter with the help of sterile 10 ml syringe.

Procedure:1) Preparation of inoculums:-

 Incubate the contents of a freeze dried culture suspended in nutrient broth overnight at 37 ºc ±1 ºc for bacteria and 22.5 ºc ± 2.5 ºc for fungus.
 Inoculate the incubated culture onto nutrient agar slopes. These slants can be stored

up to 4 months at 2-8 ºc.  Subculture from agar slopes in to 10 mL of growth medium. Incubate at 37 ± 1ºc for ± 2hr for bacteria and 22.5 ºc ± 2.5 ºc for 120 hrs for fungus.

 Subculture from the medium into fresh medium, using an inoculating loop. Inoculate at 37 ºc ± 1 ºc for 24 ± 2 hr for bacteria and 22.5 ºc ± 2.5 ºc for 120 hrs for fungus.  Repeat the above step daily. For the test procedure use only those cultures which have been sub culture for at least 5 times and not more than 14 times.  Centrifuge all cell culture until cells are compact, and remove the supernatant with a sterile pipette.
 Re-suspend the test organism in 10 mL of growth medium and shake well for 1

minute with few sterile glass beads.
26

 Enumerate the inoculums before testing using 10 fold dilutions and pour plate technique on SCDA plate at 30-35˚C for 48 hrs for bacteria and 22.5 ± 2.5˚C for 120 hrs for fungus.

Acceptance criteria:The number subsequently counted must represents not less than 2×108 or not more than 2×109 organisms per ml. Retain 1 dilutions as control. Test Procedure: Add 3 mL diluted disinfectant solution to a capped sterile test tube.  Start a timing device, immediately inoculate disinfectant solution with 1 mL of culture and mix by swirling.
 After appropriate contact time (as recommended by manufacturer), subculture 1 drop

into each of 5 tubes containing Dey-Engley broth.  To ensure delivery of 0.02 mL into first tube of Dey- Engley broth at exactly time, it will be necessary to withdraw a suitable amount from the disinfectant test mixture shortly before hand. This must be immediately preceded by vortexing.  Mix the contents of all tubes of Dey- Engley broth by vortexing. Inoculate at 37 ºc ± 1ºc for 48 hrs for bacteria and 22.5 ºc ± 2.5 ºc for 120 hrs for fungus.  To observe the recovery of microorganisms, after incubation take 1mL from each Dey-Engley broth tubes, add into sterile petri plates and pour 20 mL SCDA (45 ºc) in to each plate.  Mix well the contents of plate. Incubate at 37 ± 1 ºc for 48 hrs for bacteria and 22.5 ±2.5 ºc for 120 hrs for fungus.

Acceptance Criteria: The test passes if there is no apparent growth in at least three out of five Dey-Engley broth tubes.

Testing of control:-

1) Dey-Engley broth contamination:Incubate one un-inoculated tube of Dey-Engley broth at 37 ± 1ºc for 48 hrs for bacteria and 22.5 ºc ± 2.5 ºc for 120 hrs for fungus and examine for growth. No growth should occur. 2) Disinfectant contamination:27

To 1 tube of Dey-Engley broth, add 0.02mL of disinfectant solution. Inoculate at 37 ± 1ºc for 48 hrs for bacteria and 22.5 ± 2.5 ºc for fungus and examine for growth. If growth occurs, the test is considered invalid. 3) Fertility test:To 1 tube of Dey-Engley broth, add 1.0 mL of 107 dilution of culture and incubate at 37 ±1 ºc for 48 hrs for bacteria and 22.5 ± 2.5 ºc for 120 hrs for fungus and examine for growth. If no growth occurs, the test is considered invalid.

4) In activator efficacy:To 1 tube of Dey-Engley broth, add 0.02 mL of disinfectant solution and 1.0 mL of 107 dilution of the culture at 37 ± 1ºc for 48 hrs for growth. If no growth occurs, the test is considered invalid.

Purpose of Using Dey-Engley neutralizing Broth: Dey-Engley neutralizing broth is a universal neutralizer broth. Dey-Engley (D/E) broth contains 0.5% polysorbate 80, 0.7% lecithin, 0.1% Sodium thioglycolate, 0.6% Sodium thiosulfate, 0.25% Sodium biosulfate, 0.5% tryptone, 0.25% yeast extract, and 1.0% dextrose; letheen broth contains 0.5% polysorbate 80, 0.07 % lecithin, 1.0% peptamin, 0.5% beef extract, and 0.5% Sodium chloride; and Tryptone-Azolecithin-Tween (TAT) broth base contains 4.0%(v/v) polysorbate 20, 0.5% lecithin, and 2.0% Tryptone. These ingredients are acts as a universal in activator for the inhibitory compounds. So DeyEngley broth is used to recover microbial inoculum after disinfectant exposure.

28

RESULTS D I S I N F E C TA N T S E F F I C ACY R E S U LT S

29

30

OBSERVATIONS

Disinfectant
Manufacturer Manufacturing Date Expiry Date Batch no Recommended contact time Concentration

Incidur.
Universal Healthcare Silvasa. March 2008. Feb 2010. 803 60 minutes. 1% v/v

31

Disinfectant Microbial culture

Contact time (minutes) 30 minutes 60minutes 90 minutes

Dey-Engley broth tubes
Tube 1 Tube 2 Tube 3 Tube 4 Tube 5

Incidur

S.aureus

√ × × √ √ √ √ × × √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √

√ × × √ √ √ √ × × √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √

√ × × √ √ √ √ × × √ √ √ √ √ √ √ √ × √ √ × √ √ √ √ √ √

√ × × √ × × √ × × √ √ √ √ × × √ √ × √ × × √ √ √ √ √ √

√ √ × √ × × √ √ × √ √ √ √ × × √ × × √ × × √ √ √ √ √ √

P.aeruginosa

30 minutes 60 minutes 90 minutes

Bacillus subtilis

30 minutes 60 minutes 90 minutes

Escherchia coli

30 minutes 60 minutes 90 minutes

Candida albicans

30 minutes 60 minutes 90 minutes

Salmonella abony

30 minutes 60 minutes 90 minutes

E. I E. coli

30 minutes 60 minutes 90 minutes

E.I B. subtilis

30 minutes 60 minutes 90 minutes

E. I S. aureus

30 minutes 60 minutes 90 minutes
32

Sign: √ = Growth.
× = No growth.

Test Controls:S.NO Control 1) 2) 3) 4) Dey- Engley Contamination Disinfectant Contamination Inactivator Control Fertility Control × × √ √

Sign: √ = Growth.
× = No growth.

Enumeration of Microbial Inoculums on SCDA:S.No Microbial culture Cfu/mL 106 1) 2) 3) 4) 5) 6) 7) 8) 9) Staphylococcus aureus Pseudomonas aeruginosa Bacillus subtilis Escherchia coli Cabdida albicans Salmonella abony Environmental isolates of Escherchia coli Environmental isolates of Bacillus subtilis 120 TNTC TNTC TNTC TNTC 140 TNTC TNTC 107 20 75 67 110 65 16 15 25 26 108 2 2 3 2 3 2 3 3 2 109 × × × × × × × × × 1010 × × × × × × × × ×

Environmental isolates of Staphylococcus TNTC
33

aureus

Recovery:S.NO Microbial culture Contact time Cfu/mL(Dey –Engley tube) Tube 1
TNTC TNTC × TNTC TNTC TNTC TNTC × × TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC

Tube 2
TNTC TNTC × TNTC TNTC TNTC TNTC × × TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC

Tube 3
TNTC TNTC × TNTC TNTC TNTC TNTC × × TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC × TNTC TNTC ×

Tube 4
TNTC TNTC × TNTC

Tube 5
TNTC × × TNT C × × TNTC TNTC × TNTC TNTC TNTC TNTC × × TNTC × × TNTC × ×

1)

Staphylococcus aureus

30 minutes 60 minutes 90 minutes

2)

Pseudomonas aeruginosa

30 minutes 60 minutes 90 minutes

× × TNTC × × TNTC TNTC TNTC TNTC × × TNTC TNTC × TNTC × ×

3)

Bacillus subtilis

30 minutes 60 minutes 90 minutes

4)

Escherchia coli

30 minutes 60 minutes 90 minutes

5)

Candida albicans

30 minutes 60 minutes 90 minutes

6)

Salmonella abony

30 minutes 60 minutes 90 minutes

7)

E.I E.coli

30 minutes 60 minutes 90 minutes

34

8)

E.I Bacillus subtilis

30 minutes 60 minutes 90 minutes

TNTC TNTC TNTC TNTC TNTC TNTC

TNTC TNTC TNTC TNTC TNTC TNTC

TNTC TNTC TNTC TNTC TNTC TNTC

TNTC TNTC TNTC TNTC TNTC TNTC

TNTC TNTC TNTC TNTC TNTC TNTC

9)

E.I aureus

Staphylococcus 30 minutes
60 minutes 90 minutes

*TNTC- Too numerous to count.

× = No growth.

OBSERVATIONS
Disinfectant Manufacturer Manufacturing Date Expiry Date Batch no Recommended contact time Concentration Levermed. Universal Healthcare Silvasa. March 2008. Feb 2010. 803 5 minutes. Not available(ready to use)

35

Disinfectant Levermed

Microbial culture

S.aureus P.aeruginosa Bacillus sutilis Escherchia coli Candida albicans Salmonella abony E. I E. coli E.I B. subtilis E. I S. aureus

Contact time (minutes) 3 minutes 5 minutes 7 minutes 3minutes 5 minutes 7 minutes 3 minutes 5 minutes 7 minutes 3 minutes 5 minutes 7 minutes 3 minutes 5 minutes 7 minutes 3 minutes 5 minutes 7 minutes 3 minutes 5 minutes 7 minutes 3 minutes 5 minutes 7 minutes 3 minutes 5 minutes 7 minutes

Dey-Engley broth tubes Tube 1 Tube 2 Tube 3
× × × √ √ √ × × × √ √ √ √ √ √ √ √ √ √ √ √ × × × √ √ √ × × × √ √ √ × × × √ √ √ √ √ √ √ √ √ √ √ √ × × × √ √ √ × × × √ √ √ × × × √ √ √ √ √ √ √ √ √ √ √ √ × × × √ √ √

Tube 4
× × × √ √ √ × × × √ √ √ √ √ × × × × √ √ √ × × × √ √ √

Tube 5
× × × × × × × × × √ √ × √ √ × × × × × √ √ × × × √ √ √

Sign: √ = Growth. × = No growth. Test Controls:
S.NO Control 1) 2) 3) 4) Dey- Engley Contamination Disinfectant Contamination Inactivator Control Fertility Control × × √ √

Sign: √ = Growth.
× = No growth.
36

Enumeration of Inoculums on SCDA:
S.No Microbial culture 106 1) 2) 3) 4) 5) 6) 7) 8) 9) Staphylococcus aureus Pseudomonas aeruginosa Bacillus subtilis Escherchia coli Cabdida albicans Salmonella abony
200 50 TNTC 240 TNTC TNTC

Cfu/mL 107
19 9 60 16 22 18 25 25 26

108
3 2 3 2 3 3 2 2 3

109
× × × × × × × × ×

1010
× × × × × × × × ×

102 Environmental isolates of Escherchia coli 100 Environmental isolates of Bacillus subtilis Environmental isolates of Staphylococcus 103 aureus

Recovery:S.NO Microbial culture 1) Staphylococcus aureus Contact time (minutes) 3 minutes 5 minutes 7 minutes 3 minutes 5 minutes 7 minutes 3 minutes 5 minutes 7 minutes
37

Tube 1
× × × TNTC TNTC TNTC × × ×

Cfu/mL(Dey –Engley tube) Tube 2 Tube 3 Tube 4 Tube 5
× × × TNTC TNTC TNTC × × × × × × TNTC TNTC TNTC × × × × × × TNTC TNTC TNTC × × × × × × × × × × × ×

2)

Pseudomonas aeruginosa Bacillus subtilis

3)

4)

Escherchia coli

3 minutes 5 minutes 7 minutes

TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC × × × TNTC TNTC TNTC

TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC × × × TNTC TNTC TNTC

TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC × × × TNTC TNTC TNTC

TNTC TNTC TNTC TNTC TNTC × TNTC TNTC TNTC TNTC TNTC TNTC ×

TNTC TNTC × TNTC TNTC × ×

5)

Candida albicans

3 minutes 5 minutes 7 minutes

6)

Salmonella abony

3 minutes 5 minutes 7 minutes

× × TNTC TNTC ×

7)

E.I E.coli

3 minutes 5 minutes 7 minutes

8)

E.I Bacillus subtilis

3 minutes 5 minutes 7 minutes

× × × TNTC TNTC TNTC × TNTC TNTC TNTC

9)

E.I Staphylococcus 3 minutes aureus 5 minutes 7 minutes

*TNTC- Too numerous to count, × = No growth.

38

OBSERVATIONS

Disinfectant
Manufacturer Manufacturing Date Expiry Date Batch no Recommended contact time Concentration

OXIVIR.
Johnson Diversy. August 2008. August 2010. 804 5 minutes. 2% v/v

39

Disinfectant Disinfectant

Microbial culture

Contact time (minutes) 3 minutes 5 minutes 7 minutes

Dey-Engley broth tubes Tube 1
× × × × × × × × × × × × × × × × × × × √ √ √ √ √ √ √

Tube 2
× × × × × × × × × × × × × × × × × × × √ × √ √ × √ ×

Tube 3
× × × × × × × × × × × × × × × × × × × × × × × × √ ×

Tube 4
× × × × × × × × × × × × × × × × × × × × × × × × × ×

Tube 5
× × × × × × × × × × × × × × × × × × × × × × × × × ×

Oxivir

S.aureus

P.aeruginosa

3 minutes 5 minutes 7 minutes

Bacillus subtilis

3 minutes 5 minutes 7 minutes

Escherchia coli

3 minutes 5 minutes 7 minutes

Candida albicans

3 minutes 5 minutes 7 minutes

Salmonella abony

3 minutes 5 minutes 7 minutes

E. I E. coli

3 minutes 5 minutes 7 minutes

E.I B. subtilis

3 minutes 5 minutes 7 minutes

E. I S. aureus

3 minutes 5 minutes
40

Sign: √ = Growth,× = No growth. Test Controls:
S.NO Control 1) 2) 3) 4) Dey- Engley Contamination Disinfectant Contamination Inactivator Control Fertility Control × × √ √

Sign: √ = Growth, × = No growth.

Enumeration of test inoculums on SCDA:
S.No Microbial culture Cfu/mL 106 1) 2) 3) 4) 5) 6) 7) 8) 9) Staphylococcus aureus Pseudomonas aeruginosa Bacillus subtilis Escherchia coli Cabdida albicans Salmonella abony Environmental isolates of Escherchia coli Environmental isolates of Bacillus subtilis
65 57 115 68 60 TNTC 75 102

107
20 75 67 110 65 16 15 25 26

108
2 2 3 2 3 2 3 3 2

109
× × × × × × × × ×

1010
× × × × × × × × ×

Environmental isolates of Staphylococcus TNTC aureus
41

Recovery:
S.NO Microbial culture Contact time (minutes) 3 minutes 5 minutes 7 minutes 2) Pseudomonas aeruginosa 3 minutes 5 minutes 7 minutes 3) Bacillus subtilis 3 minutes 5 minutes 7 minutes 4) Escherchia coli 3 minutes 5 minutes 7 minutes 5) Candida albicans 3 minutes 5 minutes 7 minutes Cfu/mL(Dey –Engley tube) Tube 1 × × × × × × × × × × × × × × × Tube 2 × × × × × × × × × × × × × × × Tube 3 × × × × × × × × × × × × × × × Tube 4 × × × × × × × × × × × × × × × Tube 5 × × × × × × × × × × × × × × ×

1)

Staphylococcus aureus

42

6)

Salmonella abony

3 minutes 5 minutes 7 minutes

× × × TNTC TNTC TNTC TNTC TNTC × TNTC TNTC ×

× × × TNTC TNTC × TNTC × × TNTC × ×

× × × × × × × × × TNTC × ×

× × × × × × × × × × × ×

× × × × × × × × × × × ×

7)

E.I E.coli

3 minutes 5 minutes 7 minutes

8)

E.I Bacillus subtilis

3 minutes 5 minutes 7 minutes

9)

E.I Staphylococcus 3 minutes aureus 5 minutes 7 minutes

*TNTC- Too numerous to count,

× = No growth.

OBSERVATIONS
43

Disinfectant
Manufacturer Manufacturing Date Expiry Date Batch no Recommended contact time Concentration

OXIVIR.
Johnson Diversy. August 2008. August 2010. 804 5 minutes. 2.5% v/v

Disinfectant

Microbial culture

Contact time (minutes) 3 minutes 5 minutes 7 minutes

Dey-Engley broth tubes Tube 1
× × × × × × × × × × × × × ×

Tube 2
× × × × × × × × × × × × × ×

Tube 3
× × × × × × × × × × × × × ×

Tube 4
× × × × × × × × × × × × × ×

Tube 5
× × × × × × × × × × × × × ×

Oxivir

S.aureus

P.aeruginosa

3 minutes 5 minutes 7 minutes

Bacillus subtilis

3 minutes 5 minutes 7 minutes

Escherchia coli

3 minutes 5 minutes 7 minutes

Candida albicans

3 minutes 5 minutes

Test Controls:
S.NO Control
44

1) 2) 3) 4)

Dey- Engley Contamination Disinfectant Contamination Inactivator Control Fertility Control

× × √ √

Sign: √ = Growth, × = No growth. Enumeration:S.No Microbial culture Cfu/mL 106 1) 2) 3) 4) 5) 6) 7) 8) 9) Staphylococcus aureus Pseudomonas aeruginosa Bacillus subtilis Escherchia coli Cabdida albicans Salmonella abony Environmental isolates of Escherchia coli Environmental isolates of Bacillus subtilis 65 57 115 68 60 TNTC 75 102 107 20 75 67 110 65 16 15 25 26 108 2 2 3 2 3 2 3 3 2 109 × × × × × × × × × 1010 × × × × × × × × ×

Environmental isolates of Staphylococcus TNTC aureus

Recovery:45

S.NO Microbial culture

Contact time (minutes)
30 minutes 60 minutes 90 minutes

Cfu/mL(Dey –Engley tube) Tube 1 × × × × × × × × × × × × × × × × × × × × × × × × Tube 2 × × × × × × × × × × × × × × × × × × × × × × × × Tube 3 × × × × × × × × × × × × × × × × × × × ×
×

Tube 4 × × × × × × × × × × × × × × × × × × × ×
×

Tube 5 × × × × × × × × × × × × × × × × × × × ×
×

1)

Staphylococcus aureus

2)

Pseudomonas aeruginosa

30 minutes 60 minutes 90 minutes

3)

Bacillus subtilis

30 minutes 60 minutes 90 minutes

4)

Escherchia coli

30 minutes 60 minutes 90 minutes

5)

Candida albicans

30 minutes 60 minutes 90 minutes

6)

Salmonella abony

30 minutes 60 minutes 90 minutes

7)

E.I E.coli

30 minutes 60 minutes 90 minutes

8)

E.I Bacillus subtilis

30 minutes 60 minutes 90 minutes

× × ×

× × ×

× × ×

46

9)

E.I Staphylococcus 30 minutes aureus
60 minutes 90 minutes

TNTC

× × ×

× × ×

× × ×

× × ×

× ×

*TNTC- Too numerous to count, × = No growth.

47

OBSERVATIONS

Disinfectant
Manufacturer Manufacturing Date Expiry Date Batch no Recommended contact time Concentration

VIREX II 256.
Johnson Diversy. November 2008. Novemner2010. 804 10 minutes. 0.3% v/v

48

Disinfectant

Microbial culture

Contact time (minutes) 5 minutes 10 minutes 15 minutes

Dey-Engley broth tubes Tube 1 × × × × × × × × × × × × × × × × × × × × × √ √ × √ √ × Tube 2 × × × × × × × × × × × × × × × × × × × × × × × × √ × × Tube 3 × × × × × × × × × × × × × × × × × × × × × × × × × × × Tube 4 × × × × × × × × × × × × × × × × × × × × × × × × × × × Tube 5 × × × × × × × × × × × × × × × × × × × × × × × × × × ×

Virex II 256

S.aureus

P.aeruginosa

5 minutes 10 minutes 15 minutes

Bacillus subtilis

5 minutes 10 minutes 15 minutes

Escherchia coli

5 minutes 10 minutes 15 minutes

Candida albicans

5 minutes 10 minutes 15 minutes

Salmonella abony

5 minutes 10 minutes 15 minutes

E. I E. coli

5 minutes 10 minutes 15 minutes

E.I B. subtilis

5 minutes 10 minutes 15 minutes

E. I S. aureus

5 minutes 10 minutes 15 minutes 49

Sign: √ = Growth, × = No growth. Test Controls:
S.NO 1) 2) 3) 4)

Control
Dey- Engley Contamination Disinfectant Contamination Inactivator Control Fertility Control × × √ √

Sign: √ = Growth, × = No growth.

Enumeration of test Inoculums on SCDA:
S.No Microbial culture Cfu/mL 106 1) 2) 3) 4) 5) 6) 7) 8) 9) Staphylococcus aureus Pseudomonas aeruginosa Bacillus subtilis Escherchia coli Cabdida albicans Salmonella abony Environmental isolates of Escherchia coli Environmental isolates of Bacillus subtilis Environmental aureus isolates of Staphylococcus TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC 107 70 39 50 40 45 49 38 42 52 108 2 2 3 2 3 2 3 3 2 109 × × × × × × × × × 1010 × × × × × × × × ×

50

Recovery:S.NO Microbial culture Contact time (minutes)
5 minutes 10 minutes 15 minutes

Cfu/mL(Dey –Engley tube) Tube 1 × × × × × × × × × × × × × × × × × × × × × TNTC TNTC × Tube 2 × × × × × × × × × × × × × × × × × × × × × × × × Tube 3 × × × × × × × × × × × × × × × × × × × ×
×

Tube 4 × × × × × × × × × × × × × × × × × × × ×
×

Tube 5 × × × × × × × × × × × × × × × × × × × ×
×

1)

Staphylococcus aureus

2)

Pseudomonas aeruginosa

5 minutes 10 minutes 15 minutes

3)

Bacillus subtilis

5 minutes 10 minutes 15 minutes

4)

Escherchia coli

5 minutes 10 minutes 15 minutes

5)

Candida albicans

5 minutes 10 minutes 15 minutes

6)

Salmonella abony

5 minutes 10 minutes 15 minutes

7)

E.I E.coli

5 minutes 10 minutes 15 minutes

8)

E.I Bacillus subtilis

5 minutes 10 minutes 15 minutes

× × ×

× × ×

× × ×

51

9)

E.I aureus

Staphylococcus 5 minutes
10 minutes 15 minutes

TNTC

TNTC × ×

× × ×

× × ×

× × ×

TNTC ×

*TNTC- Too numerous to count, × = No growth.

OBSERVATIONS

Disinfectant
Manufacturer Manufacturing Date Expiry Date Batch no Recommended contact time Concentration

VIREX II 256.
Johnson Diversy. November 2008. Novemner2010. 804 10 minutes. 0.4% v/v

52

Disinfectant

Microbial culture

Contact time (minutes) 5 minutes 10 minutes 15 minutes

Dey-Engley broth tubes Tube 1 × × × × × × × × × × × × × × × × × × TNTC TNTC × TNTC TNTC × TNTC TNTC × Tube 2 × × × × × × × × × × × × × × × × × × × × × × × × √ × × Tube 3 × × × × × × × × × × × × × × × × × × × × × × × × × × × Tube 4 × × × × × × × × × × × × × × × × × × × × × × × × × × × Tube 5 × × × × × × × × × × × × × × × × × × × × × × × × × × ×

Virex II 256

S.aureus

P.aeruginosa

5 minutes 10 minutes 15 minutes

Bacillus subtilis

5 minutes 10 minutes 15 minutes

Escherchia coli

5 minutes 10 minutes 15 minutes

Candida albicans

5 minutes 10 minutes 15 minutes

Salmonella abony

5 minutes 10 minutes 15 minutes

E. I E. coli

5 minutes 10 minutes 15 minutes

E.I B. subtilis

5 minutes 10 minutes 15 minutes

E. I S. aureus

5 minutes 10 minutes 15 minutes 53

Sign: √ = Growth, × = No growth. Test Controls:
S.NO 1) 2) 3) 4)

Control
Dey- Engley Contamination Disinfectant Contamination Inactivator Control Fertility Control × × √ √

Sign: √ = Growth, × = No growth.

Enumeration of Test Strains on SCDA:
S. Microbial culture No 1) 2) 3) 4) 5) 6) 7) 8) 9) Staphylococcus aureus Pseudomonas aeruginosa Bacillus subtilis Escherchia coli Cabdida albicans Salmonella abony Environmental isolates of Escherchia coli Environmental isolates of Bacillus subtilis Environmental isolates of Cfu/mL 106 TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC 107 40 39 40 49 45 44 38 42 46 108 2 2 3 2 3 2 3 3 2 109 × × × × × × × × × 1010 × × × × × × × × ×

Staphylococcus TNTC
54

aureus

Recovery:S.NO Microbial culture Contact time (minutes) Cfu/mL(Dey –Engley tube) Tube 1 × × × × × × × × × × × × × × × × × × TNTC TNTC × Tube 2 × × × × × × × × × × × × × × × × × × × × × Tube 3 × × × × × × × × × × × × × × × × × × × ×
×

Tube 4 × × × × × × × × × × × × × × × × × × × ×
×

Tube 5 × × × × × × × × × × × × × × × × × × × ×
×

1)

Staphylococcus aureus

5 minutes 10 minutes 15 minutes

2)

Pseudomonas aeruginosa

5 minutes 10 minutes 15 minutes

3)

Bacillus subtilis

5 minutes 10 minutes 15 minutes

4)

Escherchia coli

5 minutes 10 minutes 15 minutes

5)

Candida albicans

5 minutes 10 minutes 15 minutes

6)

Salmonella abony

5 minutes 10 minutes 15 minutes

7)

E.I E.coli

5 minutes 10 minutes 15 minutes

55

8)

E.I Bacillus subtilis

5 minutes 10 minutes 15 minutes

TNTC TNTC ×
TNTC

× × × × × ×

× × × × × ×

× × × × × ×

× × × × × ×

9)

E.I aureus

Staphylococcus 5 minutes
10 minutes 15 minutes

× ×

*TNTC- Too numerous to count, × = No growth.

Discussion
Disinfection in Pharmaceutical practice includes disinfection of the inanimate surfaces, Instruments, articles used in aseptic processing. The process of disinfection removes most of the harmful organisms but may not kill spores. Routine disinfection of surface has been contaminated by infectious material or agents. Though cleaning of these surfaces may be sufficient in non critical as up to 95% of harmful bacteria can be removed by good cleaning with soap and water. However, it has been seen after cleaning the germs that have been left behind soon begins to grow and accumulate. Therefore, use of disinfection in critical and high risk areas like burn units and ICUs may be justified. A sound cleaning and sanitization program is needed for controlled environment used in the manufacture of pharmacopeial articles to prevent the microbial contamination of these products. Sterile drug products may be contaminated via their pharmaceutical ingredients, process water, packaging components, manufacturing environment processing equipment and manufacturing operators. Current good manufacturing practices (cGMP) emphasize the size, design, construction and location of buildings and construction materials and the appropriate material flow to facilitate cleaning,
56

maintenance and proper operations for the manufacture of drug products. When disinfectants are used in a manufacturing environment, care should be taken to prevent the drug product from becoming contaminated form chemical disinfectant as a result of the inherent toxicity of the disinfectants. The requirement for aseptic processing include readily cleanable floors, walls and ceilings that have smooth and non porous surfaces; particulate, temperature and humidity controls; cleaning and disinfecting procedures to produce and maintain aseptic conditions. The cleaning and sanitization program should achieve cleanliness standards, control microbial contamination of products, and be designed to prevent chemical contamination of pharmaceutical ingredients; products contact surfaces or equipments, packaging materials and ultimately the drug products. These principles also apply to non sterile dosage forms where the microbial contamination is controlled by the selection of appropriate pharmaceutical ingredients, utilities, manufacturing environments, sound equipments cleaning procedures, products especially formulated to control water activity, inclusion of suitable preservatives, and products packaging design. In addition to disinfectants, antiseptics are used to decontaminate human skin and exposed tissues and may be used by personnel prior to entering the manufacturing area. Chemical sterilants mat be used to decontaminate surfaces in manufacturing and sterility testing areas. Furthermore, sterilants may be used for the sterilization of pharmacopeial articles, and UV irradiation may be used as a surface sanitizer.

The effectiveness of a disinfectant depends upon its intrinsic “biocidal” activity; • • • • • • The concentration of the disinfectant Contact time Nature of the surface disinfected Hardness of water used to dilute the disinfectant Amount of the organic material present on the surface Type and number of microorganism present

Under the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA), the Environment protection agency (EPA) registers chemical disinfectant marketed in the United States and requires manufacturers to supply product information on the use dilution, and type of microorganisms killed and the necessary contact time. Certain liquid chemicals sterilizers intended for use on critical or semi critical medical device are defined and regulated by the U.S Food and Drug Administration (FDA).Selection of an antiseptic for hand and surgical site disinfection.

57

Disinfectants work by oxidizing the germs, breaking down their cell walls or by the otherwise deactivating them. Different ingredients or combination of ingredients kill the different germs, therefore, selection of the disinfectant will also depend upon the specific germs required to kill. A broad spectrum disinfectant will be ideally suited. A wide range of disinfectants is available commercially. These includes phenols, Aldehydes, Quaternary ammonium compounds (QACs), chemical based on release of nascent Chlorine, Iodophors, Hydrogen peroxide and combination of various chemicals with detergents. Though many products are commercially available, products and procedures described in trade and other literature may not adequately decontaminate items when the surface has been contaminated with highly resistant or unusual organisms, or if excessive numbers of organisms are present. Disinfection is also affected by the quality of environmental hygiene because dust and other organic matter present on the surfaces directly affect the efficacy of disinfectant. When choosing a disinfectant for specific hospitals use it may be necessary to know the number and the types of organisms likely to be present and the susceptibility of the patients involved. Products and procedures described by manufacturer may therefore not always the suitable for hospital use, and disinfectant chosen for one purpose may not be equally effective if used for another. Ideally the good disinfectant should have a broad antimicrobial spectrum and non–irritating, non-toxic, non-corrosive and least expensive. Selection decision should include effectiveness against potential pathogenic agent, safety to people, impact on equipment and the environment and expense. Although a few phenolic agents exhibit high toxicity and low biodegradability and as a result has been banned, many of the phenolics are still in use throughout the world because of their low cost. Phenol is rapidly corrosive on tissues. Keeping in view its less effectiveness and potential health hazards, better and safe disinfectants are required to place them.

Chlorine has very efficient and rapid action on bacteria, viruses, and most fungi. But on the other hand there are certain disadvantages. Those include:  Its maximum effect is at pH-7, if pH is more than 8, the effect is strongly reduced.  When pH falls down the stability of solution is reduced.  The efficacy is strongly reduced by organic matter – when it reacts with proteins, chloramines are produced which are bactericidal but work slowly.  Chlorine preparations’ mixed with acid creates very toxic vapors.  Strong solution of hypochlorites smell unpleasantly and are toxic–irritant to skin, eyes and mucous memberane.  Very corrosive, especially to aluminum but even stainless steel is also damaged. So if used in modern pharmaceutical buildings for surface disinfection of floors and walls these may corrode the aluminum doors and windows even if there are spashes of chlorine solution on them.
58

Aldehyde containing disinfectants are widely used for the chemical sterilization of heat sensitive equipment as they are non-corrosive to instruments, rubber and plastic articles. But, Aldehydes cause irritation to skin, eyes. They may cause allergy, allergic asthma and allergic contactexema. Inadequate rinsing of Gluteraldehyde causes Colitis. Keeping in view all the above side effects, the health Authorities are considering the development and use of safer alternatives to aldehydes. So it seems that the aldehyde based disinfectant is now discouraged because of its harmful effects on humans, even when its antimicrobial activity is very good.

The Iodophors provide wide germicidal activity and show limited antimicrobial effects in the presence of organic matter. They are corrosive and stain fabric and equipment. Traditionally Iodine compounds have been used for skin disinfection purposes. Scanty literature is found which shows the use of the “Tamed Iodine” compounds for surface disinfection including instruments, walls and floors. Among Quaternary ammonium compounds, two products are active against a wide range of microorganisms including yeast and moulds. These are odorless; colorless; non-corrosive; and highly stable compound over a wide range of pH (3-10.5) and temperature. They are relatively stable in presence of organic matter, have bacteriostatic residual effect on treated surfaces, caused low irritation and low toxicity. In general disinfectants belonging to QACs group were discouraged because of their selective varied antimicrobial efficacy, slow activity against Coliforms and gram negative bacteria, less effectivity on spores and bacteriophages, and their effectivity is affected by hard water salts.

Four locally available disinfectants were included for efficacy study: Levermed, Incidur, Oxivir, Virex II 256. All the disinfectants were used at their working dilutions as recommended by the manufacturer and as mentioned in manufacturer literature.

Disinfectant efficacy and their shelf life were tested by In-Use Dilution test method. In this method, we were screening disinfectants for their efficacy and shelf life at various concentrations and contact time against a wide range of standard test organisms and environmental Isolates. For the In-Use Dilution method, the test organisms are enumerated on Soyabean-casein Digest Agar by pour plate method. Neutralizers that inactivate the disinfectants should be included in either the diluent or microbiological media used for the microbial enumeration. Dey-Engley neutralizing broth is purposely used for disinfectant efficacy study. In our present Study we found that, the average reduction of test microorganisms was found to be Maximum by Virex II 256 and Oxivir. Both Virex II 256 and Oxivir found to be
59

effective against all standard microbial strain and environmental Isolates at manufacturer recommended concentration and contact time.

Therefore, the results of present Disinfectant Efficay study shows that Gluteral, Glyoxal (Incidur) and n-popano, Isopropyl, Benzalkonium chloride (Levermed) are not good nonefficient disinfectants. On the other hand, Hydrogen peroxide, Alkyl Benzene sulphonic acid, Alcohol ethoxylate, Hydorxyethene-1-1-diphosphonic acid, 2- butoxyethanol (Oxivir)and Didecyl dimethyl ammonium chloride, benzyl ammonium chloride, amine oxide,, sodium bicarbonate (Virex II 256) are good and efficient disinfectant in our findings. Phenolics and Benzalkonium Chloride are not efficient disinfectants but their formulation with other chemical derivatives increases their effectiveness. So, newer generation disinfectant such as Oxivir and Virex II 256 are more effective than traditionally used phenolics and Benzalkonium chlorides.

However, other Issues like cost, safety for humans use, and corrosiveness of metallic surfaces need to be addressed before choosing an Ideal disinfectant for Pharmaceutical use.

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