Disinfectants in Laboratory Animal Science: What Are They and Who Says They Work?

PRODUCT FOCUS Volume 32, No. 1 Lab Animal January 2003

Amy Ingraham, BA, RLATG and Tammy Marotta Fleischer, ABS, QC

Two thousand years ago—well before gov- Definitions difficult to kill, whereas the HIV virus is ernment regulations for disinfectants, steri- A disinfectant is an agent that will rather easy to eradicate on hard surfaces. lants, and sanitizers existed—the Bible, vari- destroy or eliminate from inanimate Infectious prions (e.g., the causative agent ous Eastern religious writings, and sacred objects all pathogens except bacterial in bovine spongiform encephalopathy tomes extolled the virtues of personal endospores. A sterilant will destroy or (BSE), or mad cow disease) are not includ- hygiene, dietary considerations, and “what eliminate pathogens, including bacterial ed in this group because the prion is tech- to do when all else fails” blueprints for endospores, from inanimate objects. nically not a cellular microorganism but remaining healthy. Even Greek literature Disinfectants include products that are rather a protein. At this time there are no contains precise standard operating proce- virucidal, bactericidal, and fungicidal. documented chemical or physical agents dures (SOPs) to rid inanimate objects of Sterilants are biocidal, because by defini- that will inactivate these infectious pro- microbial pathogens: In The Odyssey, tion they will kill all living organisms, both teins. Similarly, there is no registered ger- Homer refers to ‘fumigating’ Odysseus’ pathogenic and nonpathogenic. The term micide that can with certainty kill pin- house by burning sulfur; Aristotle advised, ‘germicide’ is synonymous with ‘disinfec- worm eggs, although a simple ammonia “Bury your dung.” tant’. solution will kill the ova of other parasites The entire scientific pursuit of microbi- Microorganisms have different levels (e.g., Cryptosporidium parvum2). ology was of course unknown at the time, of resistance to chemical disinfection. Table 1 lists types of microorganisms in and not until the 1400s did the invention of Resistance varies with the type of organ- descending order of resistance to chemical the microscope allow people to see the world ism. For instance, bacterial spores are very germicides. A germicide at a given level of of microbes. It was Robert Koch’s germ the- ory of disease during the 1880s that estab- TABLE 1. Types of microbes, listed in descending order of resistance to chemical ger- lished the role of microbes as the cause of micidesa,b. disease1. Although they were often effective, early Microbe type Examples Bacterial spores Bacillus subtilis attempts at disinfection were not always Clostridium sporogenes pragmatic. Sir Joseph Lister flooded the skin Clostridium piliforme (the cause of Tyzzers’ disease) of patients undergoing surgery with carbol- Mycobacteria Mycobacterium tuberculosis ic acid, causing skin burns but preventing M. bovis the development of nosocomial illnesses. Nonlipid (nonenveloped) Parvovirus Even though the use of fire was recognized or small viruses Poliovirus as a means to ‘disinfect’ inanimate objects, it Rhinovirus was not always practical to burn down a Fungi Tr ichophyton spp. (causes ringworm) building to kill the microbes it contained. Vegetative (non-spore-forming) Salmonella choleraesuis The ability to rid the laboratory or animal bacteria other than mycobacteria Staphylococcus aureus facility of potentially harmful microorgan- Pseudomonas aeruginosa isms is no less important today. In this article Lipid (enveloped) viruses Coronavirus we discuss the various types of liquid disin- Herpes simplex virus fectants that can be used on surfaces such as Hepatitus B virus Human immunodeficiency virus floors and tabletops, and clarify how these products are registered in the United States. aSee refs. 10, 11. bNote: Infectious prions and parasitic oocytes are not included.

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effectiveness should inactivate or kill all a product does not have an EPA registration penetrating the cell wall of the organisms, organisms located at that level and on the number, then it should not be used for dis- causing a disruption of the cell metabolism lower levels of resistance. Apart from pri- infection in an animal-care area. and function. Secondarily, free iodine may ons and parasite ova, bacterial spores are In 1982, the government once again bond in solution with the cell proteins to the life form that is most difficult to kill, so amended the FIFRA to clarify terminology. form salts4.Iodine compounds are poor the Food and Drug Administration (FDA) Before this clarification, a “sporicide” and a cleaners, requiring the use of another chem- uses biological indicators that contain bac- sterilant were two separate classifications. A ical to clean the surface. In addition, iodines terial spores to prove sterilizing efficacy. “sporicide” could have one failure out of 60 are relatively unstable in terms of contact Notice that this list does not contain pro- replicates in a sporicidal assay, and the “ster- time and light exposure, and can perma- tozoan cysts. Pinworm eggs are very persis- ilant” could have no failures in the same nently stain surfaces if not used properly. tent in the laboratory animal facility, and assay. These new regulations considered Extensively used during the 1960s, iodines are usually able to tolerate chemical germi- sporicides and sterilants as the same. imparted a permanent brown discoloration cides. To rid a facility of the cysts, it is The FDA classifies differently products to floors and walls. Perhaps most impor- advisable to consider ‘cooking’ them with that disinfect surfaces and those used to tantly of all, iodophors are only active with- steam heat. Like an egg, they will harden sterilize medical devices. The FDA registers in tight brackets of concentration. and become inactive when heated. products that are liquid sterilants, and the Increasing the concentration may actually EPA registers products that are disinfec- reduce the available iodine in the aqueous Delineations of Authority tants. EPA-registered disinfectants may not solution5.Additionally, microorganisms, In the US, the Environmental Protection be used as terminal sterilants on any surface specifically Pseudomonas cepacia,have Agency (EPA) registers commercially man- or instrument that is introduced directly been cultured from full-strength povi- ufactured disinfectants under regulations into the human body or that comes in con- done–iodine products6. set forth in the Federal Insecticide, tact with normally sterile areas of the body Phenolic compounds are broad-spec- Fungicide, and Rodenticide Act (FIFRA) as or the bloodstream. trum disinfectants that can kill both amended3.Chemical disinfectants are A sterilant (sporicide) is capable of enveloped and nonenveloped viruses as well referred to as “pesticides” in these federal destroying all forms of microbial life. as mycobacteria. Phenolics appear to kill acts. Sterilants are the most potent and are effec- microorganisms by penetrating the cell wall The first FIFRA, enacted in 1947, was tive against all forms of vegetative bacteria, and acting as cell poisons, or by denaturing essentially a ‘truth-in-advertising’ law, bacterial spores, fungi, fungal spores, and the cell’s proteins4.These compounds are because its goal was to protect the public viruses. An FDA-registered liquid chemical extremely corrosive and require exceptional from false or exaggerated claims made for sterilant will completely eradicate all of the precautions in handling. Phenolics are certain products that were sold across state microorganisms listed in Table 1. extremely hazardous to cats, cause skin lines. In 1972, the government extensively A high-level disinfectant should eradi- depigmentation, and have posed human modified the FIFRA so that it now requires cate all microbes except for bacterial spores. health problems4,7. a manufacturer to submit not only an appli- An intermediate-level disinfectant should In the past, the phenol coefficient has cation but also a label clearly stating all eradicate enveloped viruses and all the less- been the standard against which all other claims, test data from an EPA-certified lab- resistant microbes. A low-level disinfectant disinfectants were judged. This standardiza- oratory supporting these claims, toxicology must kill only three species of bacteria: tion included comparing germicidal chemi- data, a “confidential statement of formula”, Salmonella choleraesuis, Staphylococcus cals and provided an index to the concen- and precise “use directions”.The “confiden- aureus, and Pseudomonas aeruginosa. tration of other products. The errors inher- tial statement of formula” will include spe- Incredibly, there is also a category of disin- ent in converting phenol coefficient num- cific ranges for every ingredient included in fectants below that of a low-level disinfec- bers to effective dilutions of products for the product as well as a description of that tant. These products, called “hospital disin- practical disinfection are so great that the ingredient’s function in the formulation. fectants”,must only kill S. choleraesuis and S. procedure must be considered unsatisfacto- The confidential statement must also list aureus. ry, particularly for nonphenolic com- Chemical Abstracts Service (CAS) numbers pounds. Currently, the Association of and approved raw material vendors. A pes- Types of Disinfectants Used Official Analytical Chemists (AOAC) use ticide label must include a list of active in Laboratory Animal Science dilution test remains the standard for deter- ingredients with their amounts, storage, Iodines and Phenols mining the efficacy of disinfectants; howev- and disposal information. The label must Iodine compounds are disinfectants that er, the government has provided funding to also have precautionary statements, first-aid can kill many kinds of microorganisms, establish alternative assays, each with an instructions, the manufacturer’s complete including both enveloped and nonen- exact number of microorganisms per chal- address, and an EPA registration number. If veloped viruses. Iodines appear to work by lenge. These assays should soon be adopted.

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Chlorine Compounds bleach–water solution to maintain adequate Chlorine dioxide. Chlorine dioxide is Sodium hypochlorite. Sodium hypo- chlorine concentrations (i.e., 100 p.p.m.). available as a high-level disinfectant that has chlorite (the active ingredient in bleach) is Sodium hypochlorite is highly reactive wide applicability not only in the animal- perhaps the best example of a chlorine and degrades rather quickly. Thus, as Fig.1 care field but also in the manufacturing and compound used as a disinfectant. If used shows, bleach rapidly loses its effectiveness paper industries. Health officials used chlo- within some rather stringent parameters, as sodium hypochlorite degrades. The rine dioxide to remove the anthrax contam- bleach is a very effective and relatively inex- Quality Assurance Laboratory of Pharmacal ination in the Hart Senate Building in 2001. pensive virucidal product. The mode of Research Laboratories conducted a test that Manufacturers use it in toothpaste, for action of chlorine compounds is not fully simulated normal laboratory storage condi- instance, as well as in paper and pulp man- understood. Chlorine compounds may tions. We stored a simple solution of 18.0% ufacturing. It is necessary to mix the prod- actually cause cell death by a two-step sodium hypochlorite in an opaque bottle uct on site, and its shelf-life is inherently process, reacting with the cell protoplasm that was capped and left on a countertop in short and of mixed product efficacy. after forcing entry through the cell wall, or the laboratory to simulate a bleach bottle Chlorine dioxide is a free radical and a perhaps by inhibiting the cell enzyme reac- stored on the shelf at room temperature. powerful oxidant that kills microbes by tions and denaturing the cell proteins4,7. The overhead lights were turned on in the reacting with bacterial and viral amino People have used bleach as a disinfectant morning and off at night 5 days a week, acids. Chlorine dioxide requires a very short since the early 1800s (ref. 8). During World allowing for ∼9 h of illumination per day. contact time but does not have inherent War I, H.D. Dakin advocated the use of a We locked the laboratory and left it vacant cleaning ability, necessitating a precleaning 0.50% solution of sodium hypochlorite for during the weekends. For a 70-day period step. All chlorine dioxide products currently skin and open wound disinfection; many we analyzed daily samples from the bottle available have inherent acidity and corrosive contemporary primate laboratories have a and recorded the percentage of active sodi- properties. Chlorine dioxide’s entry into the prepared bottle of Dakin’s solution in the um hypochlorite in the sample. Our results animal-care area has allowed supervisors to emergency monkey bite kit. The Electro- showed an initial decelerating curve of use one product rather than two or three Alkaline Company (later the Clorox degradation of the percentage of active different disinfectants to carry out the same Company) began the American production sodium hypochlorite. At 5 days, the solution process in a short period. One should not of commercially available sodium measured 15.68% sodium hypochlorite; at confuse chlorine dioxide with chlorine gas; hypochlorite solution in 1913. Hospitals 20 days it was 13.72%. At 35 days, the per- they are two completely different chemicals. and critical care facilities today accept sodi- centage was 12.74% with the rate of degra- um hypochlorite solution as an inexpensive dation substantially decreased. The shelf Quaternary Ammonium disinfectant9. life of commercially available sodium Compounds Concerns about the efficacy of sodium hypochlorite mixtures (bleach) will depend Of the many disinfecting compounds hypochlorite have involved the percentage on the storage facility, temperature controls, currently available, the quaternary ammo- of active chlorine required to provide light controls, etc. Facility managers should nium compounds are the most widely used antimicrobial properties. Reports from the label and check the product regularly using in animal facilities. There are five possible field have shown that prepared bleach solu- available chlorine detection strips or test kits. modes of action for quaternary ammonium tions rapidly lose potency under ambient laboratory conditions. An organic soil load 18.0 reduces sodium hypochlorite’s effectiveness 17.2 as a disinfectant, necessitating a precleaning 16.4 step before disinfection. Bleach has an 15.6 inherent corrosive effect on certain metals, 14.8 concrete, and clothing, and can pose prob- 14.0 lems for animals and personnel. 13.2 Among these problems is bleach’s reac- tivity with acids, releasing chlorine gas, 12.4 which is highly toxic. It also reacts with 11.6 ercent of sodium hypochlorite active ingredient by analysis by ingredient active organic soils (e.g., feces) to produce poten- P 10.8 tially carcinogenic compounds. One result- 10.0 1 ing compound, trihalomethane, can be 10 20 30 40 50 60 70 released as a toxic gas. Therefore, supervi- Days from manufacture sors must carefully consider the use of FIGURE 1. Degradation of sodium hypochlorite over time. (Data from Pharmacal bleach and ensure when making up a Research Laboratories, Naugatuck, CT).

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products: denaturing cell proteins, influenc- required for high-level disinfection is quite numbers for chemical disinfectants after ing cell metabolic reactions, affecting cell long, and there are several questions con- reviewing the product’s claims of efficacy. permeability, stimulating cell glycolysis cerning the safety of these products for peo- These claims result from testing in strict reaction, and affecting cell membrane ple and animals. Glutaraldehyde is tubercu- compliance with the requirements set by the enzyme activity7,10.They are relatively cost locidal. The tendency of glutaraldehyde- EPA, which thoroughly reviews the data effective, easy to use, and, if handled prop- based sterilants to induce contact dermatitis submitted about the product. The EPA will erly, safer for animals and people than the has led health care facilities to limit its use in issue a registration number specific to the other currently available disinfectants. recent years because of liability issues4,9. particular product only if all the necessary These compounds are generally not tuber- data have been submitted to support the culocidal. The newest quaternary ammoni- Alcohols claims. Once the EPA issues a number, the um compounds generally meet most of the Alcohol products denature cell proteins manufacturer may not alter the product’s cleaning-disinfecting requirements. to cause cell death. However, they can quick- chemical composition unless it provides the The first generation of quaternary ly become ineffective in protein-rich envi- EPA with test data proving the product still ammonium compounds became available ronments. Because of their volatile character- retains the capabilities to support the origi- in 1935. Dilution with hard water or with istics, the use of alcohol products as primary nal claims. the wrong type of soap or detergent can disinfectants has declined. Contact time for The AOAC use dilution test is an exten- inactivate these earlier generation quater- alcohol products can range up to several sive evaluation of germicides that employs nary ammonium compounds. Therefore, it hours, which can be difficult considering hard water with organic serum added, in an is extremely important to obtain a quater- their highly evaporative nature. Also, alco- attempt to simulate ‘real-world’,worst-case- nary disinfectant that has been properly hols can damage rubber and plastic compo- scenario challenges. This test assesses claims compounded with compatible nonionic nents4. that a given germicide will kill vegetative detergents, which will not affect its disin- bacteria. fecting properties. The newest generation Peroxygen Compounds Testing a product for its ability to deacti- quaternary ammonium compounds are The search for the ‘perfect’ disinfectant vate spores is more extensive. After inocula- generally blends of alkyl dimethyl benzyl may lead to peroxygen products such as tion of penicylinders (porcelain cylinders) ammonium chloride (ADBAC) quaternary hydrogen peroxide, which is a sterilant at and surgical loops with specific microor- ammonium compounds, rather than the the appropriate concentrations. Peroxygen ganisms, one vacuum-dries them, making old single-chain compounds. A good qua- compounds act as rapid oxidizers to cause the pathogen even more resistant to pene- ternary ammonium compound should cell death or inactivation. Once known as tration and killing by the disinfectant. Then remain active as a virucidal, fungicidal and oxidized water or peroxymonosulfates, per- one exposes the cylinders or loops to the bactericidal agent when subjected to the oxygen compounds have come to the labo- specific product in the proposed use dilu- AOAC use dilution test, in 400 p.p.m. hard ratory animal care field as part of a search tion. After a specified period, which follows water plus 5% organic serum. The EPA reg- for products that are environmentally the manufacturer’s claims for kill time, the istration number on the label certifies only friendly and relatively low in toxicity for use testers are removed and placed in a culture that the product is a disinfectant, not that it around animals. Many of the peroxygen medium. The literature that the manufac- is a cleaning product. While quaternary products leave no residuals and have excel- turer submits to the EPA will include the ammoniums have inherent cleaning abili- lent germicidal activities. Peroxygen prod- recommended exposure time for the prod- ties, the addition of a cleaning agent ucts came to the forefront when foot-and- uct to achieve a complete kill. The sporicidal increases the ability of the product both to mouth disease appeared in the United test involves either B. subtilis or C. sporo- clean and to disinfect. Kingdom in 2000. These products are very genes. Analysts examine the resulting The newer generation of quaternary strong oxidizers, and their germicidal activ- growth for the specific microorganism. ammonium compounds will kill some ity occurs quickly, that is, only a short con- They repeat this process extensively, and the nonenveloped viruses as well as enveloped tact time eradicates microbes. Peracetic chemical being tested must prevent growth viruses. acid is a peroxygen product. Hydrogen per- in 60 of 60 tests for a total of 12 sets of tests oxide when used in the vapor phase is a involving 360 penicylinders and 360 surgi- Glutaraldehyde sterilant. cal loops, and three lots of each product A member of the aldehyde group that Critics have questioned the efficacy of includes formaldehyde, glutaraldehyde has Product Testing and these tests in recent years. Inconsistencies limited use in the animal-care facility. Registration are common within a testing lab and Glutaraldehydes work by alkylating amino A germicide’s EPA registration number between various laboratories11.Scientists acids to alter RNA, DNA and protein syn- is a ‘stamp of authenticity’ for the product. have cultured the microorganisms in labo- thesis4,7.The necessary contact time Under FIFRA, the EPA issues registration ratories for generations, and the cultures

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may not react, as would a ‘wild-type’ culture purchasing the product may not hold the Acknowledgments freshly isolated from the field. The tests master registration number. Companies The authors thank Roger Orcutt for his specify a minimum, but not a maximum can subregister the formulation from the suggestions in preparing this manuscript. number of microorganisms per assay, and company holding the master label. this can of course drastically affect the test Therefore, a registration number or part of Received 11/20/02; accepted 12/2/02. results. The result has been perhaps a false a registration number may be used on sense of security about the effectiveness of products with different names. The EPA References 1. Koch, R. The etiology of tuberculosis [Koch’s particular products. Since 1991, the EPA has must approve any subregistration, and the postulates.]. Mittheilungen aus dem been reevaluating the specific claims of subregistrant designs its own label after Kaiserlichen Gesundheitsamte 2, 1–88 (1884). many germicides. Some products that had having received EPA approval. These labels 2. Jenkins, M.B., Bowman, D.D. & Ghiorse, W.C. been listed as “sterilants” before 1991 cannot add any claims that the master label Inactivation of Cryptosporidium parvum were reclassified as “high-level disinfec- does not have and must be submitted to oocysts by ammonia. Appl. Environ. Microbiol. 64, 784–788 (1998). tants” after these reevaluations (see the holder of the master registration for 3. 7 USC Chapter 125. Federal Insecticide, www.epa.gov/pesticides). approval. Fungicide, and Rodenticide Act (FIFRA). http://www.epa.gov/pesticides/fifra.htm. An EPA registration number can look 4. Ingraham, A.S. The chemistry of disinfectants What to Look For like this: 1234-56. This number tells us that and sterilants. Contemp. Top. Lab. Anim. Sci. When evaluating disinfectants for ani- the company holding the master registra- 31, 18–23 (1992). 5. Gottardi, W. in Disinfection, Sterilization and mal-care facilities, the first concern should tion has an establishment number of 1234, Preservation 4th edn (ed. Block, S.) 152—166 be finding the EPA registration number. and the 56 identifies the product. A regis- (Lea & Febiger, Philadelphia, PA, 1991). 6. Favero, M.S. & Bond, W.W. in Disinfection, Claims for disinfectants that are unregis- tration number of 1234-56-789 indicates Sterilization and Preservation 4th edn (ed. tered may not be substantiated. For that the product is subregistered. The first Block, S.) 633—634 (Lea & Febiger, instance, household bleach is a common set of numbers (1234) is the establishment Philadelphia, PA, 1991). 7. Rutala, W.A. APIC guideline for selection and product found in many laboratory animal number for the holder of the master regis- use of disinfectants. 1994, 1995, and 1996 facilities. Only a few manufacturers of tration, the 56 identifies the product, and APIC Guidelines Committee. Association for Professionals in Infection Control, Inc [see household bleach have EPA-registered the 789 is the subregistrant’s establishment comments]. Am. J. Infect. Control 24, numbers for their products. The other number. 313–342 (1996). 8. Dychdala, G.R. in Disinfection, Sterilization companies are manufacturing a product A product’s EPA registration number and Preservation 3rd edn (ed. Block, S.S.) that will clean and brighten your clothes, allows the user to obtain any necessary 157–172 (Lea & Febiger, Philadelphia, PA, but may not disinfect your facility. In fact, information on the product by consulting 1983). 9. Rutala, W.A. Draft guideline for selection and the percentage of active ingredients in the list of registered pesticides in the use of disinfectants. Am. J. Infect. Control 17, these products may be too low for effective United States. From there, one can deter- 24A–35A (1989). 10. Mlynarcik, D., Denyer, S.P.& Hugo, W.B. A sanitizing. mine who holds the master registration, study of the action of a bisquaternary ammo- Within the field of laboratory animal whether it is subregistered, contact nium salt, an amine oxide and an alkoxy science, the same registration numbers can addresses and phone numbers, and all phenylcarbamic acid ester on some metabolic functions in Staphylococcus aureus. appear on different chemical vendors’ active ingredients and their percentages. MICROBIOS 30, 27–35 (1981). products. The vendor from whom you are 11. Orcutt, R.P.Evaluation of disinfectants and sterilants. Contemp. Top. Lab. Anim. Sci. 30, 15–18 (1991). 12. Spaulding, E.H. Chemical disinfection and antisepsis in the hospital. J. Hosp. Res. 9, 5–31 (1972).

The authors are affiliated with Pharmacal Research Laboratories, 33 Great Hill Rd., Naugatuck, CT 06770. Please send reprint requests to Fleischer at the above address, or email: [email protected].