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In laboratory animal science, there are visible soils (proteinaceous soil, urine scale, oils, etc.) and a myriad of microbial clutter that, while not visible to the naked eye, are extremely important to eradicate from the environment.

A controlled and reproducible environment is essential to the efficacy of reliable research and to the maintenance of high quality animal care. The animal facility personnel must be exceedingly vigilant to monitor and eliminate zoonotic and contagious infectious agents from the laboratory animal’s environment.

The Guide for the Care and Use of Laboratory Animals 1996, pg. 42, states:

“The frequency and intensity of cleaning and disinfection should depend on what is needed to provide a healthy environment for an animal, in accord with its normal behavior and physiological characteristics. Methods and frequencies of sanitation will vary with many factors….”

Not only is it important to maintain a sanitary and controlled environment, but also it must be done with products that are as safe as possible for animals and personnel. Finally, these products must meet the basic concerns of efficiency, cost effectiveness, and quality control. Animal facilities are high density housing areas. While we use modern cleaning and application equipment (cage washers, high pressure washers, foggers and sprayers, etc.) we rely most heavily on chemicals. Liquid cleaners, disinfectants, powdered products that are mixed on site, and gaseous products that are used in specialty applicators, are in the sanitation arsenal. The ultimate goal is the reduction or elimination of microbial clutter in the animal environment (Figure 1).

This article provides the laboratory animal care professional with a basic review of chemicals and their applications.

A brief review of terms:
Cleaning Agent: A chemical agent that, when used in conjunction with some form of agitation, will aid in the effective removal of soils from an inanimate surface. These are called detergents.

Sanitizer: An agent that reduces the number of bacterial contaminants on inanimate surfaces to levels prescribed by the Public Health Service’s rules and regulations.

Disinfectant: An agent that destroys or eliminates specific infectious agents from a surface.

Sterilant: Any physical or chemical agent that inactivates or kills all form of life, especially microorganisms.

Cleaning Agents are generally classified based on their pH (with pH 7 being neutral). There are acid cleaning agents (acid detergents – in solution, the pH of the product is below 7) and there are alkaline cleaning agents (alkaline detergents – in solution the pH of the product is above 7). Cleaning Agents do not carry anti microbial claims. Their role is to remove soils from the environment.

These products are considered “detergents” in that they are soluble cleaning agents that usually are not inactivated by hard water and contain wetting agents and emulsifying agents. The classifications are based on the types of soils they are geared to attack and remove.

Figure 3 shows general guidelines for using acids versus alkalines.

Acid Detergents are recommended for cleaning urine scale from the lab animal environment (generally associated with rabbit and primate animal housing areas). The acidic nature of these detergents reacts with the calcium deposits in the urine to emulsify and remove the scale. Acid products are also used in hard water areas to neutralize and remove the calcium, magnesium, and other mineral deposits found in the incoming water. Hard water scale (also called “lime deposit”) is formed when calcium carbonate (CaCO₃) and magnesium carbonate (MgCO₃) precipitates out of the water, and is deposited on the surface of caging, flooring, etc. When the solution hits a hot surface, the water is vaporized off and the mineral deposits are precipitated out. This explains why cage washers show a white haze of hard water on the walls and ceilings. In hard water areas, this is an extremely aggravating problem that generally affects the rinse chamber of tunnel washers (the areas where no chemical treatment occurs). If left untreated, the washers have a shorter useful life. The components of the machine, such as the heat exchangers, conductivity probes, temperature sensors, fill level probes, and manifold spray systems, will fail at a faster pace than a well maintained and scale-free machine.

Acid detergents are formulated with corrosion inhibitors, surfactants (surface acting agents), and other chemical enhancing properties. Organic and mineral acids are the backbone of these products. Their chemical formulations are carefully compounded so the ingredients are stable at the high wash temperatures used in laboratory animal care facilities. Acid blended with surfactants can efficiently remove urine scale and proteinaceous soils if the latter’s level of contamination are low to moderate. While there are many acids available, organics such as citric and acetic, and minerals like phosphoric acid, are most widely used. Acids such as sulfuric, hydrochloric, and nitric are considered highly reactive acids that can be very volatile and unstable and are usually not recommended for safety reasons.

Alkaline detergents are targeted to remove proteinaceous soils like oil, body excrements, etc. Alkaline detergents used in the cage and tunnel washers are usually formulated with surfactants, builders, chelating agents and other additives to increase their efficiency. While they serve the same purpose, not all detergent products are created equally. A higher pH detergent may not necessarily mean it cleans “better.” The pH reading itself does not imply a better detergency ability.

Protein soil levels, water conditions, and other factors may require different types of detergents. For instance, some detergents contain either sodium or potassium hydroxide in their formulations. Hydroxides are strong bases and are considered caustics. Caustic base compounds usually are higher pH formulations. When properly formulated with a compatible surfactant, chelating agent, or builder, they work well at removing oils and heavy protein based soils, but they may discolor aluminum.

Chlorinated detergents contain sodium hypochlorite, which is an oxidizing agent. These products are formulated to work on soils associated with animals on high fat or high protein diets, or heavily greasy soils such as from pigs or dogs, animals that tend to rub and mark the caging with their body oils. Chlorinated products are also recommended in glass and bottle wash operations where a high clarity factor is desired.

Alkaline detergents are formulated as either phosphate or phosphate-free products. Phosphate builders boost the cleaning process by sequestering the hard water minerals and holding them in a suspension, which then is not re-deposited on the surfaces being cleaned. Phosphate use has been linked to eutrophic water systems (characterized by an abundant accumulation of nutrients that support the dense growth of algae, causing the decay in the oxygen in the water system).

Phosphate-free detergents are recommended where wastewater discharge is a concern. Phosphate-free detergents formulated with a combination of chelating agents and other binders will solublize soils, prevent precipitation of calcium and magnesium minerals, and aid in soil dispersion, but they do not generally clean as well as products containing phosphates. To improve the cleaning abilities of these products, longer wash times, and increased concentrations may be needed. Depending on the formulation, phosphate-free detergents may be “eco-gentle” but not so “eco-nomical.” Products listed as “phosphate-free”may have low levels of phosphates in their formulations. Carefully reading the label will determine whether the product contains phosphorous or other phosphated compounds.

Animal facilities generally will require both acid and alkaline detergents to work in synergy to clean the animal environment. The ideal situation is for mechanical wash machines to use each detergent in a dedicated cycle. When that option is not available, each product may be injected separately into a wash cycle, but only when compatibility of the products has been established. At times, a third product such as a neutralizing agent may be recommended as a buffer between the two products, and to increase the efficacy of each working compound.

There are specialty additives like chlorine bleach, enzymes, glycol ether, etc. that will target specific types of soils. The type of soils in each facility must be addressed to determine the cleaning products best suited for the site.

Effluent Concerns
Local ordinances may dictate that the effluent (waste water) conforms to specific pH guidelines. Because mechanical washers may discharge several gallons of chemically treated water at high temperatures pH adjustment may be necessary. The most common treatments are aimed at reducing or enhancing the pH of the water at the point of drain. There are several avenues to achieve these pH stabilizations. While it is perhaps logical that acids and alkalines should “balance” one another’s pH, these chemical groups frequently are incompatible and cause secondary off gassing of released by-products. Care must be taken to ensure compatibility of chemical agents when approaching effluent treatment.

Chemical Disinfection
Anti microbial agents must be registered with the United States Environmental Protection Agency (EPA) under the terms stated in the Federal Insecticide, Fungicide, and Rodenticide Act as amended (FIFRA). As microbial clutter cannot be easily visualized, neither can the results of using a chemical disinfectant. Thus, the EPA is charged with oversight and evaluation of the anti-microbial claims of chemical disinfectants. In addition, all registered products must carry EPA establishment (EPA Est. No.) and EPA registration numbers (EPA Reg. No.). The EPA uses the Association of Official Analytical Chemists (AOAC) dilution test to determine the efficacy claims of the disinfectant.

Discussions of the germicidal activity of chemical disinfectants generally refer to cell death (in the case of bacterial cells) and inactivation of the microbe (for viral cells). Modes of action of germicides are usually based on observations of cell disruption, such as membrane damage, after contact with the chemical germicide.

Iodines
The Iodines are naturally occurring and are among the oldest chemical disinfectants still in use. Iodine is a solid at room temperature and usually is mixed with another agent to produce Iodophors, which are then used as the chemical disinfectant. Iodines appear to work by quickly penetrating the cell wall of the microbe, causing complete cell disruption and subsequent death. The anti microbial activity of iodine can be slowed by a heavily proteina- ceous environment, which will decrease its killing ability.

Phenolics
Phenolics are broad-spectrum disinfectants that appear to penetrate the cell wall or work to denature the proteins in the cell. In the early tomiddle 1900s, these products were used everywhere as chemical disinfectants, but have fallen out of favor because of their high corrosive nature, and the side effects of contact with them (the phenols are very hazardous to cats for instance).

Chlorine Compounds
To begin to discuss chlorine compounds (“bleach”), it is hard to find a primate facility today that does not have a recipe for Dakin’s solution in the emergency monkey bite and exposure kit. During the First World War, H.D. Dakin used sodium hypochlorite for open wound disinfection, saving many soldiers’ limbs and lives. Today it is the first line of treatment when exposure to old world primate’s bodily fluids has occurred. Although organic soils quickly deactivate the effectiveness of these chemical agents, chlorine compounds are fast acting and broad-spectrum products. Bleach products are highly reactive. They have inherent corrosive and caustic properties and when they come in contact with acids, they will release chlorine gas. Chlorine bleach (sodium hypochlorite) also degrades very rapidly as Figure 5 represents.

Quaternary Ammonium Compounds
The quaternary ammonium products are the first “man-made” chemical disinfectants. “Quats” appear to denature cell proteins and affect the cell’s permeability, which is why they are effective as disinfectants. Most of the quat products available today are dual chain blends of ADBAC (alkyldimethyl benzyl ammonium chloride) quats that have been compounded with non-ionic detergents so that they can be cleaner-disinfectant products.

Chlorine Dioxide
Chlorine dioxide has been called an “ideal biocide.” Liquid chlorine dioxide is a chemical disinfectant (also a sterilant) that is generated on site. Chlorine dioxide is mostly used as a molecule encapsulated in water so that it is applied as a liquid, it is a powerful oxidant that appears to eradicate microbes by reacting with the amino acids of the cell. It works very quickly and is rather aggressive.

Peroxygen Disinfection
Peroxygen disinfectant (Potassium Peroxymonosulfate) is a very rapid acting biocide that came to the attention of the lab animal community after the foot and mouth outbreak in the United Kingdom in 2000.These products are very strong oxidizers enabling a rapid microbe kill in a short period of time. It appears that the mode of action is the release of hypochlorous acid which combines with the cell protoplasm or inhibits enzymes from working properly, causing rapid cell death.

Efficacy of Products over Time
Both chlorine dioxide and the potassium peroxymonosulfates are compounds generated onsite (mixing with water to create the anti-microbial activity of the product). The generation of the active ingredients is a continuous process. While both compounds are stabilized, the reactive properties slow as the mixed product ages, reducing its efficacy as a disinfectant over time.

Figure 6 shows the various microbial activities of the disinfectant classes mentioned above, and the modes of action. In addition, it lists the advantages and disadvantages of the various compounds discussed in this review.

Click Image For a Larger Version

Surface Substrate Compatibility
Sanitation chemicals remove soils, clean the physical environment, and eliminate microbial clutter that can potentially exterminate the animal population within the facility, and yet there is always the issue of “what about the surfaces in the facility” that must be addressed.

Few sanitation chemicals are so benign that they do not have some corrosive or caustic properties. Most of these properties are generally associated with the concentrated product, and once mixing on site occurs, the caustic or corrosive issues are diminished. Much has been written about polycarbonate and polysulfone caging and the results of hydrolysis with certain compounds. Thorough rinsing with clean water in mechanical washers is highly recommended. Of more recent interest has been the caging degradation caused by so called “dirty steam” autoclaving.

Changing stations and hoods may require special cleaning after exposure to high-level disinfectants to prevent softening and rusting of the steel material.

The golden rules of thumb are:

1. Read the MSDS and all literature that comes with the product.
2. If there is a contact time required, follow that carefully and do not allow for prolonged exposure beyond the manufacturer’s listed time.
3. Use a potable water rinse to remove residual substances when ever possible.
4. Observe the safety and PPE requirements of the product.

Failure to follow these requirements is not a “do-over” if exposure occurs. Until the perfect product has been built, using care will result in a sanitary environment that is safe for people and animals.

References

• “Disinfectants in Laboratory Animal Science: What Are They and Who Says They Work” by A.S. Ingrahamand T.M. Fleischer, LabAnimal Volume 32,No 1, Jan 2003.
• “The Chemistry of Disinfectants and Sterilants” by A. S. Ingraham, Contemporary Topics, Volume 31 No 2, March 1992.
• “A Program for Proper Handling and Utilization of Chemicals in a Laboratory Animal Care Facility” by F.E. Lynch, A.S. Ingraham, and K. B. Shapiro; Pharmacal Research Laboratories, 1992.
• “Disinfection, Sterilization and Preservation” Edited by Seymour S. Block (fourth edition), Lea and Febiger, publishers, 1991.
• “The Chemistry of Cleaning” by Charles Jones, Rohm and Haas Company, (date unknown).
• “A Focus on Chlorine Dioxide: The ‘Ideal Biocide’” by G. D. Simpson, R.F. Miller, G.D. Laxton,W.R. Clements; Unichem International Inc. The Guide for the Care and Use of Laboratory Animal, 1996.

Ken Shapiro is President of Pharmacal Research Laboratories, Inc. He has over 28 years of experience in developing sanitation programs for animal care facilities. He can be reached at kshapiro@pharmacal.com.

Frank Lynch is the Mid-Atlantic Sales Rep for Pharmacal Research Laboratories, Inc. Frank has 27 years of experience in chemical sales to the Lab Animal Community. Frank can be reached at flynch@pharmacal.com

Amy S. Ingraham, BA, RLATG, Northeast Sales, Pharmacal Research Laboratories. Amy has been with Pharmacal in a sales and service capacity for 21 years. Previously she worked as a laboratory animal care technician and manager for 12 years. Amy can be reached at aingraham@pharmacal.com

Pharmacal Research Labs, Inc.; P.O. Box 369, Naugatuck, CT 06770; 800- 243-5350; www.pharmacal.com