Clean Environments for Sterility Testing

If manufacturers are marketing a product or device as sterile—a sterile label claim—they are going to want to show that it is indeed sterile. To do so, products go through a serious of steps to test for sterility and part or all of those tests may need to be completed in a clean environment.

There can be several levels of clean environments. Two of the most common used at Nelson Laboratories include clean rooms and isolators. Which of the two used depends most frequently on the size of the device or the packaging used.

A clean room environment is just that—a room fitted for sterility testing that provides a clean environment. People and devices go through a fairly complex series of entry points to enter the room ensuring the environment remains clean. Basically we want to make sure we do not contaminate the product or device. The clean room has all of the monitors, cleaning procedures and air filtering necessary to maintain the clean environment for testing.

Isolators on the other hand are environments created out of stainless steel and glass that are accessed by the tester with attached arm length gloves. Testers put their hands into the gloves to access the products to be tested.

Pros and Cons

To determine which clean environment to use is determined by the product and its packaging. Clean rooms have the advantage of space. Large products or products with complex packaging needs a lot of space. It also allows the tester to be in the room and in many situations more than one tester in the room. The biggest drawback is the fact that the largest risk for contamination is the presence of personnel even with the complex clean room entry procedure.

Isolators on the other hand have the advantage of eliminating the risks associated with a human presence. A product or device is placed in the isolator after which the entire environment is sterilized with high concentrations of vaporized hydrogen peroxide forced into the isolator chamber. Testers place their hands and arms into the gloved sleeves to perform the sterilization tests. The isolator is the best for choice for small products or a small number of samples. If packaging is permeable it may not be as good an option as the clean room since the sterilizing gas can penetrate the packaging compromising the sterilization test.

Whether an isolator is used or a clean room, the standards for sterility testing include USP <71> and ISO 11737-2. Under the standard clean rooms are rated between 1 and 8 with the lower rating indicating a cleaner room

The Role of Cleanrooms in Sterile Pharmaceutical Manufacture and Sterility Testing

Introduction
An important aspect of the use of cleanrooms (Figure 1) is the ability to monitor the quality of the manufacturing environment and thus detect changes, which may be deleterious to product quality. Those aspects of product quality, which concern us here, are the levels of microbial and particulate contamination at successive stages in the manufacturing procedure. To a certain extent, particulate levels in a product reflect the particulate burden in the manufacturing environment. Additionally good manufacturing practice (GMP) requires that certain minimum environmental standards be maintained in certain manufacturing areas. Products that are terminally sterilized are required to be manufactured under environmental conditions equivalent at least to class C (MCA, Rules and Guidance for Pharmaceutical Manufacturers, 1997, often described as the Orange Guide), and products that are aseptically manufactured are filled under class A conditions, situated in a class B environment [1]. In the United Kingdom, the details of construction of rooms that will meet these conditions are stipulated in British Standard 5295. Some information will also be found in Annex 1 of the Orange Guide. Table 1 lists the relevant information for particulate and microbiological contamination.

Particle Monitoring
Particulate levels in the atmosphere of the cleanrooms was determined by using a particle counting system which continuously
samples air at a constant flow rate through a light-scattering sensor.The degree of scatter is proportional to the size of the particles present so that processing of the signals from the scattering unit provides a figure for the number of particles sampled in a known time and also the size of these particles. Normally the filling and sterility testing rooms are class B and the preparation room is class C. Class A conditions are achieved locally by the use of laminar air flow (LAF) hoods which are used for aseptic manipulations and filling operations. Sampling of the filling and sterility testing room atmospheres was done from preparation room. The air flow rate into the microcount sensor is 1 ft/min-1 so that the sampling time gives a measure of the volume of air sampled and the particle number at the 5.0 and 0.5 µm levels. A sampling time setting of 1 min is suggested so that the display gives particles per cub. ft. Convert this value to particles per cubic meter using the conversion.
1 m³ = 35.3 ft³

Microbial Monitoring
Two types of sampling method, active and passive, were used to determine the microbiological quality of the atmosphere. Both methods rely on the fact that microbial cells, which are invisible to the naked eye, will reveal themselves as colonies after incubation of the nutrient agar plates used for sampling. A colony may form as the result of the growth of a single organism. A single organism colony will also arise from a clump of a few organisms of the same strain. Because of this, the results of colony units are noted as colony-forming units (CFUs) and not as simple numbers of organisms. Two types of agar used in these tests, Nutrient agar for the detection of bacteria after incubation at 31°C and malt extract agar for the detection of bacterial and fungal organisms (moulds and yeasts) after incubation at 25°C.
The simplest method of enumeration is passive sampling by the use of a settle plate. This involves exposing the plate at the location of interest for a known period of time (sampling time), which is terminated by replacing the lid of the plate. The result is expressed as a number per unit area per unit time (cfu/cm2/h). The second technique is active sampling, which involves measuring the number of microorganisms in a known volume of air, e.g. the concentration of microorganism in the air. Generally, a measured volume of air is drawn through the sampling apparatus and the microbes present impinged on or into a nutrient media usually agar. The result can therefore be expressed as a number of cfu's per unit volume of air. It is usually considered that this is a more precise way of estimating the microbiological quality of the environment and is the measure for example in Table 1.
Surface sampling: Contact plates (containing either type of agar, nutrient or malt extract) for surface contamination. As with the passive and active sampling choose sampling sites of interest or area that present a microbiological risk to the product. (Note: ensure that any excess agar is removed from the surface after sampling using an alcohol wipe. It should be noted that the greatest microbial risk to a product comes from the operators). Contact plates taken before and after hand washing and putting on gloves can be used to assess the efficiency of clothing procedures prior to entry into the cleanest, critical areas. The efficiency of any sterilization process is directly proportional to the bio burden or challenge to that process. An increasing approach to sterilization in the pharmaceutical industry is the use of F° to control a sterilization process, rather than the set cycle times listed in British Pharmacopoeia (BP). To employ control via F° implies that the bio burden and likely heat sensitivities of any organisms contaminating the product before sterilization are known. One feature of microbiological monitoring is therefore the measurement of microbiological contamination during the production process. This provides information on the quality of raw materials, handling procedures and the ability of the various steps employed (e.g. filtration) to remove any contaminating organisms present.
Additional Measures of Environment Quality
Before, during and after use of the class A rooms (e.g. filling and sterility testing rooms) record values for temperature and humidity within the rooms and also values for over-pressure. The required values are laid down in British Standard (BS) 5295 and are temperature, 20°C ± 2°C; humidity, 35-50%; over-pressure, 25Pa (2.5 mm water gauge).
Sterility Testing of Pharmaceuticals
Sterility testing attempts to reveal the presence of viable forms of bacteria, fungi and yeasts which may occur in or on pharmaceutical products, and in simple terms this is done by exposing portions of the material under test to media capable of supporting the growth of these different microorganisms. After 14 days incubation at a suitable temperature, the media are examined for the presence, shown by turbidity or pellicle formation, or absence of growth. If no growth occurs in the test media, the material is assumed to be sterile. Since this is a destructive test, only a sample of the batch can be analyzed, and this introduces the problem of sampling statistics. Regulatory or Licensing Authorities require a product contamination rate within a batch of 1 in 1,000,000 or less, it can be easily demonstrated that the sensitivity of the sterility test is several orders of magnitude lower than this. The sterility test is therefore suspect and of little value in products that are terminally sterilized where the possible contamination rate (based on an Fo approach) is very much lower than 1 in 1,000,000. Terminally sterilized products can therefore be exempt from the test as long as bio burden and autoclave monitoring (physical measurements) are applied. However, the sterility test is still mandatory for aseptically produced products. One additional problem is contamination of test material during the sterility test itself, this invalidates the results and it is vitally important that the procedure is carried out under specific conditions. Usually under class A environmental standards or increasingly in isolator units which can be maintained to levels of microbiological cleanliness far greater than class A.
As stated above, the test requires the product to be exposed to a suitable nutrient medium to support microbial growth; this is difficult if large volumes (500 ml) are being tested. There are currently two approaches to this problem. The classical technique was to use direct inoculation (BP method 11) of the product into the medium, but this limits the volume that may be tested and can be problematical if the product contains antimicrobial agents (e.g. preservatives). The modern method of handling large liquid (or readily soluble) samples is to use membrane filtration (BP method 1). In this procedure, the entire contents of the containers under test are divided and passed through one of two sterile bacteria-proof membranes (0.45µm pore size) set up in a special filtration assembly. After filtration of the contents of all the test containers through the filter units these latter become containers for sterile media (one for fluid thioglycollate and the other for soya casein digest) and are then incubated accordingly. This has the dual advantage of testing the complete contents of the container and eliminating any antimicrobial substances, which may have interfered with the test. If viable organisms had been present in any of the test containers, the growth of these in the units can be detected in the usual manner. There are several feathers of the sterility test that are common irrespective of the method used and these are considered below.

Fabric and Garment Testing for Cleanrooms, Flame Resistance, and Sterlization Compatibility

FABRIC TESTING
The tests performed on non-woven fabrics manufactured in the U.S. are similar to the tests performed on all woven fabrics manufactured worldwide. Some fabrics are calendared, which means they are treated with heat and pressure.Calendared fabrics feel lighter and are softer to the operator’s skin.
Some of the most common tests and the standards applicable to those tests are:

• Weight	ASTM-D-3776
• Thickness	ASTM-D-1777
• Grab Tensile	ASTM-D-1682
• MVTR	ASTM-E-96B
• Air Permeability	ASTM-D-737
• Pore Size	Coulter porometer
• Abrasion tests	Wyzenbeek/Taber
• Suter Hydrostatic	AATCC-127
• Spray Rating	AATCC-22
• Flammability	ASTM F-1506, 16CFR Part 1610 or NFPA 70E
• Surface Resistivity	ASTM-D-257 or AATCC-76
• Static Electricity Decay	FTM 4046
• Bacterial Filtration Efficiency	Modified Ford Peterson Method

What these test results indicate to the end-user:
Weight: Heaviness in ounces when one square yard is measured. A lighter fabric contributes to operator comfort.
Thickness: Measurement of fabric width in millimeters. A lower thickness is preferred because thickness directly correlates to weight.
Grab Tensile: Measures the durability of the fabric by measuring the breaking strength of the yarns.
Moisture Vapor Transmission (MVTR): Describes the amount of water in grams that passes through one square meter of fabric in 24 hours. More moisture passing through the fabric translates to more comfort for the operator. Moisture build-up causes the operator to feel hot because of the increase in humidity between the fabric and the body.
Air Permeability: The ability of a fabric to allow air to pass through it, which is quantified by a volume:time ratio per area. Air flow in heating and cooling processes, such as the cooling process of the body, contains contaminants that can be transferred to the product. The lower the permeability or transfer of air from within the garment to the outside, the lower the contamination to the product.
Mean Pore Size: Describes the average size, in microns, of spaces between the weave of polyester in fabric. Particles emitted from the operator (from skin and clothing) can be reduced with a smaller pore size.
Wyzenbeek Abrasion: Measures the abrasion resistance of a fabric by rubbing the fabric against a wire screen.
Taber Abrasion: Measures the abrasion resistance of a fabric by rubbing the fabric with abrasive wheels.
Suter Hydrostatic: Measures the amount of pressure in centimeters of water needed to force three drops of water through any fabric. This test relates to the pore size of the fabric. Fabrics designed to have higher hydrostatic values will allow less particles to pass from operator to product.
Spray Rating: Measures the ability of a fabric to resist wetting. Fabric performance may be enhanced with agents other than polyester. In the case of the Integrity series, a thin film of Teflon molecularly bonds with the polyester threads. This becomes another layer of protection between the operator and the environment. Fabrics designed to have higher hydrostatic values will allow less particles to pass from operator to product. This protective layer reduces absorption of liquids and qualifies this fabric as splash resistant. Optimum spray ratings are between 80 and 100 (maximum) because these fabrics absorb little to no water when sprayed (hydrophobic).
Flammability: Refers to special cleanroom flame-retardant fabrics and garments below.
Water Impact: Method of testing splash resistance or the ability for fabric to resist absorption of liquids measured in grams of liquid penetrating fabric. The lower the mass of liquid allowed through the fabric, the better protected the operator will be from spills in the cleanroom environment.
Static Electricity Decay: Quantifies the ability of a fabric to reduce an electrical charge, measured over time. The less time it takes a fabric to reduce an electrical charge of 500 volts to 50 volts, the better its performance in a cleanroom environment. Static electricity in the working environment due to instrumentation, movement, etc. can cause two forms of product failure. Charged particles around the operator will migrate and cause particle contamination, and a charge will reach a critical point and then discharge from the operator to the product in the form of a spark.
Surface Resistivity: Quantifies the resistance of a fabric to pass an electrical charge through it measured in Ohms from point to point. The resistivity of a garment must be within the electrostatic dissipative range which is 105 ohms/sq. to 1011 ohms/sq. Within the ESD range of 105 ohms/sq. and 1011 ohms/sq., any electrical charge will be broken down to 0 volts over a small period of time and no electrical charge will enter the product. Fabrics outside this range may cause electrical discharge and product failure.
Bacterial Filtration Efficiency: Assesses the ability of the fabric to contain viable particles.
Additionally, some cleanroom fabric manufacturers have developed “in house” test methods, such as testing for the presence or absence of an antimicrobial if the fabric has an antimicrobial agent suffused within the weave. A fabric with an antimicrobial agent controls the growth and transference of living particles to an operation. A general decrease in microbial population, cleaner (on particulate level, due to a reduction in the number of living particles), and less bioper-meation of microbes through the pores of the garment will result with the addition of an antimicrobial agent. The efficacy of the antimicrobial is directly proportional to the durability of the antimicrobial agent in the fabric.
GARMENT TESTING
The most important, universal specification for cleanroom apparel is that it is appropriate to protect the cleanroom process or product manufactured in the cleanroom and that it is always worn correctly.
There are many associations worldwide that recommend tests for non-woven and disposable products, including INDA (United States), EDANA (Europe), and ISO (international). However, none of these groups sets criteria for material consideration in cleanrooms. For example, the Gelbo test is a method described in INDA IST 160.1 1995, EDANA Method 220.0 1996, and ISO 9073-10-2003 (each method being slightly different but with the same basic approach), yet none of these tests contains specifications for cleanroom apparel for use in certified cleanroom classifications. Members of the IEST working group 3 are evaluating and creating recommendations for cleanroom non-woven garments for the IEST-RP-CC003.4 revision.
HELMKE TUMBLE TEST
Only IEST-RP-CC003.3 recommends that apparel used in cleanrooms meet Category I particle cleanliness derived from the Helmke Tumble test. The classification table describes cleanliness categories in size ranges of 0.3 microns and0.5 microns.
Both the ISO 9073-10-2003 and IEST-RP-CC003.3 documents stress ranking of all results to create more reproducible data. The IEST-RP-CC003.3 Working Group performed a statistical, round-robin Helmke Tumble test in 1999 and established the ranking of woven cleanroom garments (medium coverall, frock, or five hoods) into categories I, II, and III at both 0.3 and 0.5 microns per cubic foot minute of air cumulative.1
ADDITIONAL PARTICLE TESTING METHODS
There are other recommended tests for cleanroom apparel listed in IEST-RP-CC003.3. In addition to the Helmke Tumble test, which tests a cleanroom garment for the cumulative number of particles 0.5 micron and greater per cubic foot of air per minute, the particle dispersion test (body box test) specifically addresses the particle filtration containment of the full garment system in a 100% HEPA- (or ULPA-) filtered environment. The releasable large particles test, similar to the method outlined in ASTM F-51 Appendix X1, evaluates the fabric of the cleanroom garment microscopically (100 ) for fibers and larger particles (5 microns and greater). The microbial penetration test assesses the garment fabrics ability in preventing penetration of both viable and non-viable particles. The combination of the results of all these tests provides a comprehensive evaluation of the barrier and shedding propertiesof the cleanroom fabric or garments.
ESD TESTS
A cleanroom fabric or garment that contains a conductive yarn in a stripe or grid pattern is tested for its ability to dissipate static electricity. Conductive yarn reduces any electrical charge and attraction between particles around the operator. Particles [living (viable) and non-living (nonviable)] are small and will be affected by electrical fields created by operator movement. An electrical field will cause a particle to be charged and move toward oppositely charged particles. Particle movement is reduced if the electrical field is reduced. Beltron is the most frequently used cleanroom-/gamma-compatible conductive thread. Conductive thread is woven in a grid or stripe patternto dissipate static electricity.
The most common ESD tests required for cleanroom fabric and apparel are:

• Static Electricity Decay	FTM 4046
• Surface Resistivity	ASTM-D-257
• Surface Resistance @ 50%	RH ESD STD 2.1

Additional ESD testing may be specified in the scope of work in a customer’scontract based on the protection of the product or process in the cleanroom.
TESTING FOR RESIDUAL ELEMENTS
There are some applications that are sensitive to residual elements if present in the cleanroom garment items. Extraction tests can be performed to determine the qualitative presence or absence of anions or cations, nonvolatile residues, volatile residues, silicone, or antibiotics and if residual elements are present in the cleanroom garment items, the concentration can be determinedin subsequent quantitative analysis.
DEVICE BIOBURDEN/AAMI STERILITY TESTS
All cleanroom materials used in sterile cleanroom applications must have a quarterly dose audit test performed and be validated for sterility assurance levels using ANSI/AAMI/ISO 11137 – 2006 Parts 1, 2, 3, if the sterilizationmethod is gamma or e-beam radiation.
Typically, an FDA-regulated manufacturer that is producing a product in an aseptic cleanroom with a final product sterility level of 10-6SAL will specify that all materials required during the manufacturing process be validated and certified sterility to 10-6SAL. However, an FDA-regulated, terminally sterilized, non-implantable medical device manufacturer in a non-sterile cleanroom may specify that all cleanroom materials used in the process be validated and certified sterility to 10-3SAL.
It is in the best interest of the cleanroom materials manufacturer to reduce the average device bioburden levels during the manufacturing process to in turn reduce the level of gamma radiation required to terminally sterilize the product to the sterility assurance level required by the customer. It is also most cost effective to radiate the manufactured materials to the same sterility assurance level. Therefore most suppliers of sterile cleanroom materials validate their processes to deliver sterile clean-room reusable or disposable materials at 10-6SAL (sterility assurance level) to serve a broader, universal market. ANSI/AAMI ST67:2003 “Requirements for Products Labeled “Sterile” addressesrecommendations for garment sterility.
TEST METHODS FOR ETO/STEAM STERILIZATION
The preferred sterilization method for apparel in the U.S. is gamma sterilization based on validation using the ANSI/AAMI/ISO standards mentioned above. However,all the components used in the manufacturing of the cleanroom apparel must be compatible with the sterilization method chosen as well as be cleanroom compatible. Presently, gamma sterilization is the most cost effective method of sterilization for cleanroom materials. However, cleanroom apparel may be validated andsterilized using E-beam sterilization using the same ANSI/AAMI/ISO standards.
Cleanroom apparel may be sterilized using ETO. Sterility validation is performed using ANSI/AAMI/ISO 11135:1994.
Cleanroom apparel used in the United Kingdom and Europe may also be steam sterilized. However, steam sterilization of cleanroom apparel causes shrinkage and wrinkling of the reusable garment system. Sterility validation of steam sterilization is performed per ANSI/AAMI/ISO 11134:1993 for industrial facilities or ISO 13683:1997 for health care facilities.
TESTING OF FLAME RETARDANT FABRIC AND GARMENTS
Traditional flame resistant clothing is constructed of flame retardant treated cottons or an inherently flame resistant material, like DuPont Nomex®fiber. These garments will shed particles that will compromise the integrity of the cleanroom and contaminate the products and processes in the cleanroom. The FDA regulated industries mandate that if these garments are worn in a sterile cleanroom environment, they must be validated for sterility compatibilityas well as cleanroom compatibility.
NFPA 70E
Compliance to NFPA 70E in cleanroom environments requires that all personnel working on electrical equipment operating at greater than 50V wear arc-flash protective garments to prevent injury. Polyester is specifically prohibited under any circumstances when exposed to live electrical parts operating at greater than 50V. The automotive industry has been using cleanroom FR garments meeting ASTM F1506 for workers exposed to electric arc for several years in their cleanrooms and recently the pharmaceutical and semiconductor industrieshave begun wearing these clean-room FR garments in their manufacturing cleanrooms.
CLEANROOM FLAME RESISTANT FABRIC
Dupont’s filament Nomex®is used to create the flame-resistant characteristic in fabrics for cleanroom applications. Normal woven Nomex®yarn generates particles in the cleanroom; however, the filament Nomex®used in clean-room FR fabrics uses the same Nomex®chemical structure in a filament form to replace the fibrous forms used in most Nomex®fabrics. In most of the FR cleanroom fabrics filament Nomex®and carbon yarn is combined and woven into fabric. This resulting fabric is flame resistant, clean-room compatible,and static electricity dissipative.
CONSTRUCTION OF CLEANROOM FLAME-RESISTANT GARMENTS
Typical cleanroom garments constructed of cleanroom FR fabric meet NFPA 70E Category 1. Seam construction of cleanroom FR garments must comply with IEST-RP-CC003.3 (i.e., 100% Nomex®filament thread for sewing, serging of all rough edges and flat-feld seams, etc.) to assure cleanroom compatibility, durability of the seams, and encapsulation of particles. All other components (i.e., zippers with protective tape, protective snaps, tunnelized neoprene wrist closures, etc.) in the garment must be cleanroom compatible and flame resistant as well. Flame-resistant cleanroom garments must meet ASTM F1506 and be labeledas such to meet NFPA 70E.
VALIDATION OF CLEANROOM FR GARMENTS
The validation of the cleanroom flame-resistant garment system includes all the results of the tests performed to confirm cleanroom compatibility, gamma compatibility, and flame resistance. Testing of cleanroom FR garments must be performed to validate arc-flash resistance per ASTM F 1959 to determine the arc rating. The sterility of the garment per ANSI/AAMI/ISO 11137-2006 over time must be validated in the FDA-regulated industries as well as thedurability of flame resistance after many exposures of gamma radiation.
ADDRESSING THE COMPROMISE OF CLEANROOM PROTOCOL AND FLAME RESISTANT PROTECTION
The specifications of cleanroom compatibility, sterility compatibility, and flame resistance (both HRC Categories 1 and 2) characteristics are clearly defined by industry standards. Constant research and development of flame-resistant fabrics and the construction of flame-resistant cleanroom garments is being conducted by fabric and garment manufacturers worldwide and new dual-layer systems are available now that offer cleanroom compatibility, sterility compatibility,HRC 1 and 2 compliance, and wearer comfort.
A more in-depth presentation of this information and discussion of fabric and garment tests will be performed by a panel of industry experts at a technical session at ESTECH 2007, the annual technical meeting of the
References
1. “Improving the Repeatability and Reproducibility of the Helmke Drum Test Method” and “The Size Distribution of Particles Released by Garments During Helmke Drum Tests”, Journal of the IEST 44, no.44 (Fall2001).

Understanding Key Issues Affecting Long-term Wall System Performance

Jim Hendley

Impact, thermal shock, abrasion, chemical staining agents, water exposure, and UV lights are a few of the things that can damage vivarium walls. Selecting the right high performance coating can improve the appearance and longevity of wall finishes in these demanding environments.
When you look at your vivarium walls and ceilings, it probably isn’t apparent that a lot of work goes into selecting those finishes. Most people talk about what type of paint they have on these surfaces when in fact, there is more to it than that. The high performance coatings used in vivariums withstand a wide array of operating conditions. This article reviews useful information affecting the selection and longevity of your high performance wall and ceiling coating systems.
Paint versus High Performance Coatings – the Differences
Protective coatings come in many chemistries, performance properties, and thicknesses. For the sake of this article, I’ve divided them into two classifications, paints and high performance coatings (HPCs). Changing from light to moderate duty surface protection to more demanding environments requires the use of HPCs to insure adequate performance.
Paints are thin-mil (a mil is a thousandth of an inch) systems designed to provide uniform pigmentation/color/hiding of the color beneath it. They typically are between 0.5 to 3 mils in total film thickness. Paints are typically used in offices, break rooms, and other areas not subjected to abuse, wash down, or regular water exposure. They are normally standard acrylics or water-based acrylic epoxies. Thin-mil coatings offer a cost effective solution in less demanding environments.
HPCs range from 5 to 50 mils, and often involve multiple coats and a range of reinforcement options. Materials range from 100% solids epoxies, high build hybrid acrylics, water-based, and/or high solids urethanes. HPCs are used in wet areas, spaces subjected to wet sanitization, impact zones, surfaces subject to thermal shock, and walls and ceilings subjected to flexing.
Paints are typically in the “catch-all” paint section of architectural specifications. HPCs often have their own architectural specification section because of the special skills required to install them. When HPCs are specified, one contractor is often responsible for the installation of the resinous floor, wall, and ceiling systems. This helps insure all of the architectural detailing associated with these systems are installed properly.
Substrate Considerations
What you put your paint or HPCs on top of matters. Vivarium substrates can consist of standard drywall, moisture resistant board products, cast in place concrete, precast concrete, and concrete masonry units (CMU). Standard drywall should only be used in dry areas with minimal risk from impact damage. Moisture and impact resistant board products are good for occasionally wet applications. When board products are hung on the wall, the screw heads and seams need to be filled with special drywall compound to cover these imperfections. The degree of fill and number of steps taken is referred to in the industry as level of finish. Level of finish is an important consideration for both standard and moisture resistant board products. Too low of a level of finish can result in telegraphing of the screw heads and seams through paint or HPC systems. Too high of a level of finish can cause delamination of wall and ceiling systems with even light impact or thermal shock.
For areas subject to frequent wash down and wet conditions, concrete masonry units (CMU), or cast in place and/or concrete are used. CMU substrates require correction of surface voids and imperfections prior to paint or HPC application. Filler options include: pure acrylic resin coatings applied in multiple coats, acrylic modified cement, epoxy cement, or epoxy gel.
Selection of the CMU filler depends upon the degree of fill, smoothness, and levelness required along with consideration of impact, wash down, and thermal shock exposure of the area. Typically, the higher the level of cleanliness required, the more filled the surface of the CMU must be and the higher level of skill required to install it.
Below grade poured concrete substrates can be placed utilizing self-compacting concrete to minimize bug holes. These substrates should be tested for moisture content prior to application of non breathing paint or HPC systems. If moisture content is high, topical treatments can be utilized prior to application of the protective coating to address this condition.
Detailing for Success
Surface intrusion of water or sanitizers into and behind wall and ceiling protection systems can cause substrate damage, peeling, blistering, and, in certain cases, back-side mold growth. In addition, proper allowance for building movement and expansion and contraction of dissimilar materials can prevent cracking. To address these conditions, proper treatment of architectural details is critical. Areas of concern include: proper detailing of expansion joints, utilizing appropriate caulk, treatment of inside corners, wall ceiling transitions, termination at door frames and embedded fixtures and penetrations, and top of cove base transition. Standards for proper treatment of these conditions should be clearly documented in the architectural drawings as part of the contractor bid package. A final check point to insure the subcontractor selected for your project is capable of properly installing these details is the project mockup. Project representatives should inspect and sign-off on the mockup prior to installation of significant square footage of the HPC on your project.
Project Planning – the Cross Functional Team
Understanding how your current vivarium wall and ceiling finishes are performing is an excellent starting point in planning a new or renovation project. A project team consisting of facilities planning and design, vivarium management, space users, and maintenance staff can document both pros and cons of the systems currently in use. Gather historical information on the type and location of each finish and how they have performed. Note cracking, peeling, blistering, loss of gloss, pin holing, chalking or yellowing, problems with impact damage, cutting, gouging, and staining with the corresponding coating used in each type of space (clean corridor, dirty corridor, specific types of holding areas, necropsy, etc). Maintenance department input concerning difficulties associated with certain finishes due to cure time, odor, complexity of repair, need to recoat an entire room due to discoloration, etc. should be taken into consideration.
Anticipated usage condition information for your current project can also be gathered. This can include anticipated impact, thermal shock, abrasion, chemical and staining agents, and water exposure, UV lights, and possible damage from certain types of animals, caging systems carts, etc. The more detail by area the project team can provide to your outside design team, the better. For example, typically, the cage wash is considered separately because it is one of the toughest performance environments for wall and ceiling systems. Compilation of the information the team has gathered, including must-have and “would like to have” HPC performance requirements by area, prepare the project team for discussion with the design team.
Conclusion
Wall and ceiling coating systems in vivariums can provide lasting protection and aesthetics. A critical look at your current situation and the use of the areas can dramatically impact the type of system needed to completely protect your wall. Remember the difference between paint and high performance coatings—it will impact your decision how to best guard against mold, cracking, or any other areas that would jeopardize a safe, clean workplace.

Advances in Laboratory Animal Infusion and Sampling

Paul Loughnane

Andrew Jacobson

Recent technical updates include smart, networked systems and refined models for catheterization.
In both clinical and pre-clinical settings, researchers often need to infuse compounds or withdraw samples of blood or other body fluids. While there is some overlap, notably the infusion pumps, for the most part the devices used with laboratory animals are completely different from those used with humans. In 1999, Jacobson noted in Healing and Smith’s Handbook of Pre-Clinical Continuous Intravenous Infusion,¹ “the tethered infusion systems of today look remarkably similar to those of 1970.” However, as we report in this paper, the pace of technological change has increased remarkably over the past few years. While the infusion models from the 1960s through the 1990s have generally held up, the devices used in these models are in the midst of a substantial transformation.
Systems that automate both infusion and sampling can improve accuracy, operational efficiency, and animal welfare. New methods of connecting fluid lines to experimental animals are reducing failure rates and are much easier to use. Finally, new resources for training and collaboration will continue to develop and spread infusion and sampling best practices in the future.
All of these advancements are aligned with, if not driven by, the 3R principles of reduction and refinement.
Automation
Researchers in the fields of toxicology and safety pharmacology are the most demanding users of infusion equipment. Running a conventional infusion study with dozens or even hundreds of animals is highly labor intensive. For example, infusion rates must be calculated to adjust the dosage to each animal’s weight, infusion pumps must be programmed individually, pump alarms must be monitored and responded to, and data must be collected and input into laboratory information management systems (LIMS). Each manual step—and there can be tens of thousands of keystrokes to program a study’s worth of conventional pump data—is an opportunity for an error. Furthermore, the extended time technicians must spend in the animal room working with the equipment can stress the animals.

Product Type	Product Name	Company
Infusion Study Automation Software	AVA WIFI™	AVA Biomedical
Infusion Study Automation Software	Orchesta™	Instech Solomon
Infusion Study Automation Software	Axios™	Strategic Applications
Automated Blood Sampler	Culex®	Bioanalytical Systems
Automated Blood Sampler	AccuSampler®	CMA Microdialysis
Automated Blood Sampler	ABS2™	Instech Solomon
Dried Blood Spot Cards	Whatman® FTA® DMPK	GE Healthcare

Table 1: Automation Products
Systems that combine smart, networked pumps with centralized control and monitoring software are now available to automate this process (Table 1). A researcher will set up the program for an entire study just once. The system calculates flow rates for each pump using imported weight tables, and then sends the information to each pump
 via a wireless or wired network. The central monitoring PC displays alarms, such as occlusions, and can send that information to remote employees via email or text message. It automatically documents events and user interventions,
replacing the vast majority of paper documentation with electronic data.² These systems have the potential to revolutionize the processes of large scale infusion studies (Figure 1).
Automated systems to withdraw and store blood samples from rats were first developed in the mid 1990s and have already had a significant impact in the departments that
have implemented them. A computer-controlled set of pumps and valves that withdraw precise samples at programmed time points without disturbing the animal replaces the labor-intensive and stress-inducing process of manual blood withdrawals. These systems require researchers to redesign procedures, but nonetheless acceptance has grown steadily over the past decade, particularly in drug metabolism and pharmacokinetics (DMPK) research.As use has become more widespread, the systems have evolved from initial designs to more reliable, easier-to-use second- and third-generation machines. Some of the new systems can sample from mice or large animals in addition to rats (Figure 2).
One of the most exciting developments in blood sampling is the dried blood spot (DBS) technique.³ Vials of 150-500μL of blood which must be refrigerated and spun down are
replaced with a 15μL drop placed on special paper. DBS samples can then be stored at room temperature and shipped in conventional envelopes. In addition to operational efficiency, this technique’s small blood volume
can mean more samples per animal and thereby a reduction in the number animals needed for a given study. This technique is currently available with cards for manual sampling and will be combined with automated sampling in the near future (Figure 3).
Improved Connections
In a traditional tethered infusion model, an animal is catheterized in an initial procedure and the proximal end of the catheter is plugged and tucked under the skin while the animal recovers. In a subsequent procedure, the catheter is exteriorized, a harness or jacket is placed around the animal, and the catheter is routed through a spring tether up to a swivel for further connection to an infusion pump outside the animal’s cage. Related models use implanted buttons, with the catheter externalized through the center of the button, or tail cuffs for externalized tail vein catheters.

Product Type	Product Name	Company
Rat Tethers	FastTether2™	AVA Biomedical
Rat Tethers	Vascular Access Harness™, Vascular Access Button™	Instech Solomon
Rat Tethers	Quick Connect™ infusion system	Strategic Applications
Programmable Implantable Pump	iPrecio®	Primetech Corporation
Large Animal Port	InLine™ Port, OmegaPort™	Access Technologies
Large Animal Port	Cath-In-Cath™	AVA Biomedical
Large Animal Port	PortHold™	Instech Solomon

Table 2: Recent Connection Products
Several new systems have been designed for rats that refine these models (Table 2). In each, the catheter is attached to a special harness or button at the time of catheterization, eliminating the second procedure and associated recovery time. These new harnesses or buttons feature connectors such that a mating tether can simply be plugged in when the infusion study is to begin. Similarly, the tether can be easily disconnected when the animal needs to be weighed or when the infusion study is complete. Some of these systems are further designed so that the fluid path seals off when not connected, reducing the chance of contamination and infection and preventing retrograde flow that can lead to an occluded catheter.
Most recently, versions of these harnesses are available with connections for two catheters, simplifying procedures such as simultaneous infusion and blood sampling, or bile sampling, where bile can be routed through an external loop in the harness back to the duodenumunder resting conditions, but then is easily diverted for sampling by connecting a mating two-channel tether (Figure 4).
Ideally, an experimental animal would not be tethered, removing the physical restraint and permitting multiple housing. The implantable Alzet® osmotic pump (DURECT Corporation) has long been an option for rodent infusion for those that can adapt their protocols to the fixed flow rates and reservoir volumes that are available from the manufacturer. The recently introduced iPrecio® pump offers a flexible alternative. The 8g pump is small enough for implantation in rats, but includes a miniature peristaltic mechanism and 900μL reservoir that can be refilled through the skin. The pump is programmed prior to implantation and can administer simple or complex flow profiles with rates from 1 to 30μL/hr (Figure 5).
Larger animals can wear a jacket with a pocket for an ambulatory infusion pump. In this case, vascular access is often gained via an implanted port similar to those used in human medicine. Here too there have been advances. While implanted ports can remain patent for years, failures can occur when the animal dislodges the needle from the port.The PortHold™ features a titaniumplate under the septum with holes that are slightly larger than the needles used to access the port. These holes prevent the needle from working it sway out of the port as the animalmoves. The Cath-In-Cath ™ port and the InLine™port prevent needle dislodgements by advancing a percutaneous, through the- needle catheter (instead of a port needle) several centimeters through and past the port septum (Figure 6).
Training and Collaboration Resources
Researchers that are new to animal infusion or sampling now have many resources to draw on. Hands-on training courses in microsurgery are held in specially designed facilities, or instructors can develop a custom course to be held at your site. Online courses are available to users around the world in subjects as specific as rodent catheterization and osmotic pump implantation.
For those that do not have the surgical expertise in-house, most animal vendors offer catheter implantation as a surgical service. Furthermore, these vendors can also install the new harnesses discussed above, reducing the burden on the researcher when the animal arrives to a simple connection of a mating tether.
Experienced researchers also have new resources (Table 3). Discussion groups on internet sites such as LinkedIn are now commonplace. Harlan Laboratories has held several focused “Infusion Seminars” in Europe over the past several years, with participants from pharmaceutical companies, contract research organizations (CROs), academia and industry presenting and sharing ideas. For example, Harlan Laboratories Switzerland presented their efforts to optimize their equipment and procedures, focused primarily on long-term catheter patency, in order to extend their infusion studies out to 13 weeks.⁴

Product Type	Organization
Seminars; Training Courses; Publication	Academy of Surgical Research
Training Courses, live	Charles River Laboratories
Training Courses, live	Columbia University Medical Center Microsurgery Research & Training Laboratory
Seminars; Training Courses, live	Harlan Laboratories
Seminars; Publication	Infusion Technology Organisation
Training Courses, live	René Remie Surgical Skills Centre
Training Courses, online and live	Veterinary Bioscience Institute

Table 3: Training and Collaboration Resources
Taking this concept one step further, in 2009 a group of researchers in Europe working in different, and sometimes competing, companies formed the Infusion Technology Organisation. Its goals are broad: “to provide a forum by which information and data generated from infusion technology can be shared on a global basis.”
These advances in equipment and techniques are already producing results. For example, Dr. Russell Bialecki at AstraZeneca has combined infusion, automated blood sampling, radio telemetry (measuring parameters including blood pressure, heart rate, temperature, and ECGor EEG), and collection ofmetabolicwaste.With this “integrative pharmacology” model they collect data in a single experiment that would traditionally require several. In addition to the obvious benefits of a reduction in the number of animals and the time and expense to conduct separate studies, often the availability of the simultaneously-collected data can help researchers understand variability that would have otherwise been interpreted as noise.⁵
Researchers that are able to achieve long-term catheter patency not only have the option to run longer studies, but also to reuse animals after a wash-out period. Refinement methods such as remote control of infusion pumps, video assessment, and perhaps even robotic removal or introduction of fluids from the animal room can further reduce animal stress while streamlining research processes.
As researchers continue to make individual improvements and then share them in the various forums for collaboration—prompting manufacturers to develop new products in the process—the devices and techniques used in laboratory animal infusion and sampling will continue to advance in the future.
References

1. Healing, G. and Smith, D. Handbook of Pre-clinical Continuous Intravenous Infusion. Taylor & Francis, 2000. ISBN 978- 0748408672.
2. Haas R., Jacobson A., Sommers, J., Pawl S., Agate J., Brooks A., Alexander A. “Cooperative development of an automated syringe infusion pump for preclinical toxicology.” Poster at 2009 Society of Toxicology Meeting.
3. N. Spooner, R. Lad, M. Barfield (2009) Anal. Chem. 81 1557-1563 http://www.ncbi.nlm.nih gov/pubmed/19154107
4. Kaiser, S. “Continuous Infusion Toxicity Studies in Rats: Experiences and Developments at Harlan Laboratories Switzerland.” Presentation at Harlan Infusion Seminar, Paris France, 11 May 2010.
5. Kamendi, H.W., Brott D.A., Chen, Y., Litwin, D.C., Lengel, D.J., Fonck, C., Bui, K.H., Gorko, M.A. and Bialecki, R.A. “Combining Radio Telemetry and Automated Blood Sampling: A Novel Approach for Integrative Pharmacology and Toxicology Studies.” Journal of Pharmacological and Toxicological Methods (2010), doi: 10.1016/ j.vascn.2010.04.014

The 3 Green Rs - Reduce, Reuse, Recycle

Jack Metterville

Unwanted and unused vivarium equipment doesn’t have to sit idly taking up space. Stainless steel equipment and plastic caging can have a second life.
It remains topical and important to continue the dialogue concerning the initiatives of “going green.” We are reminded daily of the significant environmental and economic issues that face each of us. The conditions of the economy and environment challenge our ability to reach and maintain an enjoyable and healthy quality of life. We try to use less, reuse what we can, and recycle what we cannot. How we observe and react to economic and environmental challenges effects the manner in which we develop as individuals. It also impacts how we develop and implement our business strategies and the likelihood of success. Going green, zero carbon footprint, carbon neutral, carbon credits, greenhouse effect, global warming, sustainable energy, and green building are all terms that have become common and integral part of our lives.As such we are challenged to consider our buying decisions and conservation practices.
In some regards the current conditions of the economy force upon us the need to recycle. The world economic climate of the past few years has impacted our spending habitats dramatically. We have limited budgets and are forced to make our dollar go further. We recycle and reuse because it is the right thing to do while at the same time protecting the limited budgetary funds that are available to us. With this said, research must continue!
The animal research community is significantly concerned and invested in resource conservation. We are impacted by the struggles of the economy while continuing the effort to promote and support the initiatives of research for the purpose of developing novel human therapeutics. The efforts associated with the discovery and development of new drugs is among the most important endeavors we can pursue. These initiatives lead to remedies or cures of conditions and diseases that enhance or prolong our human experience. These efforts, however, are not undertaken without significant energies and investments in time and resources.
Waste Minimization Initiatives
Research institutions by necessity are large users of energy, construction materials, water, chemicals, and laboratory equipment and supplies. Government, university, hospital, and pharmaceutical research facilities are composed of millions of square feet worldwide. Hundreds of thousands of individuals spending hundreds of millions of dollars yearly comprise the workforce involved in animal research and drug discovery. The use of energies and products by these groups though important and critical is enormous. The good news is that even a slight decrease in the use of exhaustible resources by our members has a significant and positive impact on the overall resources not utilized.
Most organizations worldwide have implemented waste minimization programs. These programs are designed to conserve resources, create healthy and enjoyable work places, and save research dollars. The U.S. General Services Administration implemented the Resource Conservation and Recovery Act a decade ago. They mandated that federal members follow particular guidelines to ensure the appropriate purchase, disposal, and recycling of many different types of products. Search the Internet for major universities across the globe and you will be able to read how they have and are continuing to implement procedures to ensure resource and energy efficiencies. Pharmaceutical companies like Merck & Co., Pfizer, Sanofi-Aventis, Bristol-Myers Squibb, GlaxoSmithKline, Novartis to name just a few are fully invested to cut energy consumption, reduce waste, and promote recycling. These initiatives include how they buy and dispose of new and unwanted vivarium equipment.
Animal facilities are equipped with products such as cages and rack systems, animal transfer stations, waste disposal stations, metro rack storage systems, cage washers, autoclaves and sterilizers, bedding dispensers, water fill stations, load carts, and the list goes on. These are all products made from large quantities of stainless steel and plastic materials. Each of these animal facilities have support equipment that is extremely expensive to produce in terms of time and labor along with the costs of the raw materials required to produce the equipment. The processes used to manufacture these equipments also generate significant waste and by-products that must be handled safely and disposed of properly.
Stainless Steel
There is a predominant use of stainless steel in animal research for many reasons including durability, rust and stain avoidance, ease of cleaning, and protection from contaminants including bacteria. High quality stainless steel is resistant to breakdown caused by continuous use as well as sterilization by chemicals or autoclaving by high temperature steam. Proper storage and maintenance of stainless steel can basically avoid the induction of corrosion or pitting and extend its half life for a very, very long time. The process of stainless steel production requires large amounts of bulk products such as iron ore, silicon, chromium, nickel, and manganese. Through the use of large amounts of energy, water, and chemicals, the final product of high quality stainless steel is achieved. High quality stainless steel maintained under ideal conditions can be pit free for many hundreds of years. It does not leave much to the imagination what can happen to equipment made up of stainless steel that is not recycled appropriately. It sits and sits and piles up and piles up. Reusing of this important, expensive, and necessary material is an essential component of any responsible recycling initiative.
It should be noted that 100% of stainless steel can be recycled by melting and made into new stainless steel. Currently any new stainless steel product is made up of approximately 60%recycled stainless steel. The ability to make new products totally out of recycled stainless steel is limited only by the amount of recycled materials that are available. Therefore, no stainless steel product should ever end up in a landfill and just as important, no stainless steel product should go unused as it can be easily salvaged. Stainless steel salvaging and recycling is a large and efficient business worldwide.When you get stuck with unwanted products you can sell them, trade them, give them away but in the end you can always scrap it knowing it will be completely recycled. Scrap metal dealers are readily available to help you clear out your inventory and pay cash!
Plastic Caging
Plastic animal caging and various accessories are generally durable, scratch and break resistant, and tolerant of high temperatures (to avoid hazing and crazing)—all traits that support a long shelf life. Standard types of plastics such as polycarbonate, polystyrene, polysulfone, and various other high temperature cages are highly regarded and used because of their durability. New alternative single use or disposable plastic cage products are now available and promote their environmental and energy use friendliness. They are characteristically more readily biodegradable and eliminate the need for energy use such as water, heat, and chemicals. Individual institutions are left to establish their own opinions on the appropriateness and suitability of these disposable products for use in their facilities. The real challenge is to evaluate how we currently use our plastic products today, consider if we can do things differently, and always incorporate recycling into our work practices. Left to stockpile or disposed of improperly, plastics can become a significant toxic pollutant. Proper disposal and recycling of these items is not that hard! We just have to consciously decide to do so and make recycling a part of our standard operating procedures in the research laboratory as we often do in our own households. In most cases you would want it to be a natural instinct for our employees and our colleagues to recycle.
Though it is certainly possible, and the recycling of plastics is increasing, the challenges can be significant. Different types of plastics cannot be mixed during recycling. After grinding and during the melting process even small amounts of different types of plastics will ruin the end product. As in the stainless steel business there are many resources to assist you with the proper disposal and recycling of plastics. You may ask one of your vendors to assist you or you can find many resources on your own that will do a good and responsible job for you. However, unlike the stainless steel business, the value of plastics to be recycled is minimal. The value in recycling plastics is to understand that these unwanted products will not end up in the landfill but will be salvaged for another use.
New Equipment
There are many instances where the implementation of new products with newer and more efficient technologies such as ventilated animal rack systems, biological work stations, cage washers, or whatever makes us consider throwing out the old and bringing in the new. It is the throwing out part that should make us pause and think. For those that have the budgets and can afford the newer options, all the better. Hopefully, efficiencies are gained and resources conserved with the purchase of new and better products. But please remember the proverb, “One mans junk is another man treasure.” New and better doesn’t mean the old stuff is bad or unusable. Conversely because of the quality of the stainless steel and plastic of the previously used equipment, it is still usable in many instances. A challenge is created as to what to do with the “newly” unwanted cages and accessories. The answer today is to make the effort to find a means to recycle and reuse!
We have discussed the practicality of recycling unwanted stainless steel and plastic items. An alternative is to seek out reputable vendors who buy and sell animal equipment on the secondary market. It may be the case where these companies will pay more than the scrap value of the equipment you no longer need. Such companies could have the resources to restore damaged products and the contacts or distribution means to ensure that the cages and accessories receive a second life.When you are buying restored and reconditioned equipment by an experienced vendor you are receiving a quality product at a significant cost savings. Such a resource can be invaluable to those that did receive the “new budget” or just want to make sure their dollars go far.
Summary
The space along with the resources required to store and maintain unwanted cages and accessories can be huge. Every research facility should assign a cost required to inventory, maintain, and store equipment. When this is done or when you associate a dollar amount it starts to become obvious that the storage of no longer needed or wanted equipment is very expensive and unnecessary. Why tie up equipment and space for products you no longer have a need for or the space to store it in? You think you’re going to use it? Look at your own cellar or garage, what do you think now? Do something useful with it! Sell it, exchange it, donate it and at the very least scrap it but do something. Any and all of these options will result in recycling and perhaps extend the product life and hopefully benefit an organization or someone that did not get their “new budget” approved. Broken stainless steel can be rebent, cut, welded. Casters can be replaced. Motors can be exchanged. Filters can be replaced. Electrical components can be upgraded. What was broken can be fixed or reconditioned. Plastic can be ground, melted and reused to make new cages.
In today’s climate of economic and environmental concerns the need for conservation of resources dedicated to research is as critical as ever. All of us providing support need to continuously evaluate our responsibility to promote practical solutions to economize our budgets while maintaining the integrity of our surroundings. Recycling conserves exhaustible resources, promotes social well-being and “it just feels good to do the right thing.” Environmental responsibility and economical practicality are mutually beneficial and save time, money and resources. The practicality feature of recycling reduces energy and water use, cuts labor and material costs, increases efficiencies and productivity and can promote meaningful research.

Laboratory Animal Diets Formulated with Fish Meal

Carrie Schultz, Ph.D.

Kristi Thompson, Ph.D.

Liz Koutsos, Ph.D.

The benefits of supplementing diets with fish meal have been demonstrated in a number of species. Immunity and overall health are among some of the potential positive effects.
Fish meal has historically been used as a protein source in manufactured diets for animals ranging from beef cattle to poultry to laboratory animals. Fish meal is a concentrated source of high quality protein composed of highly digestible essential amino acids. Fish meal has one of the best overall amino acid profiles of any single protein source. In addition, fish meal is also an excellent source of omega 3 fatty acids, like DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid), which have been shown to improve health by preventing cardiovascular disease, lowering serum triglycerides, potentially stabilizing atherosclerotic plaques, improving tolerance to a variety of stressors, and playing important roles in the development and maintenance of neural and retinal tissues and cognitive function.^1,5,12,20,22
In recent years, fish meal has been removed from some laboratory diets. Animal proteins generally cost significantly more than plant proteins; thus, pressure to decrease ingredient costs has driven the removal of animal-derived proteins from some formulations. Concern that fish meal is contaminated with mercury and nitrosamines has led some researchers to believe experimental results could be confounded by diets. However, as detailed below, fish meal and fish oil used in animal diet production contain very low levels of this metal or N-nitroso compounds. Further, research conducted in the last twenty years has proven that nearly any ingredient in a diet can potentially alter experimental results, depending upon the nature of the experiment and the ingredient; soybean meal and casein have been the most often cited protein sources shown to influence study outcomes.
Sourcing of Fish Meal
The primary source of fish oil and fish meal in laboratory animal diets is derived from the menhaden fish. Menhaden are short-lived omnivores that feed primarily on phytoplankton, thus, they accumulate very little methylmercury or other heavy metals, unlike carnivorous predatory fish. Currently, two companies in the U.S. supply fish meal and fish oil from menhaden. Fish meal is produced from freshly harvested menhaden that are promptly delivered chilled and whole, in refrigerated vessels, to the processing facilities. Processing at the five plants in the U.S. is immediate to ensure product quality and minimize the presence of non-nutrient factors like nitrosamines.
Fish Meal in Animal Diets
Animals do not require protein per se, but instead utilize essential and non-essential amino acids. Thus, the quality of a protein source is positively correlated with the digestibility, bioavailability, and proportions of the amino acids in the protein source.6 Animal proteins are the gold standard for protein quality because they provide the closest amino acid composition relative to an animal’s requirements. Furthermore, the nutritional quality of diets combining grains and animal protein sources is greater than diets containing grains alone.^4,9
To date, very little information is available on the effects of incorporating fish meal into grain-based diets for rodents; however, the advantages have been published in other laboratory species such as dogs, poultry, and swine. Whole menhaden fish meal has been shown to increase the number of live pigs per litter at birth as well as the overall body weight of pigs at birth and two weeks post parturition.¹⁴ Similar results have been observed in laying hens fed a diet containing 3% dietary fish meal for a 12 week period. Hens receiving the fish meal diet had higher egg production rates, egg weights and egg volume (p<0.05) compared with hens fed a corn-wheat-soybean meal (SBM) diet.¹⁸ Or-Rashid et al¹³ evaluated the effects of providing fish meal to ewes during late gestation and early lactation on the proportion of DHA and EPA in colostrum and milk as well as the subsequent effect on the plasma fatty acid profile of nursing lambs. Ewes fed a fish meal supplemented diet had greater (p<0.013) percentages of EPA, DHA, total n-3-PUFA, total CLA and total very long chain n-3-PUFA in colostrum and milk compared to control ewes receiving a SBM based diet. At birth, lambs born to fish meal supplemented ewes had greater concentrations of plasma EPA, DHA, and total very long chain n-3-PUFA compared to lambs born to control ewes. Fatty acid concentrations also increased over time for lambs nursing ewes supplemented with fish meal.
Dietary fish meal may also be advantageous for immunocompromised animals. In poultry, both chronic and acute infections induced by coccidiosis (Eimeria acervulina) decrease growth performance and MEn and AA digestibility in chicks fed a corn-SBM diet. However, this same effect was not observed when chicks were fed a diet containing 15% dietary fish meal.¹⁵ The authors concluded the observed positive effects on growth and nutrient digestibility may be attributed to decreased intestinal inflammation caused by increased levels of dietary omega 3 fatty acids provided by the fish meal. Although concentrations of EPA and DHA are greater in fish oil, fish meal remains a good supplier of these omega 3 fatty acids as well as an excellent concentrated source of high quality protein.
Heavy Metal and Non-nutritive Contaminants
Mercury
Mercury occurs naturally in the environment and is an industrial pollutant that accumulates in bodies of water in the form of methylmercury (MeHg).²¹ It can be found in most species of fish and shellfish, but bioaccumulates in larger, longer living deep water species like shark, tuna, swordfish, and king mackerel. Methylmercury toxicity in humans has been associated with neural dysfunctions and cognitive deficits.
MeHg-induced dysfunctions and deficits observed in humans have been reproduced in laboratory animals and were observed as decreased motor abilities, coordination, and overall activity in C57/B6 mice¹¹ as well as impaired temporal and spatial visual function in primates.¹⁷ The degree and severity of neurotoxicity is dependent upon the dose and duration of exposure as well as the stage of development at which the animal is exposed.⁷ Roegge et al¹⁹ provided Long Evans rats 0.5 ppm MeHg via drinking water, continuously for 4 weeks prior to conception, during pregnancy and through postnatal day 16. MeHg did not significantly affect motor tasks or morphological parameters in the cerebellum of the weanling or adult rats. Colomina et al³ gavaged mice with 2 mg/kg BW/d MeHg chloride from days 15-18 of gestation and did not observe any significant effects on postnatal development in the offspring, even when the dams were exposed to additional stress during pregnancy. Laboratory diets routinely tested for mercury are typically below the limit of detection (0.025 to 0.1 ppm). Thus, an average size rat consuming 15 grams of diet per day could consume up to 0.00125 to 0.005 mg/kg MeHg BW/d, which is less than the known no-effect levels (NOELs) for neurotoxicity. However, published research findings indicate that both low- and high-dose MeHg can affect the behavior of many species. Thus, researchers investigating oncology or behavioral teratology, for example, may choose to use specially designed diets such as purified diets or grain based diets containing no fish meal. Additional information regarding the effects of MeHg on the neurodevelopment of laboratory animals can be found in the review paper authored by Castoldi et al.²
Nitrosamines
Nitrosamines were identified as carcinogens in a rodent model in 1956,¹⁰ and in the early 1960s sheep in Norway died of liver toxicity after eating herring treated with sodium nitrite. Nitrosamines are produced from nitrites and secondary amino compounds, and can be formed endogenously or prior to ingestion. Oxides of nitrogen formed during food processing, preservation and preparation can react with amino compounds and other nucleophiles to produce N-, C-, O-, and S-nitroso compounds.
A number of studies have demonstrated carcinogenic effects of N-nitroso compounds in a variety of animal species, suggesting that specific N-nitroso compounds are carcinogenic in all species. However, the affected organs and cellular targets within those tissues vary with species, dose, frequency of dose, and route of administration.¹⁰ The minimum dietary dose of N-nitrosodimethylamine (NDMA) that induces a detectable increase in cancer incidence in rats (which are more sensitive to NDMA than other rodent or lab species) is 2 mg/kg, and oral NDMA doses as low as 1 ug/kg BW are absorbed intact and reach the liver and kidney of the rat.⁸ At levels of <109 ug NDMA/kg BW/day (via drinking water) most rats survived to old age (28-31 months), with a tumor incidence of 3-13%. At levels of >109 ug NDMA/kg BW/day, most animals died of tumors.16 Concentrations (ug/kg) of NDMA in fish meal average 254 ± 16.4, whereas the average levels in most fish meal-containing diets are only 8.17 ± 0.40, suggesting some loss or degradation during the manufacturing process. This is also well below the dietary limit for carcinogenicity (2 mg/kg limit vs 8 ug/kg average concentration).
The two major sources of nitrogen oxides are from the addition of nitrate and/or nitrite to foods (e.g., curing of meat products and cheeses, pickling of vegetables) and from the heating and/or drying of foods in combustion gases (e.g., smoked foods, dried malt, dried milk products, dried spices). Prior to the use of refrigerated vessels, preservatives such as sodium nitrite were used to prevent spoilage of harvested fish for use in the animal feed industry. Of course, these additives are no longer used in menhaden fish ingredients. Laboratory animal diets are an essential and important component of animal research. If non-nutritive compounds in diets are of concern to you, consult with a nutritionist as other diet options may exist.
Conclusions
The advantages of supplementing diets with fish meal and/or fish oil have been demonstrated in a number of species due to the positive effects of omega 3 fatty acids on performance, immunity and overall health. Fish meal is a highly digestible protein source with a high content of amino acids, minerals, and vitamins. Under certain circumstances, researchers may prefer using diets without fish meal, but for most types of research the benefits of fish meal (amino acid composition, protein digestibility and contribution of omega 3 fatty acids) warrant its dietary use.
References

1. Bruner-Tran, K.L., Crispens, M.A., Ong, D.E., and Osteen, K.G., 2008. Dietary supplementation with omega-3 fatty acids reduces the reproductive impact of developmental TCDD exposure in mice. Fertility Sterility. 90: S48-49.
2. Castoldi, A.F., Onishchenko, N., Johansson, C., Coccini, T., Roda, E., Vahter, M., Ceccetelli, S. and Manzo, L. 2008. Neurodevelopmental toxicity of methylmercury: Laboratory animal data and their contribution to human risk assessment. Regulatory Toxicol. Pharma. 51: 215-229.
3. Colomina, M.T., Albina, M.L., Domingo, J.L. and Corbella, J. 1997. Influence of maternal stress on the effects of prenatal exposure to methylmercury and arsenic on postnatal development and behavior in mice: a preliminary evaluation. Physiol. Behav. 61(3): 455-459.
4. Edmonson, J.E., and Graham, O.M. 1975. Animal protein-substitutes and extenders. J. Anim. Sci. 41:698-702.
5. Federova, I, and Salem, N. Jr. 2006. Omega-3 fatty acids and rodent behavior. Prost. Leuk. Essent. Fatty Acids. 75: 271-289.
6. Food and Agriculture Organization/World Health Organization. 1991. Protein quality evaluation: Report of the joint FAO/WHO expert consultation, FAO Food and Nutrition paper 51. FAO, Rome, Italy.
7. Gilbert, S.G. and Grant- Webster, K.S. 1995. Neurobehavioral effects of development methylmercury exposure. Environ. Health Perspect. 103(Suppl 6): 135-142.
8. Gomez, M. I., P. F. Swann, and P. N. Magee. 1977. The absorption and metabolism in rats of small oral doses of dimethylnitrosamine. Implication for the possible hazard of dimethylnitrosamine in human food. Biochem J 164: 497-500.
9. Hernandez, M., Montalvo, I, Sousa, V., and Sotelo, A. 1996. The protein efficiency ratios of 30:70 mixtures of animal:vegetable protein are similar or higher than those of animal foods alone. J. Nutr. 126:574-581.
10. Lijinsky, W. 1999. Nnitroso compounds in the diet. Mutat Res 443: 129-138.
11. Montgomery, K.S., Mackey, J., Thuett, K., Ginestra, S., Bizon, J.L. and Abbott, L.C. 2008. Chronic, low-dose prenatal exposure to methylmercury impairs motor and mnemonic function in adult C57/B6 mice. Behav. Brain Res. 191: 55-61.
12. Muldoon, M.F. , Ryan, C.M., Sheu, L., Yao, J.K., Conklin, S.M., Manuck, S.B. 2010. Serum phospholipid docosahexaenoic acid is associated with cognitive functioning during middle adulthood. J. Nutr. 140:848-853.
13. Or-Rashid, M.M., Fisher, R., Karrow, N., AlZahal, O. and McBride, B.W. 2010. Fatty acid profile of colostrum and milk of ewes supplemented with fish meal and the subsequent plasma fatty acid status of their lambs. J. Anim. Sci. 88: 2092-2102.
14. Palmer, W.M., Teague, H.S. and Grifo, Jr., A.P. 1970. Effect of whole fish meal on the reproductive performance of swine. J. Anim. Sci. 31: 535-539.
15. Persia, M.E., Young, E.L., Utterback, P.L. and Parsons, C.M. 2006. Effects of dietary ingredients and Eimeria acervulina infection on chick performance, apparent metabolizable energy and amino acid digestibility. Poultry Sci. 85: 48-55.
16. Peto, R., Gray, R., Brantom, P., Grasso, P. 1991. Dose and time relationships for tumor induction in the liver and esophagus of 4080 inbred rats by chronic ingestion of Nnitrosodiethylamine or Nnitrosodimethylamine. Cancer Research. 51:6452-6469.
17. Rice, D.C. and Gilbert, S.G. 1990. Effects of developmental methylmercury exposure on special and temporal visual function in monkeys. Toxicol. Appl. Pharmacol. 102: 151- 163.
18. Rowghani, E., Boostani, A.D., Fard, H.R. and Frouzani, R. 2007. Effect of dietary fish meal on production performance and cholesterol content of laying hens. Pak. J. Biol. Sci. 10(10): 1747-1750.
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How Clean is Clean?

Steven Feinstein

Understanding the various cleaning levels can help you identify an appropriate safety and decontamination plan for your facility.
These days, issues of contamination control have been thrown into the daily spotlight, whether from the news reporting on a Norovirus outbreak on a cruise ship, a MRSA outbreak in the local hospital, or your fear of catching a cold from a co-worker or family member. These same issues exist in today’s laboratory, research, and production environments as well. This article will focus on the various levels of cleanliness as it applies to the laboratory area, and the appropriate methods of biological contamination control.
Cleanliness Defined
The various levels of biological cleanliness can be broken down into three categories: sanitization, disinfection, and sterilization. Sanitization and disinfection are both described as the destruction of “most”microorganisms on a surface, whether by heat or chemicals.When we take a closer look at the two, we are able to further define them quantitatively by the bio-burden reduction that each provides. The bio-burden is defined as the degree of microbial contamination or microbial load; or the number of microorganisms contaminating an object. To evaluate the bio-burden reduction, we start with a known number of spores and expose them to the agent and then evaluate if we have successfully destroyed all or some of the spores. Typically we start with a population of 4 log or 6 log (10⁴ or 10⁶) spores of a specifically known spore that is resistant to kill, such as Geobacillus Stearothermophilus or another similar spore.
Sanitization will offer a contamination reduction or bio-burden reduction of 99.9% or 3 log (10³). This means that we can expect that out of one million microorganisms, a sanitizer will destroy approximately 990,000 of the organisms leaving behind many viable microorganisms to reproduce. Sanitization is accomplished by utilizing chemicals and gels to achieve this level of cleanliness.
Disinfection will offer a bio-burden reduction of 99.99% and up to 99.999% or up to 5 log (10⁵). This means that we can expect that out of one million microorganisms, a disinfectant will destroy up to 999,990 of the organisms leaving behind very few, but still some, viable organisms. Disinfection is accomplished by utilizing many different chemicals or ultraviolet light.
Sterilization is the statistical destruction of all microorganisms and their spores. This is defined as 6 log (10⁶) or a 99.9999% reduction. Statistically, this definition is accepted as zero viable organisms surviving. Sterilization is accomplished via several methods including ionized hydrogen peroxide or other hydrogen peroxide based solutions, high heat, ultraviolet light, ozone, radiation, and chemicals (chlorine, formaldehyde, glutaraldehydes, etc.).
Now we can apply these definitions to their applications in today’s laboratory animal facilities. Here we find many types of surfaces, equipment, materials, people, and animals—all of which have contamination control challenges. You need to protect your lab workers, your animals, and, hopefully, the environment. To accomplish this, a detailed contamination control plan should be in place for all laboratory facilities.
Methods of Providing the Appropriate Level of Cleanliness
Sanitization
Sanitization can be accomplished in a very easy and inexpensive manner. We’ve all been trained from the time we were small children to wash our hands before eating. Washing your hands often will help protect you from germs. The Centers for Disease Control and Prevention (CDC) recommends that when you wash your hands with soap and warm water, that you wash for at least 15 to 20 seconds.When soap and water are not available, alcohol-based disposable hand wipes or gel sanitizers may be used. If using gel, rub your hands until the gel is dry. The gel does not need water to work. The alcohol in it kills the germs on your hands. Washing your hands before entering or leaving a laboratory should be a key component of any contamination control plan. By doing this, you will protect both your work from any germs that may have been on your hands prior to entering the laboratory, as well as protecting you from taking any of the germs outside of the laboratory. Hand sanitizing should be done each and every time you enter or leave the laboratory or any other critical area.
Disinfection and Sterilization
Decontamination is any activity that reduces the microbial contamination of materials or surfaces to prevent inadvertent infection. The appropriateness of a decontamination procedure depends on your goal.Do you wish to disinfect or sterilize?Will you be using the disinfectant on hard surfaces, in a biosafety cabinet, on instruments, or waste? Disinfection results in destruction of specific pathogenic microorganisms and refers to the elimination of virtually all pathogenic organisms on inanimate objects and surfaces thereby reducing the level of microbial contamination to an acceptably safe level.When choosing a disinfectant, one should consider the organism, the item to be disinfected, and the cost and ease of use of the disinfectant. Disinfection should be performed on all work surfaces and high touch areas including benches, countertops, bench top equipment, door and cabinet knobs, work surfaces in biosafety cabinets, incubators, etc., and will provide a higher level of cleanliness than sanitization.
Microorganisms vary in their resistance to destruction by physical or chemical means. A disinfectant that destroys bacteria may be ineffective against viruses or fungi. There are differences in susceptibility between various bacteria, and sometimes even between strains of the same species. Bacterial spores are more resistant than vegetative forms, and non-enveloped, non-lipid-containing viruses respond differently than do viruses which have a lipid coating. Information on the susceptibility of a particular microorganism to disinfectants and physical inactivation procedures can be found in the material safety data sheet (MSDS) for that agent. MSDSs provide additional details such as health hazards, containment requirements, and spill response procedures.
Direct contact between the disinfectant and microorganism is essential for disinfection. Microorganisms can be shielded within air bubbles or under dirt, grease, oil, rust, or clumps of microorganisms. Agar or proteinaceous nutrients and other cellular material can, either directly (through inactivation of the disinfectant) or indirectly (via physical shielding of microorganisms) reduce the efficacy of some liquid disinfectants. The majority of chemical disinfectants have toxic properties. Follow the manufacturer’s directions for use and wear the appropriate personal protective equipment (e.g. gloves, eye protection, apron), especially when handling stock solutions.
Sterilization refers to the destruction of all microbial life, including bacterial endospores. For example, surgical instruments must be sterile, but what about your facility and equipment? From an operational standpoint, a sterilization procedure cannot be categorically defined. Rather, the procedure is defined as a process, after which the probability of a microorganism surviving on an item subjected to treatment is less than one in one million, or a reduction of the bio-burden by 106. This is more commonly known as a six log reduction and often referred to as the “sterility assurance level.”¹
Traditionally, sterilization is best achieved by physical procedures such as steam autoclaving, which is the most practical option for the majority of laboratories for both sterilization and decontamination purposes of small equipment, supplies, and waste. However, steam sterilization is not practical in all applications. Instruments or materials which cannot withstand sterilization in a steamautoclave or dry-air oven can be sterilized with many different solutions, including ionized hydrogen peroxide or other gaseous sterilants. Ionized hydrogen peroxide is one of the newest technologies providing sterilization for rooms and buildings as well as providing excellent results for use on equipment, such as isolators, incubators, etc.
Hydrogen peroxide has long been known as an effective sterilant both in liquid and vapor forms.The process of aerosolizing and ionizing the aerosol positively charges the mist droplets causing them to disperse more like a gas than a non-ionized droplet because the droplets are now mutually repulsive, having the same polarity. These active droplets are attracted to negatively charged surfaces. Once the droplet attaches to the surface, the attachment point is no longer available for other droplets causing them to search for and attach to an unoccupied point.This process continues until the surface is uniformly covered by a very fine layer of sterilant, even covering hard-to reach areas such as the underneath side of ledges and small cracks and crevices.
A second result of ionizing the fine mist is the breaking apart (disassociation) of the constituents of the hydrogen peroxide into reactive species such as hydroxyl radicals, reactive oxygen species (ROS), and reactive nitrogen species. Micro-organisms (proteins, carbohydrates, and lipids) are destroyed by these reactive species through a process called lysing, or disintegration of the cell wall causing the exposure and killing of the cell nucleus.
The ionized hydrogen peroxide process offers many advantages—there is no precipitate or any other chemical residue as the only byproducts of the process are oxygen and water, which both safely evaporate into the atmosphere, making it a truly environmentally friendly “green” choice.
Developing a Decontamination Plan
If your laboratory is certified ABSL-3 or ABSL-4, you must pass several rigorous testing requirements as set forth by the CDC and the NIH. In order for you to meet those certification requirements annually, you will likely need to decontaminate your facility prior to the certification process. In addition to the annual certification, you may choose to decontaminate your facility in order to eliminate any risks of cross contamination between differing experiments, or have the need once a contamination problem has affected your work, animals, facility, or staff. Regardless of the reason, there will be a time when decontamination is required.
Part of your safety plan, contamination control plan, and laboratory certification will require you to have a decontamination plan in place.When evaluating and choosing a decontamination method, several factors should be considered:

• Will it be effective in killing the contaminants in the lab?
• Will it reach all surfaces, cracks, and crevices?
• Will it be safe on your equipment and surfaces, without causing any damage to them?
• Will it be able to be performed within your time constraints?
• Will it be environmentally friendly?
• And lastly, do you want to own your own equipment for this process, or contract the service to a competent company?

If you choose to purchase equipment, the purchase price should not be the only consideration. Maintenance costs and the training requirements should be considered. Do you have the appropriate personal protective equipment (PPE)? Is your staff appropriately trained to operate the equipment safely and effectively? Do you have the room to store this equipment?
If you choose to contract these services out to a competent supplier, consider the response time to an emergency (total time to perform the service and the technology utilized by the contractor). Be sure to confirm they have the appropriate insurances in place. Price should be an important factor, but decisions based on price alone usually turn out to be bad decisions.
It is always best to make these decisions prior to having an emergency need. Being prepared is a key component to a good safety plan and decontamination plan. Consult with a professional today so you can be prepared when the need arises, making an emergency or scheduled decontamination process easier to overcome.
Reference

1. Favero M. “Sterility assurance: concepts for patient safety.” In: Rutala W, editor. Disinfection, sterilization and antisepsis: principles and practices in healthcare facilities. Washington, DC: Association for Professionals in Infection Control and Epidemiology, Inc.; 2001. p. 110 9.)