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Biodecontamination is an imperative step to avoid cross-contamination, especially in functional animal facilities with high experimental turnover. Any contamination from animal rooms or outside sources can be transmitted to other areas that potentially affect animal health status, thus compromising re-search outcomes. A wide range of agents including quaternary ammonium compounds, phenol-based products, and alcohol are commonly applied for decontamination of animal rooms through various methods such as manual wiping, fogging, and vapor and gas exposure. In addition, H2O2 is commonly used as biocide agent; it decomposes readily to form water and oxygen, which are nontoxic3,30 and safe to use for effective biologic in-activation.11 In particular, vaporized hydrogen peroxide (VHP) has been recommended for the biodecontamination of a large variety of materials such as biologic safety cabinets,12 laborato-ries,13,16,25,28 and pharmaceutical contexts including production filling-line rooms, sterility testing environments, sealable enclosures, and lyophilizers.19 VHP-based decontamination methods are widely used as alternatives to formaldehyde24 because of their ease of use, higher levels of sterility assur-ance, and overall cost savings to a facility. Moreover, VHP is a broad-spectrum antimicrobial25 with virucidal, bactericidal, fun-gicidal, and sporicidal activity.15,19,26,27 Because the International Agency for Research on Cancer17 and Environmental Protec-tion Agency7 have classified formaldehyde as carcinogenic for humans, alternative chemical liquids and vapors for use as

decontamination agents are necessary in light of safety aspects. Consequently, research laboratories and hospitals must find a suitable decontamination method; VHP provides an alternative to formaldehyde fumigation because of VHP’s biologic efficacy against various microorganisms.10,15,22,28 Adding gaseous H2O2 to a standard low-temperature sterilization process may provide a useful method for prion inactivation,10 is residue free,22 and can be released into the atmosphere.

Chlorine dioxide is a greenish yellow, single-electron-transfer oxidizing agent that has a chlorine-like odor. Pure ClO2 is an unstable gas at room temperature and considered as true gas,4 therefore, it is generated as needed before decontamination and used in animal research facilities with appropriate sealing to avoid leaks from the enclosure. However, ClO2 is sensitive to decomposition by light and must be stored and used in ways that prevent direct exposure to sunlight.3,4,40 Unlike H2O2, ClO2 is a very selective oxidant42, with 2.5 times oxidizing power of chlorine. Due to this strong oxidizing ability, the gas is effective against a wide variety of organisms; it has shown sporicidal20 activity against Bacillus subtilis spores45 and is effective against Bacillus thuringiensis14 as well as Syphacia spp. ova.6

In light of these considerations, VHP or ClO2 might be espe-cially effective for biodecontamination after the completion of animal experiments.18 In addition, both agents have excellent material compatibility and have been tested in diverse ap-plications under laboratory conditions and used effectively in high-level containment facilities.8,27,31,42 To minimize con-tamination and achieve a 6-log reduction in microbial counts, a strategic plan was proposed, in which multiple H2O2- or ClO2-based decontamination units replaced the existing traditional

Improvement of Vivarium Biodecontamination through Data-acquisition Systems

and Automation

Shakthi RK Devan,1,* Suresh Vasu,1 Yogesha Mallikarjuna,1 Ramkumar Ponraj,1 Gireesh Kamath,1 and Suresh Poosala2

Biodecontamination is important for eliminating pathogens at research animal facilities, thereby preventing contami-nation within barrier systems. We enhanced our facility’s standard biodecontamination method to replace the traditional foggers, and the new system was used effectively after creating bypass ducts in HVAC units so that individual rooms could be isolated. The entire system was controlled by inhouse-developed supervisory control and data-acquisition software that supported multiple cycles of decontamination by equipment, which had different decontamination capacities, operated in parallel, and used different agents, including H2O2 vapor and ClO2 gas. The process was validated according to facility map-ping, and effectiveness was assessed by using biologic (Geobacillus stearothermophilus) and chemical indicator strips, which were positioned before decontamination, and by sampling contact plates after the completion of each cycle. The results of biologic indicators showed 6-log reduction in microbial counts after successful decontamination cycles for both agents and found to be compatible with clean-room panels including commonly used materials in vivarium such as racks, cages, trol-leys, cage changing stations, biosafety cabinets, refrigerators and other equipment in both procedure and animal rooms. In conclusion, the automated process enabled users to perform effective decontamination through multiple cycles with real-time documentation and provided additional capability to deal with potential outbreaks. Enabling software integration of automation improved quality-control systems in our vivarium.

Abbreviations: BI, biologic indicator; CI, chemical indicator; SCADA, supervisory control and data acquisition software; VHP, vaporized hydrogen peroxide

Received: 22 Feb 2017. Revision requested: 15 Mar 2017. Accepted: 27 Oct 2017.1Syngene International and 2Council Member, AAALAC International, Bangalore, India.

*Corresponding author. Email: sakthi.devan@syngeneintl.com

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foggers; replacement equipment varied depending on the size of area for decontamination and the type of work performed therein. The primary objectives of this project were to develop customized monitoring and operational software, automate HVAC units, and validate the effectiveness of H2O2- and ClO2-based decontamination systems in animal rooms, procedure rooms, corridors, and other areas within the vivarium facilities.

Materials and MethodsVivarium overview. The Syngene Laboratory Animal Research

facility at Syngene International (Bangalore, India) has been AAALAC-accredited since 2009 and provides discovery and development support for various therapeutic areas of preclini-cal research. The facility was built by using clean-room panels with an epoxy floor, and the provision of 26 HVAC units provide more than 90 animal rooms and procedure areas with 15 to 20 air changes hourly (100% exhaust). The barrier facility uses a dual-corridor system, with 2 cage washers and 2 autoclaves as redundant backups. The targeted areas for decontamination were categorized according to volume (small, 1000 to 5000 ft3; medium, 5000 to 10,000 ft3; and large, 10,000 to 25,000 ft3), and a unidirectional airflow pattern with pressure gradient was maintained.

The facility imports both rodents (transgenic, immunocom-promised mice) and nonrodents from approved vendors (United States, Europe, India) on weekly basis in multiple shipments. Although this facility procures animals from clean sources (barrier-bred) and categorized as class I (that is, negative for all excluded organisms), incoming animals are considered to be class II (positive or negative for organisms) regarding their health status at the facility, due to potential exposure during transportation (air and truck). Incoming animals are thus housed under stringent quarantine measures (as long as 21 d) followed by microbiologic screening, parasitologic examina-tions, environmental monitoring, and routine clinical checks, all of which are performed in addition to the established sentinel program.

Central decontamination system. The driving force was to establish a customized state-of-the-art decontamination sys-tem that could be used in individual rooms at any given time without affecting neighboring areas. Considering the need and operational challenges, facility engineers and experts realized the scope required a customized supervisory control and data acquisition software (SCADA) system. The project was accom-plished in 4 phases, comprising SCADA creation, HVAC duct isolation and installation of bypass dampers; sealing of the room enclosure, and implementation and validation of the equip-ment and process. The isolation of ducts and modification was executed from the least critical areas to the most complex areas (for example, procedure rooms, corridors, surgery suite, isolator areas, quarantine, breeding area, and oncology) by maintaining the barriers of adjacent areas. Considering the physical facility, other critical areas included long corridors with independent HVAC units located both parallel to the scientist corridor (pro-cedure rooms) and to service corridor (animal rooms), which acts as a dual corridor; therefore, multiple units were planned to provide effective decontamination throughout this large area.

SCADA development. SCADA is a software-application program for process control that is customized to the needs of decontamination. Generally, SCADA gathers real-time data from remote locations to control equipment and its operational state. The interactive development process was carried out accord-ing to the documented design of facility’s operational needs. The ability to provide 61 cycles was developed initially, with

further scope to add additional cycles as and when required. The software is password-protected at different levels, includ-ing facility engineers and user groups, so that the system can be operated and monitored from different floors of the facility. The software developer and facility engineering team optimized the system under test conditions and then the system operators and other users at the facility.

Isolation of HVAC ducts and installation of bypass damp-ers. Each HVAC unit in the facility serviced 4 to 6 clusters of rooms, and return air was conveyed through a single duct that had temperature sensors for capturing real-time data by the Building Monitoring System. However, an individual room could not be isolated for decontamination because shutting down the particular HVAC unit would affect adjacent rooms. Therefore, to isolate individual rooms and enclosures, bypass dampers were installed in the supply and exhaust ducts; ac-tuators present in the supply and exhaust dampers control the bypass dampers. The online execution of damper opening and closing occurs within 1 min, regardless of the rooms planned for decontamination. All commissioning activities were carried out systematically through a documented verification process to ensure that all components operated according to specifications.

Equipment validation and preparation of sealed enclosures. Areas were constructed by using clean-room panels, and ad-ditional efforts to seal the areas included the replacement of light fixtures and other utilities to avoid potential leaks from the animal rooms by considering safety aspects. To supply air at adjustable pressure (0.5 to 3.0 psi) during decontamination by the Minncare dry-fog equipment, a system controlled by solenoid valves was designed to deliver compressed air from central cylinder banks. The decontamination equipment was procured in parallel with modifications and software crea-tion, and subsequent validation was performed according to the facility set-up, which considered the room volume, agent used for decontamination, and calculated time. To achieve the desired concentration, oscillation fans (2 to 8) were placed for uniform distribution of agents to all corners and surfaces of a room. Portable sensors and indicators were used to monitor the relative humidity and concentration of decontaminant agent during the process.

Minidox-M ClO2 system. The Minidox-M system (ClorDiSys Solutions, Somerville, NJ) generates pure ClO2 gas which is injected into the sealed room or enclosed area. The decontami-nation phases for this unit comprise precondition, condition, charge, exposure, and aeration. The exposure concentration (360 ppm at1 h, 720 ppm at 2 h, and 1080 ppm at 3 h) is calculated automatically according to the room volume, to a maximum of 70,000 ft3. The equipment holds cylinders containing 2% chlo-rine, 98% nitrogen at the back side of the unit, and the gas passes through a regulator and 3 sets of cartridges prior to delivery into rooms programmed for decontamination. All activities and pa-rameters of the Minidox-M were monitored, and a portable gas leak detector (PortaSens II Model C16 - Analytical Technology) was used to measure potential leaks during decontamination and to verify the appropriate residual ClO2 concentration at the end of each cycle. In addition, this system has an automated process for controlling humidity levels regardless of ClO2 gas concentration during the entire decontamination cycle.

VHP-Victory biodecontamination unit. The VHP-Victory biodecontamination unit (STERIS, Mentor, OH) generates H2O2 vapor by using a stabilized aqueous solution of 35% H2O2 and can cover an area up to 20,000 ft3 in a single cycle. The operator uses a programmable logic controller touchscreen to select a fac-tory-programmed cycle, and the provided SmartPhase software

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automatically runs the cycle selected for biodecontamination. During dehumidification, the relative humidity was reduced to 50% to 60%, and the vapor generated from liquid H2O2 is intro-duced into the enclosure to achieve the desired concentration rapidly. The residual level was measured after the aeration phase at the end of the cycle, and data-log output was stored on a USB memory stick. VHP-Victory cycle phases include dehumidifica-tion, conditioning, decontamination, and aeration.

HaloFogger disinfection system. The HaloFogger system (Halosil International, New Castle, DE) uses a dry-mist dispens-ing device that delivers aerosolized disinfectant (5% H2O2 and 0.01% silver) and can cover a room volume up to 3700 ft.3 This equipment was used for smaller areas and can support other high-capacity units when a large area (particularly corridors) is targeted for decontamination.

Minncare dry-fog system. The Minncare dry-fog system (MAR COR Purification, Plymouth, MN) was operated by air pressure and designed to produce ultrafine atomized dispersion of cold sterilant (22% H2O2 and 4.5% peracetic acid) for decontamina-tion of up to 35,000 ft.3 Manufacturer-provided software was used to calculate the amount of water and cold sterilant were required according to the room volume and time needed to achieve desired concentration; the recommended concentration was 1.5 mL/m3 (operating range, 0.5 to 3 mL/m3). The residual concentration was measured by using an Accuro gas-detection tube (Draeger Safety, Leubeck, Germany), which measures acetic acid (range, 5 to 80 ppm) and H2O2 (range, 0.1 to 3 ppm), to confirm completion of the decontamination cycle.

Quality-control methods. The effectiveness of decontamina-tion process was assessed by using biologic indicator (BI) strips impregnated with a particularly hardy organism (Geobacillus stearothermophilus spores; Salesworth Synergies, Kamataka, India; Spordex [STERIS]; NAMSA, Crosstex, Maumee, OH) and chemical indicator (CI) strips (Steraffirm [STERIS]; H2O2 Strip Tests [Halosil International) prior to and after cycles. BI strips (4 to 15 strips per area) were positioned strategically at the extreme corners of rooms depending on the type of equipment to be used and the volume of the area targeted for decontamination. At the end of each cycle, BI strips were retrieved, opened, and the contents transferred aseptically into tryptic soy broth and incubated at 56° C; in addition, an unexposed BI strip was in-cubated as a positive control. All cultures remained negative for growth, except for the positive control, which became positive after overnight incubation. In addition, soybean casein digest agar contact plates (Fitech Bioscience, Bangalore, India) placed before and after decontamination were used as a secondary veri-fication method; plates were maintained for 7 d and observed for the presence of bacterial growth. All BI strips and contact plates were incubated in Syngene’s mutagenicity laboratory, and all results were evaluated and compared.

ResultsBefore initiation of biodecontamination, the customized SCA-

DA software system was configured for all cycle combinations, after which decontamination was executed for the scheduled areas as indicated on the facility maps (Figures 1 through 5). Relative humidity was maintained successfully at ≤70% during decontamination by the Minidox-M unit to achieve the desired ClO2 concentration (360 ppm at 1 h or 720 ppm at 2 h) for the exposure time based on the room volume. In addition, humidi-fier units were placed to raise humidity to the desired levels and were automatically controlled by Minidox-M system. For effec-tive decontamination by using the Minncare dry-fog system and HaloFogger, a relative humidity of 65% to 85% and H2O2 contact

time of 1 h were maintained regardless of the room volume. Conversely, dehumidifier units were used to decrease humidity levels and maintained between 50% to 60%, according to the VHP-Victory preset (H2O2 concentration, 150 to 300 ppm; Figure 6). The validation cycles performed in animal rooms, procedure rooms, and other areas demonstrated successful decontamina-tion in 84%, where they achieved 6-log (106) sporicidal reduction, based on the lack of growth from BI strips. The remaining 16% of decontamination cycles appeared to be unsuccessful, in light of growth from 1 or 2 of the 4 to 15 BI in several areas.

Similarly, none of the contact plates from rooms that were successfully decontaminated showed any growth, except for a few unsuccessful cycles for which sample enumeration showed colony growth at an average 1 or 2 cfu per plate. The unsuc-cessful cycles were rescheduled, and decontamination criteria were met thereafter (data not shown). During the validation process, a total of 265 BI strips, 195 CI strips, and 483 contact plates were used to assess decontamination of 86 different areas in the facility (Table 1). The results from BI strips and contact plates were compared and showed that no growth determined successful decontamination and cycle validation; in addition, 92 positive-control BI strips were inoculated concurrently, and all yielded appropriate turbidity within 24 h. In Figure 7, we note the pros and cons of each method we used. Overall, multiple cycles were performed for rooms with different dimensions and volumes of 1481 to 16,017 ft3 and demonstrated success-ful decontamination associated with integral control of HVAC operation through the interactive process achieved by using SCADA software.

DiscussionThe facility presented operational challenges regarding pe-

riodic decontamination as well as contact plate sampling, due to many ongoing experiments, which ranged from short-term to chronic studies, thus limiting free access and the ability to empty any particular room for decontamination. Given the complexity of the HVAC units, traditional foggers had previ-ously been used in multiple units after covering doors and supply and return ducts. However, the entire current project was systematically implemented in a fully functional research facility, without compromise of regular operations, ongoing experiments, or cross-contamination. The SCADA system was controlled by trained operators who manipulated the HVAC units as needed to isolate the rooms for decontamination, and the system used equipment with built-in programmable logic controller monitoring to collect real-time data. Automation of the centralized system enabled the performance of as many as 4 independent decontamination cycles daily, in any chosen area and with options regarding using either H2O2 or ClO2.

Decontamination of enclosed areas such as isolation units and rooms is important in many industrial, research, and healthcare facilities.34 In particular, VHP technology has been used for many years as an alternative to formaldehyde or other liquid or gaseous methods for isolator decontamination as well as larger areas, such as animal rooms, research areas,27 ventilation pipes, HEPA filters,21,35 IVC blower units,27 cage-changing sta-tions, and a variety of lab equipment.2,39 In addition, successful VHP biodecontamination was demonstrated in fully equipped transgenic and embryo-handling laboratories.27 Another study addressed VHP-based biodecontamination for space biodecon-tamination of a BSL3 laboratory suite under negative pressure.28 Parameters including temperature, relative humidity, VHP concentration, and pressure within the laboratory suite were monitored, and the authors concluded that VHP was compatible

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with a variety of routine laboratory equipment and electron-ics.28 VHP was reported to be noncorrosive and compatible with electronic components, achieved 4.5-log reduction in infectivity, and interpreted as an innovative decontamination approach recommended for prion inactivation, according to

experiments designed to mimic decontamination of medical or surgical equipment, including fragile or inaccessible surfaces of complex instruments (endoscopes, laparoscopes).9

The hamster bioassay model involved the use of VHP in a sealed container that was directly coupled to a VHP1000

Figure 1. (A) SCADA screenshot of the various units, which operate in parallel and perform different decontamination cycles. (B) SCADA screenshot of 61 programmed decontamination cycles identified according to area or room number (highlighted during operation).

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biodecontamination system (STERIS) to maintain a dry (non-condensing) H2O2 gas (1.0 to 1.5 mg/L for 3 h at 25 °C); stainless steel wires were exposed to VHP with or without previous treatment with an enzymatic cleaner and implanted (prefrontal subcortical region) in anesthetized Syrian golden hamsters for

bioassays.9 In a more realistic bioassay model based on trans-missible spongiform encephalopathy, infectious brain materials (suspension or dried onto the surface of stainless steel wires) were or were not decontaminated in a H2O2 gas plasma sterilizer prior to implantation in hamsters.44 Another study demonstrated

Figure 2. (A) SCADA screenshot of Minncare (H2O2) system, with independent supply and return dampers and bypass damper. (B) SCADA screenshot of Minncare (H2O2) in operation, illustrating decontamination and the passage of return air directly through bypass damper when the supply and exhaust dampers are closed.

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thorough decontamination of an entire class II biologic safety cabinet, including supply and exhaust filters; prior to decon-tamination, the cabinet was disassembled so that biologic indicators could be placed at various locations, and then the cabinet was prepared by sealing the front access, exhaust filter

openings, supply diffuser, work area grills, to ensure that VHP reached all parts of the cabinet, including filters.8 VHP exposure of spores of Clostridium botulinum (dried spores spread over stainless steel slides and exposed to VHP in a sealed glove box) was found to be effective at deactivating spores of toxigenic C.

Figure 3. (A) SCADA screenshot of VHP Victory (H2O2) system, with independent supply and return dampers and bypass damper to exhaust re-turn air to isolate the targeted area. (B) SCADA screenshot illustrating VHP Victory (H2O2) decontamination, when the supply air passes through the bypass damper and directly into the exhaust duct.

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botulinum and nontoxigenic Clostridium spp.22 A set of 3 studies applied dry-mist H2O2 diffusion technology for air and surface decontamination of BSL3 areas where M. tuberculosis strain H37Ra was used and demonstrated that viable bacteria were reduced by 5 log and no BI yielded any growth, thus suggest-

ing that this technology is an effective and safe alternative to formaldehyde.12 In addition, a report suggested that HPV was effective against bacterial spores including multidrug-resistant organisms on nonporous and porous surfaces of clinical areas29 and eliminated M. tuberculosis contamination from a room at a

Figure 4. (A) SCADA screenshot shown Minidox M (ClO2) system, where supply air passes through the room and return air leaves through the exhaust duct, due to provision of bypass damper. (B) SCADA screenshot illustrating Minidox M (ClO2) decontamination cycle, when both the supply and exhaust ducts are closed and the bypass damper is open.

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tuberculosis research laboratory.23 Furthermore, experimentally contaminated biologic safety cabinets in a room containing M. tuberculosis (3 log) and Geobacillus stearothermophilus (6 log) were decontaminated by using HPV (90-min exposure); the results revealed that all BI strips were deactivated for both organisms in all 10 locations.13 Another study similarly suggested that dried inocula of nosocomial organisms that had survived for as long as 5 wk in a 100-m3 room were effectively inactivated by H2O2.

38

The current validation results of H2O2- and ClO2-based de-contamination were compiled from a large number of cycles (86 areas), showed 6-log reduction of BI strips in most cases (84%), and corroborated other experiments reported. All 213 BI strips, 195 CI strips, and 398 contact plates used to evaluate H2O2-

based decontamination of our facility passed the test criteria. The few rooms that yielded positive BI results (16%), which appeared as mild turbidity in the media because of growth of the indicator organisms, might have been due to inadequate exposure concentration or time or to inappropriate placement of BI strips; however all CI strips, which were positioned next to the BI strips, passed the test criteria. During validation, ap-propriate residual concentration (0.1 ppm; acceptable level, 1ppm) was ensured after completion of H2O2 decontamination and provided clearance for personnel re-entry. In one study of a dry-fog disinfectant system,24 animal rooms were treated with cold sterilant solution consisting of a stable mixture of peracetic acid and H2O2. The dry-fog unit was positioned in the center of a room, after which the humidity was raised 80%; effective decontamination was achieved with no condensation of H2O2 in the room or on its components throughout the decontamina-tion process using VHP and peracetic acid, either separately or together.24 However, a separate experiment evaluated the use of VHP (32-min exposure) as a surface decontaminant and sterilant in a centrifuge application and examined its killing activity against spores of Bacillus subtilis subsp. globigii and B. stearo-thermophilus; the results revealed that VHP showed significant sporicidal capability.25 Similarly, another study revealed that rodent bacterial species (Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus) in the presence of 2% BSA on smooth surfaces were inactivated by VHP.2 Although, a new facility bio-decontamination demonstrated successful deactivation of all the BI exposed, and none of the lab materials, including electronic equipment, were affected adversely,16 thus corroborating our observations. Overall, the H2O2-based decontamination systems we evaluated were effective for large to medium enclosed areas (VHP Victory and Minncare systems) as well as small rooms, including ancillary areas (HaloFogger system), with an average cycle time of 3 h needed to complete the entire process.

Figure 5. SCADA screenshot of HaloFogger (H2O2) aeration phase, when the room air is conveyed through return ducts and exhausted through exhaust blower.

Figure 6. Temperature (T [°C]), relative humidity (RH), and H2O2 concentration (Conc) during a complete representative VHP decon-tamination cycle. ‘Return’ indicates measurements at the VHP Victory unit, whereas ‘Sens’ indicates measurements obtained by a sensor at a distant site of the room enclosure.

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After necessary conditions of humidity were met, ClO2 decontamination (400 ppm/h) of large animal and neonatal intensive care units (4800 m3) contaminated with Salmonella was monitored by using 100 BI strips (G. stearothermophilus spores, B. atrophaeus spores, or Salmonella newport vegetative cells); the subsequent analysis indicated better than 6-log reductions in viability for B. atrophaeus and S. newport and greater than 5-log reduction for G. stearothermophilus, thus demonstrating that ClO2 is an effective decontaminant under nonlaboratory conditions.32 Other studies reported that gase-ous ClO2 was effective against food-borne pathogens such as E. coli, Listeria monocytogenes, and S. enterica in the context of the food-processing industries.33,43 Gaseous ClO2 has proven effective against viruses, even at relatively low concentrations with extended exposure time.36,41 In addition, a research facil-ity performed ClO2 decontamination for 65 rooms and noted complete killing of all BI; moreover, no physical residue or material degradation was noted on any of the metal-containing equipment in the building.5

At our facility, a ClO2 concentration of <0.1 ppm typically was present after cycle completion; in few cycles, ClO2 at <0.3 ppm was detected during aeration phase, either in an adjacent area or at service floors (above the ceilings), where all the ducts for the rooms are connected. We therefore increased the aeration period of those particular cycles to reduce the con-centration, especially that for chlorine odor (the threshold for odor from ClO2 is 0.1 ppm1,37). The 52 BI strips and 85 contact plates passed the test criteria, and ClO2 (Minidox-M)-based decontamination was effective for large to medium areas of the sealed enclosure.

In a previous study,6 we investigated the efficacy of ClO2 gas for environmental decontamination of Syphacia spp. ova by using perianal cellophane tape impression of pinworm infected mice; tapes with attached ova exposed to ClO2 gas for 1, 2, 3, or 4 h and then incubated in hatching medium for 6 h to promote hatching. For controls, tapes with attached ova were maintained at room

temperature for 1, 2, 3, and 4 h without exposure to ClO2 gas and then similarly incubated in hatching medium ova viability after incubation was assessed by microscopic examination. Ex-posure to ClO2 gas for 4 h inactivated 100% of Syphacia spp. and 17% of the ova on the control slides (unexposed to ClO2) were nonviable; therefore, the results suggested that exposure of ani-mals rooms to ClO2 gas for at least 4 h was effective for surface decontamination of Syphacia ova.6 Similarly, an investigation of ClO2 gas for inactivation of 8 β-lactams involved various concentrations and overall exposure lengths and showed that 5 of the 9 inactivation cycles passed the acceptance criteria of achieving a 3-log reduction (pharmaceutical manufacturer’s required 99.9%) of all 8 β-lactams.31 Overall, the performed cycles were free of residue by the end of decontamination, thus allowing access into the animal rooms. Together these observations provide support for repeating unsuccessful cycles after rectifying all issues of enclosure containment and refining equipment parameters during the validation process.

In conclusion, the improved engineering controls involving several types of low- to high-capacity equipment provided options for effective, low-temperature decontamination of vivarium facilities by using either H2O2 or ClO2 gas. Implemen-tation of a SCADA system enabled the operation of multiple decontamination cycles in different enclosures, improved pro-cess efficiency, and reduced the turnaround time for performing all of the planned cycles within the facility. VHP biodecon-tamination is a highly effective and safe alternative, because residual H2O2 vapor catalytically decomposes into oxygen and water as its final products, which can be directly released into the environment. Similarly, the beneficial properties of ClO2 gas include excellent distribution and penetration, making it an effective biodecontamination agent for animal rooms that are proven sealed enclosures. Because both the agents have excellent material compatibility, decontamination cycles can be performed with a variety of materials generally used in labora-tory animal rooms (racks, cages, trolleys, cage changing stations)

Table 1. Comparison H2O2 and ClO2 decontamination systems

VHP delivery system

ClO2 VHP Victory Minncare HaloFogger

Biologic indicators (BI)No. used 52 77 136 NAEffectiveness of decontamination (log reduction) 6 6 6 NABiologic indicators result Pass Pass Pass NA

Contact platesNo. used 85 105 224 69Contact plate resulta Pass Pass Pass Pass

Chemical indicators (CI)No. used NA 45 83 67Chemical indicators result NA Pass Pass PassIntermediate results after first decontamination of all equipment (BI/CI/plates)

Pass Pass Pass Pass

Equipment target capacity 1982 m3 566 m3 1000 m3 104 m3

Optimal relative humidity (%) 60–75 50–65 65–85 55–85Decontamination cycle time (h; includes aeration phase) 3–4 3–3.5 3 2.5–3.0Aeration timeb (min; by HVAC; 15–20 air changes/h) 45–60 60 60 60

NA, not applicableaResults of contact plates after decontamination. Contact plates sampling was performed before and after decontamination, as a standard practice.bAeration phase can be increased, especially for large-volume enclosures and multiple-room complexes, until reduction to a safe concentration (ppm) is achieved.

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and procedure areas (centrifuge, biosafety cabinets, surgical equipment, anesthesia vaporizer, weighing balance, refrigera-tors and other equipment). There appeared to be no corrosion or any negative effects on material surfaces, and equipment continued to be functional after the successful decontamination. Nevertheless, each method has its advantages as well as limita-tions that must be considered, and a balance must be achieved that is based on facility requirements and the enclosure volumes to be decontaminated. Overall, the current automation process enabled users to perform successful decontamination with con-fidence, by saving time and resources, by generating real-time documentation of cycles, and by providing sufficient scope to deal with potential outbreaks, thus collectively improving the quality-control systems in laboratory animal facilities.

AcknowledgmentsWe thank Deepak Joshi and Anand Naidu (Engineering and Main-

tenance) for concept design; Sangeetha Shamsundar and Mahesh Muniyappa (Mutagenicity Laboratory) for microbial sampling and analysis; Sabarish Babu (Veterinary Sciences) for veterinary consultation; and team members for timely coordination during project execution.

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