HISTORICAL REVIEW OF NITROGEN DIOXIDE REFERENCES IN MICROBIOLOGY AND HEALTH CARE
In the environment, NO
2 is known as a persistent gaseous component in air pollution. In industry, NO
2 is used in a variety of industrial applications, such as bleaching flour and as a polymerization inhibitor in acrylates.
4,
5 Although the use of NO
2 as a sterilant is a relatively recent development, there is long a history of studying NO
2 as a pollutant, in medical applications, and as a biological contributor to reactive nitrogen species (RNS).
The NO
2 is found in ambient air at varying concentrations. Nitric oxide (NO) and NO
2 are produced by internal combustion engines and natural processes such as forest fires, lightning, and fermentation. NO
2 is the by-product of NO oxidation. Due to anthropogenic sources, any large city with a high density of internal combustion engines and fossil fuel power plants will have more NO
2 in the ambient air than will regions without such sources. In and near these cities, the ambient NO
2 concentration measured in 2017 ranged between 20 and 60 ppb.
6 Across the United States, the average daily maximum value (from a 1-hr average) has dropped 30% from 2001 to 2010. This drop reflects a long-term trend of NO
2 reduction, where the average daily maximum was 110 ppb in 1980 to 43 ppb in 2016.
6,
7 Historically, and before air quality standards began to improve air quality, this value could range as high as 1300 ppb (1.3 ppm), as recorded in the Los Angeles area in 1964.
8 Given the prevalence of NO
2 in the environment, and the large contribution from man-made sources, NO
2 has been thoroughly studied. Studies have evaluated the sources of NO
2, links to acid rain, and the impact of NO
2 on materials and living things. This wealth of information facilitates the evaluation of health and safety issues associated with using NO
2 as a sterilant gas and provides a technical understanding for the detailed chemical reactions in a typical NO
2 sterilization chamber.
One of the earliest mentions of NO
2 used in health care-related applications is the use of high concentrations of NO
2 for making oxidized cellulose, also called oxycellulose.
9 Since the 1940s, manufacturers have exposed cellulose preparations (initially, cotton gauze) to a high concentration of NO
2, resulting in its oxidized form.
The oxidized cellulose is used as a bioabsorbable and hemostatic wound dressing (eg, the Surgicel® absorbable hemostatic wound dressing). Additional beneficial properties of this type of wound dressing have been identified, such as antimicrobial and osteogenic properties.
10,
11 Oxidized cellulose products are still used today.
In 1962, researchers in Poltava, Ukraine, exposed
Bacillus anthracis spores to very high concentrations of NO
2.
12 Although these researchers believed they observed microbicidal inactivation of the spores and other microorganisms, repeated studies showed efficacy was due to the bacteriostatic properties of the oxidized cotton gauze inoculated in the wound dressing products studied, rather than direct NO
2-mediated lethality of the spores.
13 In another example of the microbicidal evaluation of NO
2, researchers measured the effect of NO
2 on airborne microorganisms. Atmospheric test chambers, under controlled temperature and humidity conditions, were used to simulate atmospheric conditions with gaseous atmospheric pollutants. In one test, researchers used
Rhizobium meliloti as the test organism and found that at 50% relative humidity (RH) and 3 ppm NO
2 concentration, a 2 log
10 reduction was observed over a 3-hour exposure period.
14 A similar study observed more than 1 log
10 reduction in the population of airborne Venezuelan equine encephalomyelitis (VEE) virus using a 10-ppm NO
2 concentration and 85% RH.
8 In both studies,
Bacillus subtilis spores were used as controls because they were not damaged by exposure to NO
2 under these conditions. Therefore, the population of the test organisms, either
R meliloti or VEE, was quantified in proportion to
B subtilis spores. Under these exposure conditions, NO
2 did not exhibit sterilant properties because the control organism (
B subtilis) was not inactivated. The reason that
B subtilis spores survived these exposure conditions is a result of the relatively low NO
2 concentration and the short exposure time used in these tests.
Another study of airborne NO and NO
2 tested cultured Chinese hamster cells (V79 cells) for DNA degradation after exposure to either NO or NO
2.
15 The V79 cells were exposed to NO and NO
2 in varying concentrations, from 0 ppm to 500 ppm and over varying periods (from 5 to 30 min). It was found that NO
2 led to a doseand time-dependent increase of the rate of single-strand breaks in V79 cellular DNA. However, NO treatment did not result in any detectable DNA damage. The lowest observable effective concentration of NO
2, which was statistically different from control values, was 10-ppm exposure for 20 minutes. The lack of DNA degradation with NO exposure can be understood by examining the Henry’s law coefficient of NO compared to NO
2, as discussed in the following text.
16
The DNA single strand-breaks were also observed using conditions appropriate for sterilization and area disinfection applications.
17 Purified DNA and
B subtilis spores were tested at 2.85 mg/L NO
2 and 75% RH, with varying exposure time. After exposure, the purified DNA and DNA extracted from the intact spores was evaluated using gel electrophoresis, with both denaturing and nondenaturing gel. The NO
2 was found to cause increasing DNA single-strand breaks, with breakages occurring after only 2 minutes of exposure. Complete DNA degradation was observed after 16 minutes under these conditions. The mechanism by which NO
2 can cause such damage is discussed later in this chapter.
Although there has been an interesting history of NO
2 for use in health care-related applications and in studies of microbicidal behavior of NO
2, the work to evaluate and develop NO
2 as a sterilant did not start until 2004. This work was motivated, in part, in response to the great interest in NO, which is one of the several RNS recognized as contributing to many biological processes.
18,
19 The NO is released by neutrophils and macrophages in response to inflammation and infection and thus plays a role in the immune response in vivo. Also, NO is a vasodilator used in treating cardiac and pulmonary diseases.
20,
21 In vivo, NO will autoxidize to form NO
2, and from the point of having both intercellular and intracellular NO and NO
2, subsequent chemical reactions lead to species that will cause microorganism apoptosis.
22,
23
Given this remarkable span of biological activity associated with NO, the exogenous application of NO was tested for antimicrobial properties that might be useful for sterilization and disinfection.
17 These investigations revealed that the NO cycle of in vivo chemical reactions can be reproduced in vitro. However, it was further recognized that NO
2 provides a more direct route to initiate the microbicidal chemical reactions involving RNS. Once again, the Henry’s law constant for the gases involved helps to explain the advantages of using NO
2 instead of NO as the sterilant gas.
By now, NO
2 has been thoroughly tested for its ability to inactivate microorganisms. To be registered as a sterilant with the EPA, a chemical agent must destroy or eliminate representative forms of microbial life in the inanimate environment.
24 Recommended microorganisms for testing the microbicidal activity of a sterilant are listed in regulatory documents and international standards, such as ISO 14937:2009.
2 The organisms tested with the NO
2 process included those listed in
Table 35.1.
25 For these tests, a known population of each microorganism was inoculated onto a carrier surface and allowed to dry. These were exposed to NO
2 sterilization cycles with increasing exposure time and with the concentration and RH for the cycles held constant at 2.75 mg/L NO
2 concentration (2000 ppm at 600 mm Hg) and 70% to 80% RH. The D-values for each microorganism were calculated using the fraction negative technique (single point, Stumbo-Murphy-Cochran [SMC]; see
chapter 11) and based on the first exposure time where partial exposed inoculated carriers were sterile. Whereas most microorganisms exhibited rapid lethality upon exposure to NO
2, spores of
Geobacillus stearothermophilus exhibited the greatest resistance to the
NO
2 process.
Table 35.1 shows that the D-value observed for the
G stearothermophilus was 1.3 minutes, under the conditions used for this evaluation.
Aside from demonstrating that NO
2 can kill all forms of microbial life, screening a wide range of microorganisms permits the identification of microorganisms that have higher resistance to the NO
2 process. The indicator organism in a biological indicator (BI) must be a highly resistant microorganism (typically, a spore-forming organism) that is appropriate for used in a BI.
26 The indicator organism should be more resistant than the bioburden found on medical devices or surfaces to be decontaminated. The BI consists of a known population of highly resistant spores that are inoculated onto a suitable carrier (see
chapter 65). The inoculated carrier can be placed in a permeable barrier package (eg, a small Tyvek pouch), built into a self-contained BI (SCBI), or used in a process challenge device (PCD). The PCD can provide some additional and controllable protection to the BI so that the PCD resistance to the NO
2 process is adjusted to represent the resistance of the load to be sterilized.
INACTIVATION KINETICS WITH NITROGEN DIOXIDE
The BIs, consisting of spores of
G stearothermophilus, exhibit log-linear inactivation kinetics with increasing NO
2 process exposure time. The log-linear response with increasing exposure duration is expected and is typically observed with other sterilization processes.
27 Like with other sterilization processes, analysis tools like Holcomb-Spearman-Karber (HSK), survivor curve method, and others can be used to evaluate the D-value of the indicator organism population (see
chapter 11). As with all sterilization methods, but especially when using NO
2, hydrogen peroxide (H
2O
2), and other gaseous sterilants, some mathematical tools like HSK, SMC, and most probable number are not valid when tailing occurs or when the survivor curve does not agree with the fraction negative methods.
27
Figure 35.1 illustrates the log-linear progression of population reduction as a function of exposure time to the NO
2 process. The data represented in
Figure 35.1 was determined by the direct enumeration of the surviving viable spore population on each BI and with a fraction negative method. The BIs used to measure these data were composed of
G stearothermophilus spores inoculated onto 6-mm diameter stainless steel discs. Once the inoculum is dried, the BI discs were packaged in Tyvek/Mylar
pouches, placed into a test chamber, and exposed to 2.0 mg/L of NO
2 with 70% to 80% RH. The D-value calculated by direct enumeration was found to be 26 seconds. The D-value value calculated with the fraction negative method (HSK) was 35 seconds. A log-linear response to exposure time shown in
Figure 35.1 provides a predictable model for inactivation. These results show that NO
2 can be a sterilant under the conditions tested. Predictable inactivation kinetics permit the use of the conservative overkill approach, as described in Annex D of ISO 14937:2009.
2
COMMON CHEMICAL REACTIONS
Nitrogen dioxide found in the environment is formed in most combustion processes where the temperature is high enough to cause a reaction between the N
2 and O
2 molecules. The formation of NO is shown in
equation (1):
The NO is a precursor to NO
2, and nitrogen dioxide is formed by the oxidation of NO. This reaction occurs in the air and in biological systems,
22 following the formula in
equation (2):
The hydrolysis of NO2 results in nitrous acid (HNO2) and nitric acid (HNO3):
or
This reaction is one step in the Ostwald process used in the industrial production of HNO
3 from ammonia.
31 The two isomers of nitrous acid are HONO (nitrous acid) and HNO
2 (nitryl hydride or isonitrous acid). The HNO
2 will ionize in water, like an acid, whereas HONO is more stable, will be found as a gas, and is the source of reactive OH in air pollution.
32
The HNO
3 decomposes to nitrogen dioxide, water and oxygen, as shown in
equation (5).
This decomposition occurs during evaporation and within the liquid phase of HNO3. The NO2 gives the characteristic yellow color of HNO3 and HNO3 fumes. The reactions involving NO2 will be described in the discussion of the detailed physical and chemical reactions that comprise the NO2 process.
When two sterilization molecules of NO
2 collide, either in the gas or liquid phase, there is a probability that these two molecules may bond together, forming the dimer N
2O
4. This is described by
equation (6):
The NO
2 is a radical, with one unpaired electron on the nitrogen. In molecular collisions that result in a molecular bond, these unpaired electrons form a single covalent bond between the nitrogen atoms. However, this bond is relatively weak, and at room temperature, the N
2O
4 will dissociate due to ambient thermal energy. The lifetime of an N
2O
4 molecule is short, with the half-life of the N
2O
4 being less than 10 µs at 25°C (298.15 K).
33 Therefore, an equilibrium state is described for NO
2 where molecules of NO
2 are constantly forming dimer molecules and then dissociating to return the monomer form.
When referring to a gas composed of both NO
2 and N
2O
4, the relative amounts of NO
2 and N
2O
4 are not typically mentioned because this can be readily calculated. Instead, a mixture of NO
2 and N
2O
4 is simply referred to as “NO
2 gas,” and it should be assumed that there will be some fraction of the NO
2 molecules momentarily bound in the dimer form. It is convenient to refer to the total NO
2, which includes both monomer and dimer forms of NO
2. For any quantity of gas containing NO
2, the total number of moles of NO
2 is n
total, which is the sum of the monomer NO
2 moles, n (NO
2), plus two times the dimer moles, 2n (N
2O
4), as shown in
Equation (7).
The ratio of monomer to dimer is described by the temperature-dependent equilibrium constant, K
p, shown in
Equation (8).
33
In
equation (8), p(NO
2) and p(N
2O
4) are the partial pressures of the nitrogen dioxide and dinitrogen tetroxide, respectively. The temperature-dependent fraction of dimer and monomer can be calculated from the temperature dependence of the equilibrium constant.
The equilibrium constant Kp is calculated from the thermochemical properties:
In
equation (9), ΔG° is the Gibbs free energy change per mole of reaction for unmixed reactants and products at standard conditions, R is the universal gas constant, and T is the absolute temperature of the gas.
33 At a single temperature,
equation (8) shows that the square of the NO
2 partial pressure divided by the partial pressure of N
2O
4 will be constant. The temperature dependence of K
p shows that for a given number of moles of NO
2, the number of N
2O
4 molecules will increase as the temperature decreases. The calculated temperature-dependent relationship between NO
2 and N
2O
4 is shown in
Figure 35.3. From this graph, it is clear that the partial pressure of N
2O
4 will increase exponentially with a decrease in temperature.
Another observation from the inspection of
equations (7) and
(8) is that the relative concentration of N
2O
4 will increase as the total amount of NO
2 increases. This concentration-dependent relationship between NO
2 and N
2O
4 is shown in
Figure 35.4. The rate of collisions causing N
2O
4 formation increases as the concentration of NO
2 increases, whereas the dissociation rate is only dependent on temperature and is independent of the NO
2 concentration. Therefore, as the concentration of total NO
2 increases, the partial pressure of N
2O
4 will increase more rapidly than the total NO
2 concentration.
Referring to
Figures 35.3 and
35.4 and their underlying equations, consider a liter of gas that is composed of dry air with a total NO
2 concentration (NO
2 + N
2O
4) of 10 mg/L. At 288.3 K (15°C), this 1 L of gas has 8.77 mg/L (191 µmol)
of monomer NO
2 and 1.23 mg/L (13.4 µmol) of N
2O
4. At 298.3 K (25°C), the same 1 L of dry air with 10 mg/L of total NO
2 will have 9.36 mg/L (203 µmol) of NO
2 and 0.65 mg/L (7.1 µmol) of N
2O
4. From this example, as the temperature increases from 15°C to 25°C, the equilibrium has shifted to have less dimer. Also, the pressure of the gas increases as the temperature increases from 15°C to 25°C due to both increased kinetic energy of the molecules and the increased number of gas molecules (as more N
2O
4 dissociates into NO
2). Due to the increased NO
2 partial pressure caused by dissociation of the dimer, a gas containing NO
2 cannot be considered an ideal gas. As another example, consider an increase of NO
2 concentration while the temperature remains unchanged. As described earlier, at 298.3 K (25°C), 1 L of dry air with 10 mg/L of total NO
2 will have 9.36 mg/L (203 µmol) of NO
2 and 0.65 mg/L (7.1 µmol) of N
2O
4. When the total NO
2 concentration in this 1 L of dry air is doubled to 20 mg/L NO
2 (at 25°C), this liter of gas will have 17.69 mg/L (385 µmol) of NO
2 and 2.33 mg/L (25.3 µmol) moles of N
2O
4. This is shown in
Figure 35.4. Notice that doubling the total NO
2 concentration, from 10 mg/L to 20 mg/L, more than triples the dimer concentration from 7.1 µmol to 25.3 µmol. Whereas these nonlinear examples illustrate the nonideal nature of NO
2 gas, these examples also show that the exact concentration for various conditions can be readily calculated.
DETECTION METHODS AND SPECTRAL PROPERTIES
Measuring the gas concentration in a chamber is easily done using a gas sampling circuit, where a small pump circulates a small amount of gas from the sterilization chamber, through the measurement system, and returns the gas back to the chamber. The type of sensor used for the measurement of NO2 and other gas constituents will depend on the concentration of the gas being measured. For example, at very low concentrations of NO2 (from 0 to 200 ppb), chemiluminescence is a common measurement method. Chemiluminescence is often used in environmental monitoring because ambient NO2 levels are typically between 0 and 200 ppb. For NO2 concentration ranges from 0 to 100 ppm, the electrochemical cells (EC cells) and solid-state detectors are appropriate. These types of detectors are relatively inexpensive, are available for many different types of gases (NO, NO2, H2O2, etc), sold by multiple vendors, and are appropriate for monitoring the environment around the NO2 processing equipment.
For higher concentration levels (from 100 ppm to 100 000 ppm), spectral measurement methods are appropriate using a gas cell with a short beam path. The absorption spectrum of NO
2 consists of many absorbance bands facilitating the measurements of NO
2 using standard laboratory equipment, like Fourier Transform-Infrared (FTIR) spectroscopy systems, visible light spectrophotometry systems, and photometric analyzer in the ultraviolet (UV) and visible (VIS) bands. The measurements in the infrared (IR), VIS, or UV bands will follow Beer’s law, simplifying interpretation of the measured absorbance. The NO
2 absorbance bands include the visual violet, blue and green regions, between 300 and 600 nm, as shown in
Figure 35.5. There is much less absorbance at wavelengths longer than 600 nm, resulting in the characteristic orange-red appearance of the gaseous NO
2.
34
The N
2O
4 gas will not contribute to the measured absorbance at wavelengths longer than 400 nm, so that absorbance between 400 and 600 nm can be attributed to the NO
2 monomer. Regardless, the amount of dimer component of the total NO
2 must be considered. Heating the measurement gas cell greatly reduces the amount of dimer NO
2, as indicated in the graph shown in
Figure 35.3. When 10 mg/L of total NO
2 is added to a chamber, heating the gas from 25°C to 50°C reduces the amount of N
2O
4 from 3.38% to 0.67% of the total amount of NO
2.
The choice of spectroscopic method may depend on other capabilities of the instruments and properties of the spectral bands. For example, being able to measure multiple gas constituents with the same instrument is helpful. The FTIR can measure NO2, NO, and humidity in the chamber dosed with NO2. The UV-VIS measurements will not measure water but lend themselves to simplified detector systems, such as using filtered light in the blue region of the visual spectrum.
USE OF NITROGEN DIOXIDE AS A STERILANT
When using NO
2 as a sterilant for disinfection or sterilization applications, there are three process variables that directly affect the lethal action of NO
2: sterilant concentration, exposure time, and RH. These process variables constitute the principle cycle parameters necessary for establishing the antimicrobial environment. For example,
the process parameters might be 10 mg/L NO
2 concentration, 75% RH, and 10 minutes of exposure time. However, these parameters can be changed depending on the load configuration. Loads composed of simple packages and simple device geometry might require less sterilant and a shorter exposure time. Complex device configurations or very large and dense loads may require longer exposure times. The use of NO
2 permits a wide range of sterilant concentrations. Values as low as 200 ppm and with a 3-hour dwell have been successfully tested with BIs. The maximum NO
2 concentration tested is over 20 000 ppm. This range offers ample opportunity to balance cycle time, cycle cost, and material compatibility.
Beyond the principle cycle parameters (exposure time, NO2 concentration, and RH), there are other cycle variables that can be viewed as secondary parameters. These secondary parameters are those that can be manipulated to overcome the challenges associated with specific loads. Factors like packaging, device geometry, materials, and other impedances that hinder the homogeneous achievement of the principle cycle conditions at every location within the chamber and load. These secondary cycle parameters are factors like vacuum depth and the number of sterilant exposure dwell phases, to name a few. These secondary cycle parameters are manipulated to deliver the principle cycle parameter conditions to the hardestto-reach locations, throughout the load.
Temperature is important, but it is not as critical for the NO2 sterilization process as it is with other sterilization processes. The NO2 process has no liquid-to-gas phase transition near the operating parameter set points. For example, the H2O2 sterilization process relies on the saturated vapor pressure of H2O2, which is highly dependent on temperature. When generating an H2O2 sterilant environment, the goal is to maximize the amount of H2O2 in the chamber. Operating at (and often, above) the saturation point of H2O2 applies a high sensitivity of the H2O2 process to temperature fluctuations. The NO2 process does not work near the vapor-phase transition, where a dose of 10 mg/L NO2 represents approximately 0.5% of the saturated vapor pressure of NO2 at 21°C; however, the temperature of the sterilization chamber should be controlled within a specific temperature range (chiefly for maintaining a consistent RH). Process temperatures from 10°C to 35°C have been tested and shown to provide good sterilization efficacy. Additionally, as will be shown in the following text, large temperature changes can result in relatively small changes in the D-value, compared to the temperature-dependent D-value sensitivity observed with other sterilization methods (Arrhenius temperature dependence).
Humidity is known to play a critical role in many gas sterilization processes, and this remains true for the NO
2 process. With H
2O
2 sterilization, increasing humidity at certain temperatures has been shown to reduce observed D-values by increasing condensation (so-called microcondensation) of the H
2O
2 onto surfaces.
35 With ethylene oxide, increasing humidity is proposed to increase the water content of the spore coat, thereby increasing permeability of the spore coat to the sterilant molecules.
36,
37 Similarly, humidity plays an important role in the use of NO
2 sterilant. With the NO
2 process, the influence of humidity relies on both of these previously identified mechanisms. Furthermore, RH in the sterilizing chamber creates the conditions where specific heterogeneous chemical reactions (reactions on surfaces) can occur that contribute to the sterilization process. The details of these heterogeneous chemical reactions are described in the following text.
The D-value measured at different humidity levels demonstrates that the D-value decreases as the humidity increases.
38 For example, at 40% RH and 4 mg/L, a lot of BIs is found to have a 2.4-minute D-value. At 50% RH and 4 mg/L, this same lot of BIs is found to have a 0.4-minute D-value. At very low humidity levels (below 30% RH), the measurement of BI D-values with the NO
2 process is confounded by inconsistent results. Whereas at humidity levels above 70% RH, NO
2 has a rapid microbicidal action. The mechanism by which the RH level alters the chemistry of the NO
2 process is explained later in this chapter.
Different types of cycles are possible with NO2 as the sterilant. Generally, NO2 cycles can be divided into two categories: cycles with a vacuum step and cycles without a vacuum step (completed at or near ambient pressure). The use of vacuum during the sterilization cycle can facilitate rapid distribution of the sterilant and humidity throughout the load, and vacuum rinses at the end of the cycle facilitate rapid aeration. A cycle that includes a vacuum step requires that the chamber being used is compatible with low pressures, for example, an autoclave-style sterilization chamber. Conversely, an NO2 sterilization or disinfection process that does not include an evacuation step has no such requirements on the structure of the enclosure being treated with the NO2 process. For example, a cycle without a vacuum step is appropriate for disinfecting isolators and material transfer airlocks. Sterilant gas uniformity and aeration are largely dependent on air circulation patterns and air circulation velocity. The time required for distribution of the gas throughout the chamber and the time required for aeration may be much longer when no vacuum is used because air circulation and diffusion are the main mechanisms for the gas distribution processes.
Vacuum Nitrogen Dioxide Cycles
Figure 35.6 illustrates the essential design of a sterilization system configured for vacuum cycles using the NO
2. A graph of the chamber pressure versus time for a vacuum cycle is shown in
Figure 35.7. In this graph, the pressure within the sterilization chamber is graphed as a function of elapsed time. The first step is the evacuation of the chamber to remove the atmosphere in the chamber. Next, the prechamber is filled to the appropriate pressure of sterilant gas. Then, the prechamber is opened to the evacuated sterilization chamber allowing the NO
2 to flow into the sterilization chamber. Pressure transducers, recording the pressure rise associated with the sterilant gas addition, or optical NO
2 detectors can be used to confirm delivery of the sterilant to the sterilization chamber. After the sterilant is added to the chamber, humidified air may be added to the sterilization chamber until the target RH is achieved, and dry air may be added until the target pressure for the exposure dwell is reached. The chamber pressure during the exposure dwell is typically between 500 mm Hg and 600 mm Hg (roughly between 65 and 80 kPa). These steps can be repeated, as needed, to achieve the intended sterilization process.
Figure 35.7 shows two exposure dwells, representing the first half cycle and second half cycle of an overkill approach, described in
Annex D of ISO 14937:2009.
2 After the final exposure dwell is completed, the sterilant gas is removed from the sterilization chamber, and the load therein, using repeated evacuation and rinsing with air or inert gas (eg, nitrogen).
The sterilant in the chamber exhaust can be scrubbed by chemical means. The scrubbing process removes unsafe levels of NO2 from the exhaust. The chemical removal of sterilant from the air stream may use a solid chemical scrubber material (eg, sodium permanganate) or water scrubber systems. The sodium permanganate (solid media) scrubber material neutralizes and captures the NO2 in a nonhazardous, solid material. The spent scrubber material may be disposed of as nonhazardous solid waste with no special handling measures required. With water-based scrubber systems (appropriate for larger volume systems), the NO2 is captured in water and neutralized before draining.
Nonvacuum Nitrogen Dioxide Cycles
Cycles that do not have a vacuum step are appropriate for vacuum-sensitive loads and for structures that are not designed for internal pressures that are significantly below ambient pressure. For example, the disinfection of isolators and material transfer airlocks, or the surface sterilization of packaged, prefilled syringes are cases in which a nonvacuum cycle may be appropriate. The system configuration for a nonvacuum cycle is illustrated in
Figure 35.8. Note that in this case, and throughout the rest of this document, the word “chamber” is used to refer to isolators, airlocks, sterilization chambers, and any other volume to be exposed to the process. This nonvacuum chamber is a closed-loop system for the humidity introduction, sterilant addition, and dwell phases of the process. Humidity is added to the chamber first by recirculating the gas through the humidifier path until it reaches the target level. The NO
2 sterilant is added by recirculating the humidified air through the buffer tank until the specified NO
2 concentration is reached. The order of gas additions (humidification prior to NO
2) is essential to prevent the humidification system from becoming fouled with NO
2. The NO
2 buffer tank is easily returned to a dry state after the cycle using a small vacuum pump. The dwell time begins after the NO
2 addition phase is completed and closed-loop recirculation continues to mix the gases in the chamber. The aeration phase is openloop with the exhaust gas, has the NO
2 removed by the
scrubber, and where inlet and outlet blowers can be used for pressure regulation. An example of the NO
2 and humidity concentration profiles measured in a nonvacuum cycle is shown in
Figure 35.9. Note that when the NO
2 gas is added to the humidified chamber, the concentrations of both NO
2 and humidity decrease. This is a key phenomenon in NO
2 processes because both species are deposited and react heterogeneously on surfaces. This phenomenon and how it pertains to the mechanism of inactivation will be considered later in the chapter.
In both vacuum and nonvacuum cycles, an important part of the NO
2 process is measuring and applying the NO
2 dose to a chamber. The dose is often set as the number of grams of NO
2 applied to a specific load in a chamber. One method for preparing the dose uses a small auxiliary vacuum chamber (often called a prechamber or buffer tank) for preparing and measuring the NO
2 dose, as shown in
Figure 35.6 and
Figure 35.8. With this method, the buffer tank, which is connected by valves to a vacuum pump and a source of NO
2, is first evacuated and then filled with the sterilant dose. The sterilant dose is metered by measuring the pressure rise in the buffer tank due to the addition of the NO
2 gas. The calculated pressure rise for a specific dose NO
2 must account for the relative concentration of the dimer and the monomer. After filling the buffer tank with the correct dose of NO
2, the sterilant is flushed into the chamber.