Nitrogen Dioxide

Nitrogen Dioxide

David Opie

Evan Goulet

Nitrogen dioxide (NO2) is an antimicrobial gas with many beneficial properties. In 2016, NO2 was registered as a sterilant with the US Environmental Protection Agency (EPA) under the Federal Insecticide, Fungicide, and Rodenticide Act.1 The EPA registration required the demonstration that NO2 is a sterilant, capable of destroying or eliminating all forms of microbial life; this included representative forms of vegetative bacteria, bacterial spores, fungi, fungal spores, and viruses. This registration covered uses that are not specifically applied to medical devices. The use of NO2 for the sterilization of medical devices follows International Organization for Standardization (ISO) 14937:2009 as the general sterilization standard for methods that inactivate microorganisms by any physical or chemical means and is recognized by many regulatory agencies.2 The NO2 sterilization has also been recognized by the US Food and Drug Administration (FDA) with the 510(k) clearance of a medical device sterilized by NO2 gas.3 The properties and use of NO2 as a sterilant are detailed in this chapter. The mechanism of action for the sterilant is described and is shown to act in the same way as the natural in vivo processes by which animals control pathogens. To understand the mechanism of action, the surface chemistry must be considered because only referring to the gas phase concentration of the sterilant cannot lead to a sufficient analytical interpretation of the sterilization process. This may have implications in the study of other sterilization processes. Finally, this chapter discusses material compatibility and examples of applications with the NO2 process.


In the environment, NO2 is known as a persistent gaseous component in air pollution. In industry, NO2 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 NO2 as a sterilant is a relatively recent development, there is long a history of studying NO2 as a pollutant, in medical applications, and as a biological contributor to reactive nitrogen species (RNS).

The NO2 is found in ambient air at varying concentrations. Nitric oxide (NO) and NO2 are produced by internal combustion engines and natural processes such as forest fires, lightning, and fermentation. NO2 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 NO2 in the ambient air than will regions without such sources. In and near these cities, the ambient NO2 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 NO2 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 NO2 in the environment, and the large contribution from man-made sources, NO2 has been thoroughly studied. Studies have evaluated the sources of NO2, links to acid rain, and the impact of NO2 on materials and living things. This wealth of information facilitates the evaluation of health and safety issues associated with using NO2 as a sterilant gas and provides a technical understanding for the detailed chemical reactions in a typical NO2 sterilization chamber.

One of the earliest mentions of NO2 used in health care-related applications is the use of high concentrations of NO2 for making oxidized cellulose, also called oxycellulose.9 Since the 1940s, manufacturers have exposed cellulose preparations (initially, cotton gauze) to a high concentration of NO2, 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 NO2.12 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 NO2-mediated lethality of the spores.13 In another example of the microbicidal evaluation of NO2, researchers measured the effect of NO2 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 NO2 concentration, a 2 log10 reduction was observed over a 3-hour exposure period.14 A similar study observed more than 1 log10 reduction in the population of airborne Venezuelan equine encephalomyelitis (VEE) virus using a 10-ppm NO2 concentration and 85% RH.8 In both studies, Bacillus subtilis spores were used as controls because they were not damaged by exposure to NO2 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, NO2 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 NO2 concentration and the short exposure time used in these tests.

Another study of airborne NO and NO2 tested cultured Chinese hamster cells (V79 cells) for DNA degradation after exposure to either NO or NO2.15 The V79 cells were exposed to NO and NO2 in varying concentrations, from 0 ppm to 500 ppm and over varying periods (from 5 to 30 min). It was found that NO2 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 NO2, 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 NO2, 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 NO2 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 NO2 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 NO2 can cause such damage is discussed later in this chapter.

Although there has been an interesting history of NO2 for use in health care-related applications and in studies of microbicidal behavior of NO2, the work to evaluate and develop NO2 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 NO2, and from the point of having both intercellular and intracellular NO and NO2, 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 NO2 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 NO2 instead of NO as the sterilant gas.

By now, NO2 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 NO2 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 NO2 sterilization cycles with increasing exposure time and with the concentration and RH for the cycles held constant at 2.75 mg/L NO2 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 NO2, spores of Geobacillus stearothermophilus exhibited the greatest resistance to the
NO2 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.

TABLE 35.1 List of estimated D-values observed with selected microorganisms

Organism Type

Reference Organism

D-value (min)

Bacterial spores

Bacillus atrophaeus (ATCC 9372)


Bacillus pumilus (ATCC 27142)


Clostridium sporogenes (ATCC 3584)


Bacillus subtilis var niger (ATCC 49278)


Geobacillus stearothermophilus (ATCC 7953)


Vegetative spores

Pseudomonas aeruginosa (ATCC 27559)


Salmonella enterica ser Typhimurium (ATCC 14028)


Staphylococcus aureus (ATCC 6538)



Mycobacterium terrae (ATCC 15755)



Trichophyton mentagrophytes (ATCC 18748)


Candida albicans (ATCC 10231)


Nonlipid viruses

Porcine parvovirus


Lipid viruses

Herpes simplex virus type 1


Aside from demonstrating that NO2 can kill all forms of microbial life, screening a wide range of microorganisms permits the identification of microorganisms that have higher resistance to the NO2 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 NO2 process is adjusted to represent the resistance of the load to be sterilized.


The BIs, consisting of spores of G stearothermophilus, exhibit log-linear inactivation kinetics with increasing NO2 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 NO2, hydrogen peroxide (H2O2), 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 NO2 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 NO2 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 NO2 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

FIGURE 35.1 The spore population surviving the nitrogen dioxide (NO2) exposure process is shown to decrease with increasing exposure time. The sterilant concentration (2.0 mg/L NO2) and relative humidity (70%-80%) were constant for the exposure cycles.


This section reviews some general physical and chemical properties of NO2 that are important for using NO2 as a sterilant. A more detailed discussion of chemical reactions, mechanism of microorganism lethality, and material compatibility are described later in this chapter.

Some selected and noteworthy chemical properties of NO2 include:

  • The NO2 boils at 21.2°C (294 K) at sea level to form a yellow-brown gas.28

  • The NO2 freezes at -9°C (264 K) to form a colorless solid of the dimer dinitrogen tetroxide (N2O4).28

  • The NO2 has one unpaired electron, which means that this molecule is a free radical, and sometimes the formula for nitrogen dioxide is written as NO2, with the dot indicating the radical.

  • Due to the weakness of the single N-O bond, NO2 is an oxidizer, but a relatively weak oxidizer compared to other common oxidizers (Table 35.2).29,30

The Lewis dot representation of the principle oxides of nitrogen are shown in Figure 35.2. As shown, there is an unpaired electron on the nitrogen with both NO and NO2. Therefore, NO and NO2 are radicals. However, these unpaired electrons are paired in the reactions forming dinitrogen trioxide (N2O3) and N2O4. Therefore, N2O3 and N2O4 are not radicals.

FIGURE 35.2 Lewis dot diagrams of the principle oxides of nitrogen involved in the nitrogen dioxide (NO2) sterilization process. Abbreviations: NO, nitric oxide; N2O3, dinitrogen trioxide; N2O4, dinitrogen tetroxide.

TABLE 35.2 Oxidation potential for selected gasesa

Oxidation Potential of Selected Gases


Oxidation Potential (V)



Hydroxyl radical




Hydrogen peroxide


Noxilizer’s sterilant, NO2


Abbreviation: NO2, nitrogen dioxide.

aFrom Vanýsek29 and Standard Electrode Reduction and Oxidation Potential Values.30


Nitrogen dioxide found in the environment is formed in most combustion processes where the temperature is high enough to cause a reaction between the N2 and O2 molecules. The formation of NO is shown in equation (1):

The NO is a precursor to NO2, 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):


This reaction is one step in the Ostwald process used in the industrial production of HNO3 from ammonia.31 The two isomers of nitrous acid are HONO (nitrous acid) and HNO2 (nitryl hydride or isonitrous acid). The HNO2 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 HNO3 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 NO2 collide, either in the gas or liquid phase, there is a probability that these two molecules may bond together, forming the dimer N2O4. This is described by equation (6):

The NO2 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 N2O4 will dissociate due to ambient thermal energy. The lifetime of an N2O4 molecule is short, with the half-life of the N2O4 being less than 10 µs at 25°C (298.15 K).33 Therefore, an equilibrium state is described for NO2 where molecules of NO2 are constantly forming dimer molecules and then dissociating to return the monomer form.

When referring to a gas composed of both NO2 and N2O4, the relative amounts of NO2 and N2O4 are not typically mentioned because this can be readily calculated. Instead, a mixture of NO2 and N2O4 is simply referred to as “NO2 gas,” and it should be assumed that there will be some fraction of the NO2 molecules momentarily bound in the dimer form. It is convenient to refer to the total NO2, which includes both monomer and dimer forms of NO2. For any quantity of gas containing NO2, the total number of moles of NO2 is ntotal, which is the sum of the monomer NO2 moles, n (NO2), plus two times the dimer moles, 2n (N2O4), as shown in Equation (7).

The ratio of monomer to dimer is described by the temperature-dependent equilibrium constant, Kp, shown in Equation (8).33

In equation (8), p(NO2) and p(N2O4) 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 NO2 partial pressure divided by the partial pressure of N2O4 will be constant. The temperature dependence of Kp shows that for a given number of moles of NO2, the number of N2O4 molecules will increase as the temperature decreases. The calculated temperature-dependent relationship between NO2 and N2O4 is shown in Figure 35.3. From this graph, it is clear that the partial pressure of N2O4 will increase exponentially with a decrease in temperature.

Another observation from the inspection of equations (7) and (8) is that the relative concentration of N2O4 will increase as the total amount of NO2 increases. This concentration-dependent relationship between NO2 and N2O4 is shown in Figure 35.4. The rate of collisions causing N2O4 formation increases as the concentration of NO2 increases, whereas the dissociation rate is only dependent on temperature and is independent of the NO2 concentration. Therefore, as the concentration of total NO2 increases, the partial pressure of N2O4 will increase more rapidly than the total NO2 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 NO2 concentration (NO2 + N2O4) of 10 mg/L. At 288.3 K (15°C), this 1 L of gas has 8.77 mg/L (191 µmol)
of monomer NO2 and 1.23 mg/L (13.4 µmol) of N2O4. At 298.3 K (25°C), the same 1 L of dry air with 10 mg/L of total NO2 will have 9.36 mg/L (203 µmol) of NO2 and 0.65 mg/L (7.1 µmol) of N2O4. 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 N2O4 dissociates into NO2). Due to the increased NO2 partial pressure caused by dissociation of the dimer, a gas containing NO2 cannot be considered an ideal gas. As another example, consider an increase of NO2 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 NO2 will have 9.36 mg/L (203 µmol) of NO2 and 0.65 mg/L (7.1 µmol) of N2O4. When the total NO2 concentration in this 1 L of dry air is doubled to 20 mg/L NO2 (at 25°C), this liter of gas will have 17.69 mg/L (385 µmol) of NO2 and 2.33 mg/L (25.3 µmol) moles of N2O4. This is shown in Figure 35.4. Notice that doubling the total NO2 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 NO2 gas, these examples also show that the exact concentration for various conditions can be readily calculated.

FIGURE 35.3 Graph of the temperature dependence of the nitrogen dioxide (NO2) and dinitrogen tetroxide (N2O4) concentration in a gas with 10 mg/L of total NO2. The total NO2 concentration is shown with the solid line, the NO2 monomer concentration is shown as the dotted line, and the N2O4 concentration is shown as the dashed line.

FIGURE 35.4 Graph of the concentration dependence of the nitrogen dioxide (NO2) and dinitrogen tetroxide (N2O4) concentration in a gas at 25°C. The total NO2 concentration is shown with the solid line, the NO2 monomer concentration is shown as the dotted line, and the N2O4 concentration is shown as the dashed line.


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 NO2 consists of many absorbance bands facilitating the measurements of NO2 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 NO2 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 NO2.34

The N2O4 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 NO2 monomer. Regardless, the amount of dimer component of the total NO2 must be considered. Heating the measurement gas cell greatly reduces the amount of dimer NO2, as indicated in the graph shown in Figure 35.3. When 10 mg/L of total NO2 is added to a chamber, heating the gas from 25°C to 50°C reduces the amount of N2O4 from 3.38% to 0.67% of the total amount of NO2.

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.


When using NO2 as a sterilant for disinfection or sterilization applications, there are three process variables that directly affect the lethal action of NO2: 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 NO2 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 NO2 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 NO2 concentration tested is over 20 000 ppm. This range offers ample opportunity to balance cycle time, cycle cost, and material compatibility.

FIGURE 35.5 Nitrogen dioxide (NO2) has a broad absorbance band in the UV-VIS band, absorbing violet and blue light. In the IR band, NO2 molecule has discrete absorbance bands.

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 NO2 process. With H2O2 sterilization, increasing humidity at certain temperatures has been shown to reduce observed D-values by increasing condensation (so-called microcondensation) of the H2O2 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 NO2 sterilant. With the NO2 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 NO2 process is confounded by inconsistent results. Whereas at humidity levels above 70% RH, NO2 has a rapid microbicidal action. The mechanism by which the RH level alters the chemistry of the NO2 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.

FIGURE 35.6 Block diagram of a nitrogen dioxide (NO2) sterilization system configured for vacuum cycles. When using a scrubber, the air pumped from the chamber can be safely vented.

Vacuum Nitrogen Dioxide Cycles

Figure 35.6 illustrates the essential design of a sterilization system configured for vacuum cycles using the NO2. 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 NO2 to flow into the sterilization chamber. Pressure transducers, recording the pressure rise associated with the sterilant gas addition, or optical NO2 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).

FIGURE 35.7 Pressure versus time for a typical nitrogen dioxide (NO2) cycle with vacuum. The sterilant exposure phase has an evacuation step, a gas filling step, and an exposure dwell. These phases can be repeated as needed to complete the sterilization cycle. The exposure phases are followed by an aeration phase.

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 NO2 sterilant is added by recirculating the humidified air through the buffer tank until the specified NO2 concentration is reached. The order of gas additions (humidification prior to NO2) is essential to prevent the humidification system from becoming fouled with NO2. The NO2 buffer tank is easily returned to a dry state after the cycle using a small vacuum pump. The dwell time begins after the NO2 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 NO2 removed by the
scrubber, and where inlet and outlet blowers can be used for pressure regulation. An example of the NO2 and humidity concentration profiles measured in a nonvacuum cycle is shown in Figure 35.9. Note that when the NO2 gas is added to the humidified chamber, the concentrations of both NO2 and humidity decrease. This is a key phenomenon in NO2 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.

FIGURE 35.8 Diagram of nonvacuum system.

FIGURE 35.9 Graph of the humidity and nitrogen dioxide (NO2) concentration during a nonvacuum process. Abbreviation: RH, relative humidity.

In both vacuum and nonvacuum cycles, an important part of the NO2 process is measuring and applying the NO2 dose to a chamber. The dose is often set as the number of grams of NO2 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 NO2 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 NO2, 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 NO2 gas. The calculated pressure rise for a specific dose NO2 must account for the relative concentration of the dimer and the monomer. After filling the buffer tank with the correct dose of NO2, the sterilant is flushed into the chamber.


There are several methods for storing, delivering, or generating NO2. For any given application, the most suitable method of providing NO2 depends on several factors. These include the amount of sterilant needed per cycle, the frequency of sterilant usage, transportation considerations, convenience, and single-dose or multiple-dose containers. In consideration of these factors, several methods of providing sterilant to the target chamber have been used successfully. Some of these methods are the following:

  • Delivering NO to a chamber with an ambient amount of oxygen. In the presence of ambient air, which contains about 20% oxygen, the NO will oxidize to form NO2, as shown in equation (2). This reaction is sufficiently fast that only a few minutes are needed to have a high fraction of the NO converted to NO2.

    The NO can be compressed in a cylinder to a high pressure. A 44-L compressed gas cylinder of NO pressurized to 130 atm holds 1900 g NO. This amount of NO is sufficient to fill a 1000-L volume chamber 290 times with a dose that produces 10 mg/L NO2. This method has been thoroughly tested and provides sterilization results that are not significantly different from using NO2 gas as the sterilant source.17

  • The NO2 sterilant can be generated at the time of use using a chemical reaction. For example, combining HNO3 with copper metal generates NO2 gas. This method has been developed and tested using a gas generating module that held a small amount of HNO3 an excess of copper metal, such that the reaction resulted in the liberation of a target number of grams of NO2 gas. This method is best suited for generating a single dose of sterilant gas at the point of use.39,40

  • Using a chemical reaction to make NO and adding this to a chamber with an ambient concentration of oxygen. There are many chemical reactions that will generate NO gas. For example, the first stage of the Ostwald process (in which ammonia is converted to HNO3) produces NO.31 In the first stage of this process, ammonia is oxidized by heating with oxygen in the presence of a catalyst such as platinum with 10% rhodium, to form NO, as shown in equation (10).

    Another reaction that can be used for generating NO is the use of a diazeniumdiolate.41 The NO-releasing compositions, such as the carbon-based diazeniumdiolates, release NO spontaneously under physiologic (aqueous) conditions or mixed with an acid.41

  • Another sources of NO2 are compressed gas cylinders in which NO2 gas is in a mixture of 10% NO2 and balance nitrogen. The limitation with these is that the cylinders can only hold an NO2 partial pressure of 0.5 atm. Above this concentration, there is a risk of condensing some of the NO2 into liquid, thereby disturbing the expected stoichiometry of a measure of gas taken from the cylinder. Therefore, the nitrogen serves as a carrier gas for the NO2 in the cylinder.

  • Because NO2 boils at room temperature, the NO2 sterilant can be stored and transported as a liquid. The cylinder used for storage and transportation does not need to be rated for high pressure because the vapor pressure of NO2 is 1 atm at 21.2°C. As a liquid, this is the densest form of the sterilant and is very convenient. Dosing from a cylinder of liquid NO2 is accomplished by evaporating gas from the surface of the liquid into an evacuated buffer tank. The pressure rise in the buffer tank can be directly correlated to moles of NO2 added to the tank, when equilibrium conditions are considered. Therefore, it is important to know the temperature of the gas in the buffer tank. Once the dose is prepared in the buffer tank, the sterilant can be flushed into the sterilization chamber.

  • Generating NO2 at the point of use with a plasma source is a novel method. The plasma is used to react with air, which is about 80% nitrogen and 20% oxygen, to yield NO2.42,43 This is accomplished by focusing a high-strength electric field from a microwave source on the stream of filtered air that causes the N2 and O2 molecules to dissociate. As the gas of dissociated air molecules flows away from the high-field strength region, atoms combine to form molecules, and a fraction of these molecules will result in NO2 formation.

    A microwave source can be used to generate an oscillating electromagnetic field. Using a specific configuration of waveguide, a standing wave is established so that the highest amplitude electric fields oscillate in one specific location. By placing a metal electrode in this location, the electrode acts as an antenna to couple the electric field to the gas, igniting the plasma. At the
    highest electric field amplitude, dielectric breakdown occurs, and electrons leave the metal surface, accelerating in the electric field. The electron collisions with the oxygen and nitrogen molecules results in the formation of plasma of nitrogen and oxygen ions and radicals.

    Once the dissociation of the N2 and O2 begins, many other chemical reactions occur, only some of which are shown here:

    As is apparent from equations (16), (17) and (18), there are many atomic and molecular species that are formed in the plasma.44 Ozone, nitrous oxide, and other species are formed. However, after a short time, the only stable and persistent chemical species are O2, N2, and NO2, and other gaseous species are reduced to negligible levels through chemical reaction.43,44 The rate of NO2 generation can be accelerated by mixing O2 with the air, approaching a stoichiometric ratio closer to the final NO2 composition.

    These methods of storing, delivering, or generating NO2 gas allow for the optimization of economical and convenient methods for applying NO2 to a wide range of scenarios.


The NO2 process occurs in a chamber in which a controlled amount of NO2 sterilant is combined with humidified air. Although there are chemical reactions that occur in the gas phase, the rate of these gas-phase reactions between NO2 and water (humidity) is relatively slow45 and does not contribute to the sterilizing chemical reactions. However, the rate of the reactions on the surfaces exposed to the sterilant gas and RH is much faster. Also, it is worth noting that the microorganisms that will be sterilized are on the surfaces where these chemical reactions occur. Therefore, the impact of the surface chemistry should be evaluated. To understand the chemical reactions that lead to microbial lethality, it is helpful to recall the many biological roles of NO. It was stated before that NO is produced by neutrophils and macrophages in response to inflammation and infection in vivo.19 In vivo, the process by which one cell can cause another cell’s death, extrinsic apoptosis, has several steps. These steps are summarized as follows: NO generated by a cell auto-oxidizes to form NO2; NO and NO2 combine to form N2O3; the N2O3 reacts with DNA through the nitrosation of nucleosides cytosine and guanine.46 Excluding the cytosine and guanine reactions, these steps can occur either in the extracellular region, within the cell membrane, or within the microorganism.23 It is interesting to note that the oxidation of NO and rate of reactions producing N2O3 can be 30 times faster in the hydrophobic lipid bilayer (as in the cell or spore membrane) than the same reactions in the extracellular, aqueous environment.23 The NO2 sterilization processes in vitro follow this same chemical reaction cycle found in vivo, leading to the degradation of a microorganism’s DNA. There are three scenarios where oxides of nitrogen interact with the microorganism, and these are shown in Figure 35.10. The first scenario is the known in vivo reaction described earlier, where macrophages produce NO, causing extrinsic apoptosis of target microorganisms (Figure 35.10, Reaction A). The second scenario to consider is the direct interaction of microorganisms (eg, spores) with the humidified sterilant gas (see Figure 35.10, Reaction B). The third scenario is the interaction of the microorganisms with the chemical environment that develops on the surfaces exposed to the NO2 process (see Figure 35.10, Reaction C).

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May 9, 2021 | Posted by in MICROBIOLOGY | Comments Off on Nitrogen Dioxide
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