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.

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

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 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.

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):

or

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 n_total, 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, K_p, 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 K_p 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 K_p 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.

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.

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.

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:

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).