Disinfection and Sterilization With Hydrogen Peroxide

Disinfection and Sterilization With Hydrogen Peroxide

Randal W. Eveland

Hydrogen peroxide was first isolated by Louis Jacques Thénard in 1818 from the reaction of barium peroxide with nitric acid. The first proposed disinfectant use was in 1858 by the English physician B. W. Richardson. Since that first report, hydrogen peroxide has become one of the most widely used microbiocides for antiseptic, disinfectant, and sterilization applications, with well-established effectiveness against a wide range of microorganisms.

The use of hydrogen peroxide, in liquid formulation or particularly in gas (or vapor) form, for disinfection and sterilization has continued to increase for a number of reasons. Within health care facilities, steam sterilization has been the sterilization method of choice for well over 100 years, but many electronic devices and instruments with heat- and moisture-sensitive materials cannot withstand the process conditions. Ethylene oxide gas was widely used in hospitals as a low-temperature method, but its use has decreased due to long process time (in particular for aeration or removal of toxic residues), the health hazards associated with its use (eg, carcinogenic potential of both the sterilant and its decomposition products), and government regulations. Hydrogen peroxide sterilization technologies have become popular in these cases. Similarly, gas or liquid phase sterilization processes have found applications as alternatives in various industrial processes, including medical device or component sterilization. Furthermore, gas or aerosolized peroxide processes are widely used for area disinfection (fumigation) applications (eg, pharmaceutical, animal research, production spaces, and infection outbreak remediation) due to undesired use of formaldehyde due to the chemicals’ known carcinogenicity and the long process time (see chapter 36).

Gaseous hydrogen peroxide processes can offer broadspectrum activity, short cycle times, and good material compatibility and are environmentally friendly. Hydrogen peroxide decomposition into environmentally safe and nontoxic byproducts (water and oxygen) is a major force driving the effort to use hydrogen peroxide for disinfection and sterilization applications.


The chemical formula for hydrogen peroxide is H2O2. In its pure form, it is a pale blue liquid, whereas hydrogen peroxide solutions with water are clear and colorless. Hydrogen peroxide can be mixed with water at any concentration. The chemistry of hydrogen peroxide is characterized by the reactivity of the peroxide (double oxygen) bond. This oxygen-oxygen bond is weak and will disassociate to a powerful oxidizing agent. Hydrogen peroxide decomposes into water and oxygen upon heating or in the presence of numerous chemicals, particularly transition metal salts of metals such as iron, copper, manganese, nickel, or chromium that may catalytically decompose the peroxide.

Commercial grades of liquid hydrogen peroxide typically contain stabilizers to prevent decomposition and provide stability during storage and transport. Typical stabilizers include chelants and sequestrants, with stannates and phosphates being commonly used. Stabilizers are not known to have an effect on disinfection or sterilization processes, but they are a consideration for the equipment manufacturers.

Hydrogen peroxide is a strong oxidizing agent with a standard reduction potential (E°) of 1.78 V. In comparison to hydrogen peroxide (with a larger reduction potential indicating a stronger oxidizer), ozone is a stronger oxidizer (E° = 2.07 V), peracetic acid is similar in oxidizing capability (E° = 1.81 V), whereas chlorine is a weaker oxidizer (E° = 1.36 V).1,2 The hydroxyl radical, key to efficacy, has a standard reduction potential of 2.80 V.1

The terms gas or vapor are used interchangeably when discussing vaporized hydrogen peroxide processes, and both terms refer to the same physical state of matter.
All vapors are gases, but not all gases are vapors. For a vapor, changes in pressure or temperature can allow the vapor to change to a liquid (condense). Distinctly different from a gas or vapor, an aerosol is a fine dispersion of liquid droplets. For hydrogen peroxide disinfection and sterilization processes, the control of temperature, pressure, sterilant delivery, and vaporization method is critical to ensure the gaseous hydrogen peroxide is delivered and maintained as intended (eg, at or below the condensation point) for an efficacious process.

A wide variety of hydrogen peroxide concentration solutions (with the balance as water) are used. A 3% hydrogen peroxide solution is used over-the-counter as a topical antiseptic, with a maximum of 8% hydrogen peroxide concentration recommended for home use. Various concentrations may also be used in liquid-based formulations, such as in combination with peracetic acid and under acidified formulations (see chapter 18). For disinfection and sterilization applications, hydrogen peroxide concentrations from 30% to 59% are commonly used to generate the gas under various conditions, for example, under- or oversaturated (to allow for gas condensation on target surfaces). Hydrogen peroxide concentrations from 60% to 100% are considered more dangerous to transport, for example, being classified by the US Department of Transportation (DOT) as “Packing Group 1,” the same classification used for transporting hydrogen peroxide rocket fuel. The DOT classification helps explain why concentrations at or above 60% are not routinely used as a source of hydrogen peroxide in liquid or gas disinfection or sterilization applications.


The hazards and properties of hydrogen peroxide are well established in the scientific literature.3 Hydrogen peroxide is a natural constituent of the body; it is present in exhaled breath at levels of 300 to 1000 µg/m3 and in food at levels of 3 to 7 ppm.4 The human liver, one organ that generates hydrogen peroxide, produces an estimated 3.8 g/kg of liver per day.5 In an adult man with a liver weighing 1800 g, this amounts to 6.8 g/d or 97 mg/kg of body weight per day. As the body readily produces hydrogen peroxide, the in vivo toxicity is moderated by the presence of enzymes such as catalase (an enzymatic, highly effective, catalytic decomposer of hydrogen peroxide) and metal ion sequestration that limits hydroxyl radical production.

The primary exposure routes to hydrogen peroxide are via inhalation, ingestion, skin, and/or eye contact. Hydrogen peroxide solutions of 35% and greater are severely irritating to corrosive and may result in irreversible eye damage including blindness. Hydrogen peroxide can be harmful if inhaled or swallowed. The potential for a toxic effect will vary greatly depending on the hydrogen peroxide concentration and the route of exposure. Hydrogen peroxide is not a sensitizing agent.3 There are no known effects of hydrogen peroxide to reproduction/development.3 Although there is limited evidence of hydrogen peroxide carcinogenicity with animal studies, the relevance and significance of this information for humans is unclear and therefore inadequate to characterize hydrogen peroxide as a carcinogen to humans.3 In vitro studies have shown hydrogen peroxide can have mutagenic, genotoxic, and cytotoxic effects; however, the relevance to in vivo systems is questionable due to natural mechanisms (as stated earlier) present to mitigate hydrogen peroxide.3

Although hydrogen peroxide is classified as odorless, some users have identified a nasal irritation response (likely via chemosensitive trigeminal nerve response versus an olfactory nerve interaction)6 and characterized the odor as slightly pungent or acidic. Hydrogen peroxide irritation has been identified to occur at 150 mg/m3 (108 ppm),7 but it should be noted that odor thresholds and sensory thresholds are often highly variable because methodology and individual sensitivity will vary.


Oxidizing agents are widely used for various antimicrobial medical, dental, industrial, and agricultural applications (see chapter 18). Oxidizing agents, such as hydrogen peroxide, can be powerful antimicrobial agents due to their ability to remove electrons from other substances such as proteins, lipids, and nucleic acids.8

The mechanism of action is believed to be based on the formation and subsequent reaction of hydroxyl radicals via the superoxide (O2*2) and hydrogen peroxide as first proposed by Haber and Weiss.9 The reaction to form the radicals is believed to be catalyzed in vivo by transition metal ions via the Fenton reaction. The first step in the process is the reduction of iron(III) to iron(II) (equation 1). Iron(II) can then react with hydrogen peroxide to generate the hydroxyl radical (•OH) (equation 2). The net reaction to form hydroxyl radical is shown in equation 3.

The hydroxyl radical (•OH) is a potent oxidizer to cellular components, nucleic acids, and proteins. It is the oxidation and subsequent loss in viability and function that inactivates microorganisms to provide the antimicrobial effect. The process begins on the outer surfaces and can progress, as structure damage progresses, to intercellular components.8 The oxidation (or loss of an electron by a
molecule, an atom, or an ion) and damage to these molecules will have dramatic effects on their structure and function, which culminate to give biocidal activity. Oxidation reactions with biocides have been shown to cause macromolecular unfolding, fragmentation, and crossreaction with oxidized groups.8 Proteins, carbohydrates, and lipids on the surface of microorganisms are particularly accessible targets, followed by various intercellular components, including proteins and nucleic acids, as the structure of the microorganism disintegrates.

The effects of hydrogen peroxide on nucleic acids have been well described as hydrogen peroxide, and other reactive oxygenated species (including the short-lived but extremely reactive superoxide ions and hydroxyl radicals) are formed during cellular respiration and can have detrimental effects in eukaryotic cell components; indeed, these effects have been linked to cell aging and mutagenesis.10 For example, hydrogen peroxide has a dramatic effect on DNA and RNA structures, attacking both the nucleotide bases and the sugar-phosphate backbone of these structures.11 The oxidation of these groups will lead to strand breakage and cross-reactions between converted bases/sugars, which will affect the replication, transcription, translation, and other roles of these essential structures. With respect to proteins and amino acids, a difference has been reported in the specific reactivity and modes of action between hydrogen peroxide in liquid or gas form.12,13

Overall, oxidizing agents such as hydrogen peroxide have multiple effects on proteins, lipids, carbohydrates, and nucleic acids, which leads to loss of their structure and, hence, function. Specific effects include changes in structure, breakdown of these macromolecules into smaller constituents, and transformation of structural and functional groups as well as some effects leading to cross-linking within and between these molecules under some conditions. These effects will result in the loss of viability of the microbial target. The effects are clearly significant on vegetative and actively metabolizing microorganisms, including bacteria and fungi. The overall damage to the structures of spores, cysts, and viruses also appears to cause a loss in viability presumably due to specific effects not only on surface proteins and lipids but also on penetration to the nucleic acid.


As an oxidizing chemistry, hydrogen peroxide in various forms and concentrations has demonstrated broadspectrum activity against a wide variety of microorganisms. Considering pathogens, E. H. Spaulding taught that certain classes of microorganisms can be generally classified as being more resistant to disinfection and biocides than others.14 This classification is shown in Table 32.1 and includes a summary of those microorganisms that have been shown to be inactivated by gaseous hydrogen peroxide. The list of organisms is not exclusive but provides an understanding of the potential and likelihood for hydrogen peroxide to disinfect/sterilize microorganisms in the same classification group. The most resistant organism to gaseous hydrogen peroxide is generally recognized as Geobacillus stearothermophilus spores, although spores of Bacillus atrophaeus (Bacillus subtilis var globigii) have been reported to be more resistant to liquid formulations.53 Bacterial spores are much more resistant to hydrogen peroxide than the vegetative form of the organism. The list of organisms within the resistance spectrum, from least to most resistant, is meant as a general guide. The resistance of organisms, even within the same classification, can vary greatly depending on experimental methods. Experimental conditions such as exposure time, concentration, and test system play a significant role in defining resistance, but other factors such as culture purity and the presence of organic or inorganic soil will also affect the observed resistance.

Efficacy has also been demonstrated against the mycoplasma Acholeplasma laidlawii, against Cryptosporidium parvum oocyst,54 and helminths (parasitic worms, eg, tapeworm or nematode) Caenorhabditis elegans and Syphacia muris.55 For S muris, there is research suggesting that effectivity may vary when considered in vivo versus in vitro.46

Prion disease-causing agents, for example, those associated with the diseases scrapie, Creutzfeldt-Jakob disease, and chronic wasting disease, are not strictly considered as living microorganisms because they are believed to consist solely of protein with no associated nucleic acids (see chapter 68). Although not viable, prions are transmissible and can be a significant contamination concern in the presence of high-risk tissues (eg, contaminated brain tissue). Prions are considered difficult to inactivate and are therefore ranked as more resistant than bacterial spores in Table 32.1. Inactivation of prions on contaminated surfaces has been documented for subatmospheric pressure sterilizers using 59% hydrogen peroxide gas,15 but certain subatmospheric pressure sterilizers that used plasma required precleaning with alkaline formulations prior to being effective or a process to increase gas-phase hydrogen peroxide concentration to approximately 90% to 95% for repeatable effectivity.56

When similar concentrations are considered, disinfection and sterilization processes with liquid hydrogen peroxide solutions are not nearly as rapid as those with gaseous hydrogen peroxide. The formation and subsequent reaction of hydroxyl radicals occur more readily in the gas phase because water can stabilize hydrogen peroxide in solution via hydrogen bonding. The rate of reaction differences between liquid and gaseous hydrogen peroxide has been evaluated experimentally and compared via a difference in D-value. The D-value refers to the time required to reduce the population of a microorganism by 90% (or 1 log10).
As shown in Table 32.2, the D-value for three spore species was evaluated at different hydrogen peroxide liquid and vapor concentrations (250 000 and 1.5 mg/L, respectively). Even at the concentration of 1.5 mg/L hydrogen peroxide, the gaseous sterilant has a faster D-value than aqueous hydrogen peroxide at 250 000 mg/L.

TABLE 32.1 Classification of microorganism resistance and identification of microorganisms for which gaseous hydrogen peroxide efficacy has been demonstrated

Spaulding Classification

Efficacy Demonstrated With Gaseous Hydrogen Peroxide

More Resistant


Less Resistant


Scrapie 263K strain,15 bovine spongiform encephalopathy (BSE) 6PB1 strain15

Bacterial spores

Bacillus anthracis,16,17,18 Bacillus atrophaeus (formerly Bacillus subtilis),16,18,19,20,21,22 Bacillus cereus,19,20 Bacillus circulans,19 Bacillus firmus,21 Bacillus megaterium,21 Bacillus pumilus,20,21 Bacillus thuringiensis,18 Clostridium botulinum,23 Clostridium difficile,20,24,25,26 Clostridium perfringens,20 Clostridium sporogenes,22 Clostridium tetani,20 Geobacillus stearothermophilusa (formerly Bacillus stearothermophilus)16,18,19,20,25,26,27,28


Mycobacterium avium,29 Mycobacterium bovis,20 Mycobacterium chelonae,20 Mycobacterium smegmatis,22 Mycobacterium terrae,29 Mycobacterium tuberculosis,20,30,31 Mycobacterium fortuitum32

Small nonenveloped viruses

Calciviridae (feline calicivirus, Murine norovirus, vesicular exanthema of swine virus),33,34,35,36,37 Flaviviridae (hog cholera virus),34 Paramyxoviridae (Newcastle disease virus),34 Parvoviridae (mouse and porcine parvovirus),33,38 Picornaviridae (polio type 1, footand-mouth disease virus, swine vesicular virus),20,33,34 Reoviridae (bluetongue virus),34 Rhabdoviridae (vesicular stomatitis virus)34

Gram-negative bacteria

Acinetobacter baumannii,25,26,28,39,40 Acinetobacter calcoaceticus,20 Bacteroides fragilis,20 Brucella suis,41,42 Burkholderia cepacia,26 Burkholderia mallei,43 Burkholderia pseudomallei,41 Enterobacter cloacae,40 Escherichia coli,20,22,26 Francisella tularensis,41,42 Klebsiella pneumoniae (Legionella species),22,26 Moraxella osloensis,20 Pseudomonas aeruginosa,20,21,26 Pseudomonas cepacia,20 Salmonella choleraesuis,22 Serratia marcescens,20,44 Xanthomonas maltophilia,20 Yersinia pestis41,42,45


Alternaria species,46 Aspergillus brasiliensis (formerly Aspergillus niger),20,46 Blastomyces dermatitidis,47 Candida albicans,20,46 Candida parapsilosis,20,46 Coccidioides immitis,47 Histoplasma capsulatum,46 Penicillium species,46 Trichophyton mentagrophytes19,20

Large, nonenveloped viruses

Adenoviridae (adenovirus),33,35,48 Parvoviridae (parvovirus)38

Gram-positive bacteria

Deinococcus radiodurans,20 Enterococcus faecium/Enterococcus faecalis (VRE),20,26,39,49,50 Enterococcus hirae,46 Listeria monocytogenes,20,22 Staphylococcus aureus (MRSA),20,26,28,39,51 Staphylococcus epidermidis,20,51 Streptococcus pneumoniae26

Enveloped viruses

Orthomyxoviridae (avian influenza virus, influenza A[H1N1]),34,35,52 Herpesviridae (pseudorabies virus),20,34 Poxviridae (Vaccinia)32,33

Abbreviations: MRSA, methicillin-resistant Staphylococcus aureus; VRE, vancomycin-resistant enterococci.

a Most resistant organism to vaporized hydrogen peroxide.

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May 9, 2021 | Posted by in MICROBIOLOGY | Comments Off on Disinfection and Sterilization With Hydrogen Peroxide

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