Sterilization Techniques and Regulations

1.1.1. Heat Sterilization

Moist heat sterilization, steam is used for sterilization by applying saturated steam under pressure.  This can be done for items placed in an autoclave or for vessels/pipes can be done by steaming-in-place. The autoclave, or steam line, uses saturated steam and pressure for a specific amount of time to achieve sterilization lethality, which accumulates between temperatures of 121.1-132℃.  The steam cycle will be challenged by a microorganism, biological indicator, to demonstrate lethality and will be monitored by temperature and pressure recordings. 5

            For steam sterilization of medical waste, a waste or decontamination cycle will need to be run at a minimum of 121.1℃ and 15 psi for no less than (NLT) 45 minutes.  The cycle will need to monitor and record temperature, pressure, and respective time throughout the cycle. In addition to this there will be a record of documented weekly challenges to a loaded waste cycle with biological indicator (BI) Geobacillus stearothermophilus with a spore population of NLT 1.9x104. 19

Dry heat sterilization can be achieved through static-air (oven like) or forced-air (convection) sterilizers.  Both methods have slow heat penetration, although forced-air is faster and more uniform than static-air. Dry heat and high temperatures are not suitable for most materials and is  typically between 150-170℃ for 150-60 minutes respectively. 5  

For dry heat sterilization of medical waste an incinerator is typically used.  This will require a permit for solid waste management or a permit for operation of the incinerator from the Division of Air Quality (DAQ).  Medical waste cycles will be treated at NLT 648.9℃ and the cycles will continuously record chamber temperature and time for monitoring. In addition to this ash samples will be sampled, and interlocks or other controls will need to be used to ensure operating temperatures and permitting requirements are met.  Depending on the incinerators operating age the frequency of ash sampling will vary, with more frequent sampling earlier in its lifecycle. Ash samples at minimum will record sample date, time, laboratory name, contact information, certification number and analysis results for “arsenic, barium, cadmium, chromium, lead, mercury, selenium, and silver” 19. Additional ash samples may be required of local municipal solid waste landfills in accordance with local requirements.

1.1.2. Gas Sterilization (Ethylene Oxide EtO, Nitrogen Dioxide NO2, Chlorine Dioxide, Hydrogen Peroxide, Peracetic Acid, formaldehyde)

            Ethylene Oxide gas (ETO) is used for low temperatures 37-63℃, and humidity between 40-80%, these two parameters along with exposure time and the concentration of the gas will determine the sterility.  ETO knocks down microbial activity by alkalizing protein, DNA, and RNA. 5 Typically ETO is used on materials that cannot handle heat and are moisture sensitive. 44

            Nitrogen Dioxide (NO2) gas sterilization is a low-temperature process, which makes it ideal for use with heat-sensitive devices that cannot be sterilized using high-temperature methods like autoclaving. NO2 gas sterilization works by damaging the DNA and other cellular components of microorganisms, including bacteria, viruses, and fungi. The gas is typically introduced into a sealed chamber containing the device to be sterilized, and the gas is allowed to penetrate all surfaces of the device for a predetermined period of time.  45–48

            Chlorine dioxide (ClO2) is a highly effective sterilant that is commonly used to disinfect and sterilize a variety of medical devices and equipment. ClO2 gas is highly reactive and can quickly penetrate surfaces and kill microorganisms, including bacteria, viruses, and fungi. ClO2 gas is also effective against spores, which makes it ideal for sterilization of heat-resistant medical devices. ClO2 gas sterilization is a low-temperature process, which makes it ideal for use with heat-sensitive devices that cannot be sterilized using high-temperature methods like autoclaving. The process involves introducing the ClO2 gas into a sealed chamber containing the device to be sterilized. The gas is allowed to penetrate all surfaces of the device for a predetermined period of time, after which the chamber is vented to remove the gas, and the device is allowed to air out before it can be used. 5,49–51

            Hydrogen Peroxide can be used for sterilization in its vapor phase or in a gas plasma phase.  For gas plasma Hydrogen Peroxide sterilization, the parts need to be in a vacuum pressurized closed chamber and have an electric field created from either microwave energy or radio frequency.  The electric field will charge the gas molecules, turning them into free radicals that readily disrupt and destroy cellular components of microorganisms. This sterilization cycle occurs between 37-44℃ and range from 52-73 minutes of processing time. Vaporized Hydrogen Peroxide (VHP) is also used under a vacuum pressurized closed chamber.  VHP is heated to a low temperature and pumped into the chamber.  VHP has good material compatibility, but it cannot treat cellulose and can cause some materials to become brittle, like nylon. 5,52  

            Formalin is a vaporized form of formaldehyde gas that can be used for sterilization.  Formalin at temperatures between 70-75℃ at concentrations of 8-16 mg/l is pumped into a chamber that has had air removed, similar to an autoclave. 5 The formaldehyde gas then circulates throughout the chamber, effectively sterilizing any surfaces or equipment inside. 38

            Vaporized peracetic acid (VPA) can be used to sterilize spores at low temperatures and relative humidity’s between 20-80%.  Peracetic acid is a powerful oxidizing agent that can damage microbial cell walls and other cellular components, leading to cell death. The use of VPA for sterilization has several advantages over other methods, such as steam sterilization. VPA can be used to sterilize heat-sensitive materials, such as electronic components and plastics, without causing damage. VPA can also penetrate hard-to-reach areas, such as small crevices and narrow channels, ensuring complete sterilization of the product. Low temperatures minimize the risk of damage to the product and reduces energy consumption. VPA sterilization can be performed in a closed chamber, which ensures operator safety and reduces the risk of product contamination. 5,53

1.1.3. Chemical

            Peracetic Acid can be used as a surface sterilizer through denaturing proteins, and cell wall permeability while oxidizing cellular components.  Due to its highly biocidal oxidation nature, Peracetic acid will inactivate both gram negative and positive bacteria, fungi, and yeast at concentrations of less than 100 parts per million (PPM) in 5 minutes of contact time. 5

            Chemical treatments of microbiological waste in North Carolina is required to be treated with 10% chlorine solution for NLT one hour.  The treatment also will need to be challenged weekly with a full load and a Bacillus atrophaeus BI with a population of NLT 1.0x106.  Other load testing for chemical treatments will have to be tested to compliance with the same BI. All chemical challenges will be maintained in a record with the test date, test start and stop time, BI information, and test result recorded. 54

1.1.4. Irradiation

Ionizing radiation sterilization can be achieved through cobalt 60 gamma rays, electron accelerators. 5  Gamma irradiation uses photons at energy peaks of 1.17 and 1.33 MeV for a dose of at least 10 kGy/hr to kill DNA on and within a product.55  Gamma irradiation is responsible for the sterilization of more than 40% of the worlds single use bioprocess systems materials 56. Gamma irradiation comes from Cobalt 60, which is created in a nuclear reactor and has a half-life of 5.3 years.  Because of this half-life the Cobalt-60 must be replaced annually.

            X-ray sterilization and E-beam sterilization deliver highly charged streams of electrons for a dose of at least 60 kGy/hr to a product which kills DNA. 55 E-beam technology has poor penetration of products, so surfaces and individually wrapped items can be sterilized but not large bulks of materials 56. X-ray sterilization is faster than gamma X-ray radiation has a lower penetration depth than gamma radiation and requires higher doses to achieve the same level of sterilization. 57 Gamma radiation has several advantages over electron beam and X-ray radiation for sterilization purposes. Gamma radiation can penetrate deep into materials and can be used for the sterilization of large volumes of products. Gamma radiation is also easy to control and monitor, and the process parameters can be easily validated. Gamma radiation is a well-established and widely used method for sterilization, with a long history of successful use in many industries. 58,59

            Infrared radiation can be used to sterilize products of bacterial spores but is not widely used or regulated in the pharmaceutical industry. Infrared radiation works by emitting energy in the form of heat, which can destroy the bacterial spores by damaging their DNA and other cellular components. Infrared radiation sterilization may not be suitable for all types of materials. It may be more effective for sterilizing materials that are transparent or translucent to infrared radiation, such as glass or some plastics. Infrared radiation is often used in conjunction with other sterilization methods, such as steam or chemical sterilization, to ensure complete sterilization of the product or equipment. The effectiveness of infrared radiation in sterilization depends on several factors, including the intensity and duration of the exposure, the distance between the source and the target, and the properties of the material being sterilized. 5  

1.1.5. Microwave

Microwave sterilization is a relatively new sterilization technique that has been explored for use in the pharmaceutical industry. Microwaves are radio-frequency waves and can be used to sterilize a product that is not meltable, in 1-5 minutes. 5 It involves the use of microwave energy to rapidly heat the surface of medical devices and equipment, which can then kill microorganisms that are present. Unlike traditional sterilization methods like autoclaving, which rely on high-temperature steam, microwave sterilization can be carried out at much lower temperatures, which makes it ideal for use with heat-sensitive devices that cannot be sterilized using high-temperature methods.

            Microwave treatment of medical waste in North Carolina will be applied for NLT 30 minutes at a minimum temperature of 95℃. The treatment will be continuously monitored with temperature and time recorded.  The treatment will be challenged on a weekly basis with a full load and Bacillus atrophaeus BI with NLT 1.0x106 population.  If the equipment manufacturers instructions are more stringent then they will also need to be tested. 54

1.1.6. Ozone

            Ozone sterilization is a technique that has been explored for use in the pharmaceutical industry. It involves the use of ozone gas, which is a highly reactive form of oxygen, to kill microorganisms on the surfaces of medical devices and equipment. Ozone sterilization is a low-temperature process, which makes it ideal for use with heat-sensitive devices that cannot be sterilized using high-temperature methods like autoclaving.  Ozone can be used as a sterilizer, but it is very unstable due to its high reactivity. Typically used to sterilize water, ozone cycles are about four hours and occur between 30-35℃.5,60