Sterilisation of Plastic Containers and Bottles

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Sterilisation techniques are designed to kill microorganisms. There are various sterilisation methods, however, the three basic approaches used to sterilise plasticware are:

  • Ethylene Oxide (EtO)
  • Radiation (gamma radiation, electron beam radiation)
  • Steam Autoclave

Tests should always be run on plasticware to determine suitability for a given sterilisation method.

Terms Associated with Sterilization

Bioburden

This is the number of microorganisms (bacteria, virus, fungi, etc.) present. Microbiologists can test for these. When sterilising ware, it is important to eliminate the bioburden to prevent further microbial growth.

Pyrogens

A pyrogen, which means fever causing, is a remnant of bacteria that contains chemicals called endotoxins. Endotoxins can cause fever if injected into a mammal. Several tests exist to identify endotoxin contamination. Something may be sterile, but still have pyrogens on it. Glass can be sterilised and de-pyrogenated at the same time. Exposure to high temperature (600°F or higher) will kill microorganisms and burn up endotoxins. The higher the temperature, the shorter the exposure time needed for de-pyrogenation. Most plastic ware is incapable of being exposed to these high temperatures. Therefore, plastic ware may be sterilised but, if it needs to be de-pyrogenated, it is usually washed with pyrogen free water.

RNase and DNase

Contaminating enzymes; RNase (which breaks down RNA), and DNase (which breaks down DNA), are the most critical substances influencing experimental work in molecular biology. These contaminants are one of the principle causes of failure in the manipulation and analysis of RNA and DNA in the laboratory. These enzymes come primarily from contact with skin (direct and indirect). Pipettors, lab benches, autoclaves, lab ware, doorknobs, etc. are all frequently handled without gloves. All of these items, and virtually everything in a lab setting, are contaminated with these enzymes after contact with skin. Wearing gloves only offers protection until a surface is contacted that has itself contacted skin, at which time the glove becomes contaminated. Because of the resiliency of these enzymes, maintaining a RNase / DNase – free lab is extremely difficult.

Steam autoclaving ware at 121°C for 20 minutes will destroy DNase, but will not destroy RNase. Baking ware in an oven at 300°C for 4 hours will destroy DNase and RNase. However, this method is not possible with most plastic items because of the high temperature. Alternatively, there are decontaminating cleansing solutions available in the marketplace that will destroy both of these enzymes immediately upon contact and can be used with most materials. The solution is simply sprayed onto the surface of the ware, which is then rinsed thoroughly with nuclease-free water.

Ethylene Oxide

Ethylene oxide (EtO) is a toxic, cancer causing gas. Technology and worker protection legislation allow continued EtO use. Most plastic can be EtO sterilised. EtO must contact the surfaces to be sterilised. There are several ways EtO sterilization can be accomplished.

Pure EtO

Empty ware in an open bag or ware in a sealed bag with a “breather” window, is placed in a chamber. Air is evacuated and moisture introduced (dry microorganisms are resistant to EtO sterilization).

Pure EtO is flooded into the chamber. Chamber internal pressure is kept lower than external pressure to ensure gas will not leak. Exposure time varies depending on ware and bioburden. After exposure, the chamber is purged with filtered sterile air to eliminate residual EtO.

Dilute EtO

Since it is safer than pure EtO, a 10-15% mixture of EtO with inert gas is used. Empty ware in an open bag or ware in a sealed bag with a “breather window” is placed in a chamber. Air is evacuated, and moisture is introduced (dry microorganisms are resistant to EtO sterilisation). Dilute EtO is flooded into the chamber and the chamber’s temperature increased up to 60°C (140°F). Exposure time of 4 to 24 hours varies depending on ware, bioburden, and sterilization parameters. After exposure, the chamber is purged with filtered sterile air to eliminate residual EtO.

Most plastic ware is capable of being EtO sterilised. However, zinc stearate process aid, used in injection blow moulding, can cause precipitants (particulate) to form in liquid products packaged in EtO sterilised ware.

Therefore, only special LDPE grades and colourants that do not require zinc stearate for injection blow moulded ware should be treated by EtO sterilisation processes. Additionally, tests should always be run on plastic ware to determine suitability for a given sterilisation method.

Steam Autoclave

Autoclaving can sterilize empty or filled, sealed ware. The effect of temperature and moisture kills microorganisms. Autoclaving involves exposing ware for a time to steam. The autoclave acts like a pressure cooker, allowing the steam temperature to get above the boiling point of water (100°C=212°F). Typically, autoclaving is done at 15 psi (pounds per square inch) steam being at 121°C (250°F). When autoclaving bottles always ensure the caps are loosened or removed to prevent accidental collapse or deformation. Tests should always be run on plasticware to determine suitability for a given sterilisation method.

Radiation

Ware is exposed to ionizing radiation that knocks electrons off atoms it contacts. Ionizing radiation is lethal to microorganisms because of its destructive effect upon the contents of living cells. There are two common sources of ionizing radiation used for sterilization:

  • Cobalt 60 (gamma radiation) OR
  • Electron beam or E-beam (high energy electrons)

The amount of radiation from either Cobalt 60 or electron beam is measured in MegaRads (MRads)

or KiloGrays (KGy). One MegaRad equals ten KiloGrays. Because gamma sterilisation and E-beam both use radiation, packaging materials react similarly in both systems.

Cobalt 60 Gamma Radiation

A gamma radiation sterilization facility consists of a thick walled concrete maze in a room built around a well filled with water. In the well are a number of pencil-sized steel rods impregnated with radioactive Cobalt. Articles to be sterilised are placed on conveyors that bring them through the concrete maze into the room where the radioactive rods are located. The number of rods raised from the well and the exposure time controls the degree of exposure. After exposure, ware is conveyed from the room via the maze.

A radiation dose sufficient to kill bacteria and spores is about 2.5 MRads. To minimize costs plus attain sterilisation, bioburden is determined then the minimum dosage plus a safety factor is selected.

Gamma radiation has high penetrating power (about 50 cm or close to 20 inches of the same unit-density material). Thus, many parts can be packed together for sterilisation. In this instance, the dosage reaching the centre of ware multi-packs is validated. Slightly higher doses occur at the outside edges of multi-packs.

Usually, empty packaging components are sterilised via gamma radiation. Since effects of radiation are cumulative, twice the normal dose is sometimes examined to insure minimal problems.

Listed below are thermoplastic materials that are recognized as capable of being gamma radiation sterilised, although tests should always be run on plastic ware to determine suitability for a given sterilisation method:

  • Low Density Polyethylene
  • Polyethylene Naphthalate (a newly emerging group of polymers
  • Linear Low Density Polyethylene
  • High Density Polyethylene (those containing phosphite stabilizers may yellow
  • Polyethylene Terephthalate
  • Polystyre
  • Polycarbonate
  • Nylon
  • Cyclic Olefin Copolymers (a newly emerging group of polymers)

Problems can occur when gamma radiation sterilizing polyvinyl chloride (PVC) or fluoropolymers (PTFE, etc.).

Tests should always be run on plasticware to determine suitability for a given sterilisation method.

Electron Beam (E-Beam) Radiation

An E-beam radiation sterilization facility consists of a protective maze built around an E-beam generator. The E-beam generator delivers a high dose of electrons focused in a narrow beam at the items to be sterilised. After exposure, ware is conveyed from the maze.

A radiation dose sufficient to kill bacteria and spores is about 2.5 MRads. To minimize costs and attain sterilisation, bioburden is determined and the minimum dosage plus a safety factor is selected.

Electrons from the E-beam generator have limited penetrating power (a 10-MeV E-beam will penetrate only about 5 cm or 2 inches of a unit-density material). Thus, a limited number of parts can be packed together for sterilization. The dosage reaching the centre of a ware multi-pack is validated. Higher dosages will occur at the outside edges of ware multi-packs.

Usually, empty packaging components are sterilised via E-beam. Since effects are cumulative, twice the normal dose is sometimes examined to insure minimal problems.

Listed below are thermoplastic materials that are recognized as capable of being electron beam radiation sterilised, although tests should always be run on plastic ware to determine suitability for a given sterilisation method:

  • Low Density Polyethylene
  • Linear Low Density Polyethylene
  • High Density Polyethylene (those containing phosphite stabilizers may yellow)
  • Polyethylene Terephthalate
  • Polystyrene
  • Polycarbonate
  • Nylon
  • Cyclic Olefin Copolymers (a newly emerging group of polymers)
  • Polyethylene Naphthalate (a newly emerging group of polymer)

Problems can occur when E-beam sterilising polyvinyl chloride (PVC) or fluoropolymers (PTFE, etc.)

Tests should always be run on plasticware to determine suitability for a given sterilisation method.

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