Autoclave Validation for Waste Decontamination in BSL-2 and BSL-3 Laboratories

Containment Talk 6.

Containment Talk articles discuss risk and safety issues in and around microbiological and biomedical laboratories (BSL-2, 3 and 4). Feel free to contact me via LinkedIn messaging if you have any questions or comments about laboratory safety, biosafety, biocontainment, design & equipment, engineering, etc.

Autoclave validation in microbiological and biomedical laboratories at biosafety levels 2, 3, and 4 is a requirement—but there are no current guidelines or regulations on how to validate and monitor the steam decontamination process. The focus of this article is on the decontamination of liquid and solid biohazardous waste for safe disposal into the municipal waste stream (local regulations may apply).

The sterilisation of culture media and equipment in terms of meeting specific quality criteria (ISO 17025, ISO 15189) will not be discussed as this is not covered by biosafety regulations and guidelines and good microbiological practices and procedures [1]. However, validation of these processes can be achieved by applying the same basic principles as presented in this article.

1. Definitions

Validation: Systematic and documented confirmation that the specified requirements are adequate to ensure the intended outcome or results. For example, in order to prove a material is decontaminated, laboratory personnel must validate the robustness of the decontamination method by measurement of the remaining biological agents against the detection limit by chemical, physical or biological indicators [1], [2].

Process monitoring: Process monitoring with biological sterilization process indicators is used to check a decontamination process for adequacy of decontamination [3].

Decontamination: Reduction of viable biological agents or other hazardous materials on a surface or object(s) to a pre-defined level by chemical and/or physical means [1], [2].

Sterilisation: A process that kills and/or removes all biological agents including spores [1], [2].

2. Introduction

Steam sterilisers or autoclaves are used by many industries and laboratories to eliminate the threat of pathogenic micro-organisms to the health and safety of staff, the community, patients, consumers or the environment. Steam sterilisers are the preferred method because the processes are easy to validate and monitor. Some examples of applications include:

• The food industry uses autoclaves to sterilise canned foods and food ingredients;
• The pharmaceutical industry sterilises e.g. glass vials and containers;
• Hospitals and clinics sterilise items such as clothing, surgical equipment and implants, and autoclave medical waste before disposal;
• Microbiological and biomedical laboratories sterilise culture media, all types of laboratory glassware and instruments, which may be regulated for a specific laboratory by quality standards such as ISO 17025 & 15189. They also need to decontaminate biohazardous waste before disposal.

In most industries, steam sterilisation in autoclaves is considered the method of choice to ensure sterility or decontamination where practical.

Autoclaves are used for very different processes in terms of the nature of the application and, more importantly, the quality objective. The consequences of a failed sterilisation or decontamination process are also very different. Thus, each industry has developed its own standards for validating and monitoring whether the sterilisation or decontamination process is achieving its objectives. As a result, validation and process monitoring methods vary somewhat between industries and applications. In addition, in some industries it is important that the autoclave and steam are very clean; the goods must not be contaminated by impurities in the steam or from the autoclave steam pipes and chamber.

For waste decontamination in microbiological and biomedical laboratories, there are lessons to be learned from other industries, but their standards should not be adopted uncritically (e.g. from a standard like ANSI/AAMI ST46:2002 [4]).

3. Characteristics of Waste from Microbiological and Biomedical Laboratories

Unfortunately, there is no accepted universal standard or guidance on validation for the safe disposal of biohazardous waste from microbiological and biomedical laboratories. Until about twenty years ago, there was no requirement for a method to validate a waste decontamination process. What was available was borrowed from other industries. And this is not always appropriate for microbiological and biomedical laboratories. For example, in many industries the items to be sterilised already have a low level of biological contamination. In contrast, waste from microbiological and biomedical laboratories almost always has very high levels of biological agents per weight and volume. As the decontamination process is dynamic, these differences can be significant.

4. Sterility Assurance Levels (SAL)

The sterility assurance level (SAL) is used in most processes where product sterility must be assured [5]. The SAL indicates the probability of survival of a single cell after a sterilisation process. As inactivation of micro-organisms follows an exponential function, a SAL of 10^-6 is often used to achieve a sufficient probability that all micro-organisms have been killed. A SAL of 10^-6 means that the probability of a single cell surviving is less than one in a million. A SAL of 10^-6 is used for sterilisation processes where contamination would be a significant risk, such as medical implants or surgical instruments.

For laboratory waste, where the number of bacteria and viruses per load is generally very high—it can be as high as 10^9 bacteria per ml or more—the SAL concept has its limits. However, safe decontamination of biohazardous waste is not a problem as most viruses and vegetative bacteria have a very short survival time at 121°C, known as the "decimal reduction time" or "D-value" [6].

Temperature and time of exposure to saturated steam conditions are the necessary parameters to be observed. It is a widespread misconception that a temperature of 121°C at a pressure of 104.2 kPa for 15 to 20 minutes is sufficient. In fact, the thermal resistance of microorganisms is defined by the D-value, Z-value and F-value [6].

The validation procedure and process monitoring presented in this article ensure an adequate level of safety. A biological indicator organism with very high thermal resistance (Geobacillus stearothermophilus) is used for validation.

5. Types of Loads in Microbiological and Biomedical Laboratories

In microbiological and biomedical laboratories, most autoclave loads for waste decontamination fall into one of the following four categories

1. Used clothing made of cotton or heat-resistant plastic fabric (loose or bagged dry goods).
2. Biohazardous laboratory waste such as solid culture media (agar petri dishes, agar plates, slants, etc.) in bags or containers and liquid culture broths and cell culture media in flasks, tubes and bottles.
3. Disposables, sharps and other materials (dry goods; in boxes, sharp bins, or in bags).
4. Animal carcasses (in bags or containers).

The autoclave process requires saturated steam conditions for reliable and reproducible decontamination. Saturated steam conditions are achieved when autoclaving liquid waste in bottles, tubes and vials.

Goods in bags or boxes with tightly packed garments and other dry materials can be problematic in this respect, especially in displacement or gravity autoclaves [6], where the steam, which has a lower density than air, must displace the residual air through the autoclave outlet at the bottom.

For example, in upright bags, even if the tie is loose or the bag is open, air can be trapped at the bottom of the bag. One cup of water (approximately 0.25 litre) must be added to a 30 litre bag to achieve wet conditions. Water can be safely added to waste bags in a BSL-2 laboratory. For waste generated in a higher risk environment such as BSL-3, the addition of water may be associated with aerosol formation. One option is to add the water to the bag before adding the waste. Another option is to place the waste in a bag that will melt in the autoclave. This (tied) bag is then placed in an open, regular autoclave bag that doesn't melt. In this way the waste remains sealed until the steam melts the inner bag. Water is added to the outer bag.

In vacuum cycle autoclaves it is not necessary to add water, but it is important that steam and air can flow in and out of the bag. The bag can be placed in the autoclave in an upright position, but the tie should still allow air and steam to flow in and out of the bag. The vacuum cycles will remove up to one to five percent of the air, which is sufficient to decontaminate the waste. When tightly tied bags and airtight containers are used in vacuum cycle autoclaves, they may burst or implode during the vacuum/steam cycle.

6. Bowie-Dick-Test

In hospitals and other healthcare settings where the vacuum-assisted autoclave process must conform to stringent quality and sterility requirements, the Bowie-Dick test is used to check that there is no residual air in the centre of a load of wrapped sterile instruments [4], [6].

The Bowie-Dick test is not required in microbiological and biomedical laboratories. The validation method presented here is sufficient to ensure that decontamination requirements are met.

7. Autoclave Site Acceptance Test (SAT, OQ)

Autoclave qualification consists of a chamber test and calibration carried out by the manufacturer's technician or engineer after delivery and. This is known as site acceptance test (SAT) or operational qualification (OQ).

A set of calibrated and certified thermocouples is used to test and check that the time-temperature profile is achieved throughout the chamber and to recalibrate the autoclave's internal thermocouples if necessary. The chamber test and calibration must be repeated after each service, but at least once a year. No load is present unless the users have included process validation of standard loads in the contract.

The SAT or OQ confirms the correct basic operation of the autoclave before it is handed over to the end user.

The subsequent load validation (referred to as process validation, PQ) is usually carried out by the end user.

8. Biological Indicators and Thermocouples

A biological indicator (BI) is a device for monitoring the sterilisation or decontamination process, consisting of a standardised, viable population of microorganisms (usually bacterial spores, Geobacillus stearothermophilus). Biological indicators are used to test the process’s conformance with decontamination requirements [3], [4], [6]. The manufacturer's instructions for correct use most be followed. Rapid Readout Biological Indicators allow the efficiency of the decontamination process to be monitored within one hour. This is useful in situations where each load must be cleared prior to final disposal, or where a result is required urgently.

Chemical indicators (such as indicator tapes or tablets) are used to distinguish between processed and unprocessed items (external indicators). Caveat: Chemical indicators cannot be used to determine whether the process conforms to the decontamination requirements. Chemical indicators can help to quickly identify serious malfunction of an autoclave. If chemical indicators do not respond, the process has failed.

Thermocouples or temperature loggers are helpful in determining whether the set temperature is being reached deep within bags, boxes, containers or drums. If the BI test fails, temperature logging can help narrow down the reason for the failure. In most cases, a failed BI test is due to insulation in large bags or containers, the use of waste bags that are not permeable to steam, and/or failure to displace air in gravity-assisted autoclaves. For decontamination purposes, properly performed temperature logging can be an appropriate and sufficient means of monitoring a process.

Instead of special thermocouples and loggers, the use of internal thermocouples supplied with the autoclave (included in some models to prevent overheating of liquid goods) is most helpful. If these are not available, biological indicator validation is sufficient. Successful biological indicator (BI) validation is a sufficient criterion (the BI is considered the acid test).

9. Load Validation (PQ)

The purpose of load validation is to determine and validate the programmes and load types that the end user will use to decontaminate waste. The end user (laboratory management, biosafety officer, etc.) must be able to carry out this load validation themselves. It is part of the task to set up and adopt the biorisk management system, in particular the SOPs for waste treatment and autoclave operation.

Typically, there will be two to four types of load that need to be validated. At least one dry and one liquid load will need to be validated. For large and complex microbiological and biomedical laboratories, particularly animal biosafety facilities, there may be loads of bags of cages and bedding,? bags or even drums of faeces, necropsy waste, etc.). In this case, validation and process monitoring can be time consuming and costly.

Load validation is carried out using so-called standard loads, which are characterised by the maximum number, size and mass of items to be autoclaved in a single process. The standard load describes the maximum allowable load for a particular process for which safe decontamination must be ensured. In other words, it is based on a realistic 'worst case' scenario. For routine operations, fewer or smaller loads of the same type may be used, but not larger or more compressed loads.

In particular, if garments and other compressible goods are autoclaved in bags or boxes, the number of items and the degree of compression are relevant. For example, the number of garments per bag must be limited. For liquids, the volume, wall material and thickness, and shape of the containers are relevant. For carcasses, the number, size (mass) and geometric arrangement (stacked or side by side) in bags or cartons must be noted. Photographing the standard loads is useful for specifying the standard load in an SOP and for training purposes.

The load validation is done as follows:

1. Identify the type of loads you will have for routine decontamination (bags, containers, bottles, etc.).
2. Describe the load in terms of maximum number of items, composition, largest size of bag or container, how many of these will be placed on the autoclave tray and where exactly (load geometry). For bags, describe how much the ties need to be loosened. Take photographs and make notes.
3. Validate the load using biological indicator strips (solids) or ampoules (liquids) and, if available, thermocouples or temperature loggers (sensors).
4. Two to three BI strips and sensors should be placed in the middle and/or towards the bottom third of bags and containers. For particular goods, the appropriate location of BI and sensors for validation purposes must be determined experimentally.
5. For the validation of carcass decontamination, the ampoules and sensors shall be placed in the centre of the carcasses. Validation of steam carcass decontamination requires special care and experience as carcasses can vary considerably in shape and size. The largest possible carcass size should be used. If long tubes or hoses are to be decontaminated, they must be cut in half. The strip and sensor are inserted some distance from the cut and the two ends of the tube or hose are re-joined and sealed for the process.
6. A load and process are considered validated for decontamination after three successful runs (all BIs are negative, temperature profile meets requirements).
7. For each run, a strip or ampoule of BI must be used as a control (positive growth control).
8. Load validation is repeated at least monthly, whenever loads change, or after autoclave repair or suspected malfunction.

10. Process Monitoring

The autoclave is now ready for routine use. Each individual process must be monitored using at least the physical parameters of the autoclave (temperature, time, pressure) and chemical indicators.

Biological indicators must be used at least monthly to verify the adequacy of decontamination. For higher risk laboratories, such as BSL-2 enhanced, BSL-3, or BSL-4, or if for some reason the batches show a lot of variation, BI monitoring may be required more frequently or even on every batch. Methods and frequency of BI monitoring should be evaluated by a microbiological risk assessment. If monitoring fails, the cause must be investigated and the problem resolved before routine operations can be resumed. Affected batches must be staged and re-autoclaved until successful revalidation of the autoclave.

All records must be properly maintained in accordance with the risk management system and local regulations.

11. References

[1]????? WHO (2020). Laboratory biosafety manual, fourth edition. Geneva: World Health Organization; (Laboratory biosafety manual, fourth edition and associated monographs). Internet: https://www.who.int/publications/i/item/9789240011311. Accessed January 2024.

[2]????? ISO 35001 (2019). “Biorisk management for laboratories and other related organisations”. Internet: https://www.iso.org/standard/71293.html. Accessed January 2024.

[3]????? US FDA, Title 21, Volume 8. 21CFR880.2800. Internet: https://www.ecfr.gov/current/title-21/chapter-I/subchapter-H/part-880. Accessed January 2024.

[4]????? ANSI/AAMI ST46-2002. Steam Sterilization and Sterility Assurance in Healthcare Facilities. Internet: https://webstore.ansi.org/standards/aami/ansiaamist462002. Accessed January 2024.

[5]????? Wikipedia. Sterility Assurance Level. Internet: https://en.wikipedia.org/wiki/Sterility_assurance_level. Accessed January 2024.

[6]????? McDonell (2017). Antisepsis, Disinfection, and Sterilization: Types, Action, and Resistance, 2nd Edition. Internet: https://www.wiley.com/en-gb/Antisepsis,+Disinfection,+and+Sterilization:+Types,+Action,+and+Resistance,+2nd+Edition-p-9781555819675. Accessed January 2024.