Reduced Incubation Time (RIT) for Biological Indicators

Introduction

Biological indicators (BIs) are widely recognized as the gold standard for monitoring the effectiveness of sterilization processes. Following exposure, BIs are incubated to allow any surviving microorganisms to recover and grow. Traditionally, international standards have defined seven days as the reference incubation period for established sterilization processes such as moist heat (steam) and ethylene oxide (EO). However, advances in spore production, culture media, and detection systems have enabled reliable detection of growth in a shorter time. This has led to the concept of Reduced Incubation Time (RIT).

While reduced incubation provides clear operational advantages, it cannot be assumed. Under specific conditions, formal validation of RIT is required to ensure that shortened incubation does not compromise sterility assurance.

What Is Reduced Incubation Time (RIT)?

Reduced incubation time refers to an incubation period shorter than the seven-day reference incubation time defined in ISO 11138-1:2017 for biological indicators, while still providing equivalent sensitivity for the detection of potentially injured spores that still survived after sterilization. Any incubation time claim below this reference value must be scientifically justified and validated using an accepted methodology.‑. Any incubation time claim below this reference value must be scientifically justified and validated using an accepted methodology.

Internationally, the primary standard governing RIT validation is ISO 11138-8:2021, which specifies a statistically and microbiologically sound method for establishing or confirming reduced incubation times for biological indicators used in moist heat and ethylene oxide sterilization processes.

Conditions Under Which RIT Validation Is Required

Reduced incubation time validation is required under the following conditions:

1.    When Using RIT for Moist Heat or Ethylene Oxide Sterilization

ISO 11138-8: 2021 is applicable specifically to biological indicators used for moist heat (steam) sterilization, and ethylene oxide (EO) sterilization.

If RIT is applied to these processes, validation must follow an accepted method. The standard does not currently apply to dry heat, Low Temperature Steam and Formaldehyde (LTSF), or vaporized hydrogen peroxide processes due to limited supporting data.

2.    When the Incubation Time is Shorter Than the 7-Day Reference Period

If a biological indicator is intended to be incubated for less than seven days, validation is mandatory. The seven-day period is the recognized reference incubation time in ISO 11138-1:2017, and any deviation must be justified through formal RIT validation.

3.     When a Manufacturer Claims a Reduced Incubation Time on the BI Label or IFU

RIT validation is required whenever a BI manufacturer claims or labels an incubation time shorter than seven days in the instructions for use (IFU). The validation demonstrates that the reduced time is sufficient to detect low numbers of surviving and sub lethally injured spores.

4.    When Introducing a New BI Design, Media, or Detection System

Any change in BI design such as:

• self-contained versus strip indicators,

• modified growth media,

• use of pH-based or enzymatic early detection systems, or

• changes in spore purification or sporulation methods,

may affect spore recovery and growth kinetics. In such cases, RIT validation is required to confirm that the reduced incubation time remains appropriate.

Example of pH based Biological Indicator. Image is taken from https://terragene.com/product/biological-indicator-bt10/

5.    When Required by the Quality Management System (QMS)

ISO 11138-8:2021 explicitly applies not only to BI manufacturers but also to end users who, based on their quality system or risk management requirements, choose to establish, validate, or confirm a reduced incubation time. This is particularly relevant in pharmaceutical and medical device manufacturing environments operating under GMP.

6.    When Supporting Parametric Release or Early Product Release Decisions

When BI results are used to support early release of sterilized products, especially in routine production, RIT validation becomes critical. The validation provides assurance that a negative BI result obtained at the reduced time point is equivalent to a seven-day result and does not introduce additional sterility risk.

When RIT Validation Is Not Required

RIT validation is not required when:

• the biological indicator is incubated for the full seven-day reference period, or

• the BI is used strictly according to a manufacturer’s IFU that already includes a validated RIT claim.

In these cases, the responsibility for RIT validation remains with the BI manufacturer.

Conclusion

Reduced incubation time offers significant operational and business advantages; however, it introduces potential risk if not properly justified. RIT validation is required whenever incubation times are reduced below the seven-day reference period, when reduced times are claimed or relied upon for release decisions, or when changes could affect spore recovery and growth.

Compliance with ISO 11138-8:2021 or recognized FDA guidance ensures that reduced incubation times maintain the same level of sterility assurance as traditional methods. Ultimately, RIT validation is not optional, it is a critical scientific and regulatory safeguard whenever shortened incubation is used.

Reference

International Organization for Standardization. (2021). ISO 11138-8:2021: Sterilization of health care products — Biological indicators — Part 8: Method for validation of a reduced incubation time for a biological indicator.

International Organization for Standardization. (2017). ISO 11138-1:2017: Sterilization of health care products — Biological indicators — Part 1: General requirements.