Application of the RIT protocol

Both users and manufacturers of BIs have voiced concerns about several aspects of this approach to qualifying a reduction of incubation time for BIs:

• The 30 to 80 "window" for the number of nonsterile BIs is too narrow; identifying sterilization conditions that yield such results is difficult. Often, numerous sterilization runs must be performed on a given lot to provide results that fall within this window.
• The 97.0% criterion is arbitrary and does not take into consideration the distribution of grow-out times observed for a given BI lot and the sterilization conditions used in the protocol-specified testing.
• Industry users of BIs have repeatedly challenged the necessity for protracted incubation times. Longer incubation times for BIs do not, de facto, make medical products more safe and clearly result in higher costs for users of BIs.

This RIT protocol has not been widely accepted outside the United States. The International Standards Organization (ISO) BI working group of TC198 (WG04) has been directed to develop a new RIT protocol that will be acceptable to the global community (9).

The wording of the current RIT protocol suggests that it only applies to BI manufacturers unless the user desires to apply the product in a manner other than that stated by the manufacturer. This protocol has been applied to users by some regulatory auditors and notified bodies. In such cases, users are required to expose BIs in specific product loads in production scale sterilizers. Successful execution of the RIT protocol in a production sterilizer is extremely difficult since the outcome of the sterilization exposure must result in at least 30 and no more than 80 BIs having surviving spores; i.e. nonsterile. It is important that the exposure of BIs for an RIT study be performed in a manner that ensures that they are statistical replicates. In a small research vessel, each of the 100 BIs is exposed to nearly identical sterilization conditions; in a production-scale sterilizer, there can be significant variability in exposure conditions. The less uniform conditions in the production-scale sterilizer, particularly when performing a reduced lethality exposure, can result in larger variability in the number of surviving colony-forming units (CFU) among the nonsterile BIs.

The application of the RIT protocol in this manner suggests that the sterilizer and the associated load of product in some way influence the grow-out time of spores exposed to a fractional sterilization process. There are no published data to support the notion that the molecular-level action of the sterilizing agent on the spore is influenced by the size of the sterilizer, the presence of product, or process variations that affect the rate of spore inactivation.

We should emphasize that inactivation of microorganisms by sterilizing agents generally follows first-order kinetics (10,11). In the case of BIs, spore death is the result of the destruction of a critical molecule or chemical reaction such that the spore can not develop into a replicating vegetative form (12–14). The longer a spore is exposed to a sterilization process without a lethal event, the more collateral (nonlethal) damage can occur. Collateral damage could affect the time required to complete germination and/or the time required for the vegetative cells to divide (generation time). Such germination- and generation-time effects could influence the time required to have a visible/chemical indication that a given BI had one or more surviving spores after exposure to a sterilizing agent.

Definitions

D-value. Time or dose required to achieve inactivation of 90% of a population of the test microorganism under stated dose conditions (15).

Biological indicator (BI). A test system containing viable microorganisms providing a defined resistance to a specified sterilization process (15).

Most probable number (MPN). The estimated population of maximum likelihood responsible for producing the observed combination of positive and negative outcomes (16).

Sterility assurance level (SAL). the probability of a single viable microorganism occurring on an item after sterilization. Note that the term SAL takes a quantitative value, generally 10^-6 or 10^-3. When applying this quantitative value to assurance of sterility, a SAL of 10^-6 has a lower value but provides a greater assurance of sterility than a SAL of 10^-3 (15, 17).