The historical perspective
Prior to the mid 1980s, BIs were incubated for 7 days to obtain final results. However, it was commonly observed that BIs,
particularly those used in moist-heat sterilization processes, rarely demonstrated positive results after the third day of
incubation (5). Several studies confirmed these observations and industry began to petition FDA to allow the use of shorter
incubation times for BIs (6, 7). In 1985, FDA adopted an interim position allowing for an incubation time of 5 days as the
final BI readout time (8).
On Jan. 1, 1986, the agency's Center for Devices and Radiological Health (CDRH) published the FDA Guide for Validation of
Biological Indicator Incubation Time (9). This document was initially intended for manufacturers of BIs as a guide for documenting
incubation times of less than 7 days for their products. The method consists of a statistically based sampling plan requiring
a minimum of 100 units from each of 3 different product lots, which implied three different spore crops. The sample sets are
then exposed to a sublethal cycle designed to produce 30–80% positives after 7 days incubation. Using the number of positive
BIs after 7 days incubation as the reference (or denominator in the calculation), the greatest number of days of incubation
required to obtain greater than 97% of the positive BIs in any of the partial cycles (numerator data) is the minimum incubation
time allowed per this method.
The CDRH guide may have initially been intended for BI manufacturers, but it soon became the default requirement for medical-device
manufacturers relative to less than 7-day BI release of their sterilized medical products. Around the same time as the FDA
guide was issued, the Association for the Advancement of Medical Instrumentation (AAMI) published its first BI standards through
the American National Standards Institute (ANSI). ANSI/AAMI ST19: 1986 and ANSI/AAMI ST21: 1986 characterized BIs for use
in ethylene oxide and moist heat sterilization processes respectively in health care facilities (10, 11). However, the only
guidance regarding BI incubation time was to follow the BI manufacturers' instructions. A recommendation of 7 days incubation
for BIs used in moist heat, ethylene oxide, and dry-heat sterilization processes first appeared in the United States Pharmacopoeia (USP) in 1990 and has remained unchanged per the recent 2011 USP edition (12, 13). Similar to the ISO and AAMI BI documents, the
USP contains no information on test methodology for BI incubation time determination.
The initial version of ISO 11138-1, general requirements for BIs used in the sterilization of medical products, was issued
in October 1994 (14). The European Committee for Standardization (CEN) published a similar document, EN 866-1, in 1997 (15).
Both documents included requirements for general production, testing, labeling, and performance requirements for BIs used
in healthcare sterilization processes, however, neither document contained information on BI incubation time other than to
follow "methods and conditions prescribed by the (BI) manufacturer." A nearly identical version of ISO 11138-1, ANSI/AAMI
ST59, was published in 1999; it also provided no direction regarding validation of BI incubation time (16).
During the mid-1990s, ISO initiated work to develop a BI user guidance document designated as ISO 14161. In the interest of
harmonization, AAMI elected to parallel ballot this document, meaning that it would be adopted verbatim as an ANSI/AAMI/ISO
standard following publication by ISO; the new document would also replace the ANSI/AAMI ST34:1991 standard for BI user guidance
(17). There was consensus agreement by the ISO/Technical Committee (TC) 198 BI Working Group 4, and the AAMI BI Working Group
4 that methodology for determination of BI incubation time should be addressed in the 14161 document. Accordingly, early versions
of ISO 14161 included an informative annex (Annex E) containing the 1986 CDRH Guide for validation of BI incubation time because
it was the only method available for this determination.
Subsequent ISO and AAMI BI working group discussions regarding the CDRH guide raised significant issues regarding the utility
of this methodology. Input from BI working group members indicated that it was difficult to obtain reproducible results with
the CDRH method, and that it was also cumbersome to consistently obtain between 30 and 80% BI positives after 7 days incubation.
As a result of continuing discussions, an alternative method for validation of BI incubation time was introduced to the ISO
and AAMI BI working groups. The newer methodology was based on a commercial BI manufacturer's protocol and represented a statistically
consistent but less conservative approach compared with the CDRH method for determining BI incubation times. In contrast to
the CDRH method requirement of > 97% correlation between the 7 day incubation results and the RIT, the alternative method
required a correlation of 95% but also used a larger sample size of 200 units per lot. Comments submitted to the ISO/TC 198
WG4 and AAMI WG4 on the alternative method were that it favored BI producers and that it was unproven for widespread use in
As a result of these discussions, a request was made at the June 1997 AAMI BI WG meeting that a statistical analysis be performed
comparing the operating characteristics of the two methods in question. The CDRH method was determined to be restrictive in
terms of the probability for acceptance of shortened incubation times having a Rejectable Quality Level (RQL; protects consumer)
of 0.036 at = β 0.1 and an Acceptable Quality Level (AQL; protects producer) of 0.005 at α = 0.1. The alternative method
was shown to have a significantly higher RQL and AQL and provide a higher probability for acceptance of reduced incubation
time data. Based on a statistical simulation, the CDRH guide was shown to have a probability of acceptance of 30 % when using
a theoretical "true growth readout" of > 97%, whereas the alternative method would accept this same theoretical input virtually
100% of the time (18).
Ultimately, neither method was considered to be satisfactory by the ISO or AAMI BI working groups. A joint agreement was reached
that Annex E should be removed from the 14161 draft so that the standard could move forward for publication. These decisions,
however, also included agreement that the subject was important relative to BI use and, therefore, a New Work Item Proposal
(NWIP) would be initiated to develop an ISO Technical Specification (TS) that would contain methodology for validation of
BI incubation time. This document, designated as ISO TS 16342, was developed in early 2000 and presented to the ISO BI WG
later that year (19). The ISO TS 16342 draft document contained the CDRH method and the proposed industry alternate method;
these two methods were intended to be used as a starting point for development of a new methodology. It was anticipated that
under the auspices of the ISO and AAMI BI working groups, a hybrid method acceptable to both regulators and users could be
developed based on the two statistically diverse approaches. After years of discussion, but at a low priority due to revision
of the ISO 11138 series standards, there was little movement and TS 16342 was subsequently taken off of ISO's agenda due to
the lack of progress.
Although the issue of BI incubation time was still considered relevant, there was little formal activity on the subject within
the working groups until 2005, when work began to revise ISO 14161:2000. The goal of revision was to align the standard with
the newly revised ISO 11138 BI standard series. Comments submitted on the ISO 14161 revision included requests from five different
member countries that were related to development of methodology for validating BI incubation time. At the 2007 general meeting
of ISO/TC 198 working groups in Dublin, Ireland, the BI working group agreed that the dormant NWIP to address this issue (i.e.,
TS 16342) should be re-instated as an active project. The working group also requested that any proposed methodology be supported
by actual data and published in a peer-reviewed scientific journal to support technical credibility. Due to the relatively
long time periods between ISO/TC198 general meetings, the commitment was made by AAMI members of the ISO BI Working Group
4 to take the lead on this project. Following discussions at the subsequent AAMI Working Group 4 BI meetings, an ad hoc committee was created in 2008 to identify a plan for gathering data and develop a new test protocol for RIT determination.