North American
Sterilization & Packaging
Company
17 Park Drive, Franklin, New Jersey 07416 USA
Phone:1.973.209.4388
1.800.392.6310
Fax:1.973.209.6374
Email: info@naspco.com
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METHODS FOR DETERMINING CYCLE LETHALITYCalculating D-Values. The D-value
corresponding to the survivor curve construction results is determined by either
reading it from a graph or calculating from the data the relevant time interval
for reducing the count by one log (Table I). The
performance of the fraction-negative method will generate data that are then
utilized to calculate a D-value using a method described by Pflug and Holcomb.11
This method is also referred to as the full or generic Spearman-Karber
procedure. It does not require the use of the same number of replicates nor the
same gas exposure time intervals because it involves spore strips rather than
counting CFUs.
Table I. Variables used in calculating D-values during MPQ. In Table I, t1 represents the shortest exposure time to sterilant, and all test samples run through this short time frame should show growth. The variables t2 to t5 correspond to increasing exposure times in the fraction-negative area, otherwise known as the quantal region. Exposure times t6 and t7 should represent tests in which none of the tests show growth. In the following equations, r1 is the number of test samples out of the number exposed (ni) that show no growth at exposure time t1. For each period of exposure to sterilant t1 to t6, the factors x and y are calculated as shown:
At ti, all test samples show growth, therefore,
From the calculated values of xi and yi above, the value µI (mean time to attain no growth) can be calculated for each period of exposure (t1 to t6) as follows:
The mean time to attain no growth from all of the test samples, µ, can be calculated from the sum of µI for each time of exposure t1 to t6:
Where the interval between exposure times (d) is constant and the same number of test samples (n) is used at each exposure time, the mean to attain no growth (µ) can be calculated from the equation:
The mean D-value (D) can be calculated from the equation:
where N0 = the initial number of challenge organisms per test sample. For the purposes of calculating the sterilization period using the Dcalc method below, the upper 95% confidence level for D should be used. This can be calculated from the equation:
where V is derived as follows:
In addition to the method described above, there are three other commonly used means of calculating D-values. The limited Spearman-Karber procedure requires the same number of replicates for each exposure time and equal intervals between the various gas exposure times. For example, the exposure times of 9, 12, 15, and 18 minutes provide an arithmetic series of 3-minute time intervals. The Stumbo-Murphy-Cochran procedure requires one result in the fraction-negative range, consisting of time (t), the number of units negative for growth (r) and the number of replicates (n) at each exposure time as well as the initial number of microorganisms per replicate (N0). The D-value for each exposure time is calculated from the following equation. The confidence limits of the mean may be calculated by conventional procedures using natural logs.
The factors in the equation represent the following: U = exposure interval. N0 = initial number of organisms per replicate carrier unit. NU = ln (n/r). n = total number of replicate units at exposure U. r = number of units negative for growth at U. The D-values are averaged to obtain the overall D-value for the experiment. The limited Stumbo-Murphy-Cochran procedure requires only one exposure time in the fraction-negative range. It is important to choose the most appropriate procedure of the three described to calculate the D-value and its upper confidence limit. Certain methods have limitations. For example, Graham and Boris have noted that a confidence interval cannot always be determined from the Stumbo-Murphy-Cochran procedure.12 Shintani et al. have observed that the Stumbo-Murphy-Cochran procedure can yield invalid results if r is close to n.13 They also state, however, that this procedure may be superior in situations in which the sample population is at least 50, r is at least 1, and r/n is less than 0.9. Half-Cycle Method. The half-cycle method determines the minimum time a specific product load must be exposed to an EtO process to guarantee that no survivors exist from the BIs used to monitor the cycle's efficacy. The cycle chosen is based not only on the elements discussed in the companion to this article but also on the microbiological data obtained prior to determining the parameters for the half-cycle.1 The minimum ranges of the half-cycle are based on this predetermined microbiological data, whereas the maximum ranges are dictated by the product and packaging tolerances established through functional testing. If the half-cycle method is chosen, an additional cycle should be performed, preferably before the first half-cycle is conducted. In some cases, other proportionate cycles, referred to as sublethal or fractional cycles, may be used. The gas exposure time is shortened in order to obtain survivors from the BIs used to monitor the cycles. The process parameters in a sublethal cycle are the same as those in the half-cycle with the exception of the gas exposure time period. Such cycles are conducted to demonstrate that positive BIs can be recovered and to prove the adequacy of the BI, the sterility test method, and associated equipment and materials. The sublethal cycle is not required if either the survivor curve construction or fraction-negative method is used because these methods require positive sterility test results as part of the study. A second objective in conducting a sublethal cycle is to attempt to demonstrate that the resistance of the product's bioburden is less than that of the BI. This is accomplished by using actual product test samples that have been manufactured, packaged, and handled in the same manner intended for routine production. It is imperative that these test samples were made in accordance with routine manufacturing procedures because the bioburden on the product must represent what the product would normally contain. Sometimes using a number of different product samples can yield useful information when there is concern about not only the quantity of bioburden but also where it is located. If a number of different samples have been sterility tested, those with the highest bioburden should be used for validation. After the completion of the sublethal cycle, the product samples are sterility tested. Since the objective of the test is to demonstrate sterility of the bioburden, the data can be compared to the positive sterility test results obtained from the BIs to demonstrate that the resistance of the product's bioburden is less than that of the BI. A third objective of the sublethal cycle study is to determine that a PCD used as an external monitoring device for routine sterilization cycles yields more positive sterility tests than an internal BI does. It has been suggested that it is acceptable to obtain positive sterility test results from those external PCDs exposed to the half-cycles if previous comparative resistance studies have proven that they are as resistant or more resistant than the internal product BI and if no positive sterility test results are obtained from the BIs placed in internal load locations. If total kill is obtained from all PCDs on all half-cycles, it can be assumed that the resulting validated full cycle will provide an additional safety factor that goes beyond the minimum requirements. Typically, the sterilization efficacy of the first half-cycle is determined before any subsequent cycles are conducted. If the first half-cycle experiment elicits all negative BIs, two additional half-cycles are conducted to confirm the data. All three half-cycles must employ the same parameters to demonstrate reproducibility and reliability. All three half-cycles must demonstrate the ability to elicit all negative BIs. If a SAL of 10–6 is required, a gas exposure time that is at least double that used in the half-cycle becomes the minimum gas exposure time used routinely in the full cycle. THE FULL CYCLEThe data obtained from the survivor curve construction method, the fraction-negative method, or the half-cycle method are used to design the full cycle that will be used to routinely sterilize the product. The full cycle must be capable of reliably demonstrating a required SAL that consists of the minimum time to obtain all negative BIs or CFUs with an additional margin of safety. Normally this can be expressed as 10–n where n is the cumulative probabilities of the log minimum time to sterilize and the log margin of safety. For example, if the half-cycle method was chosen to validate a given sterilization cycle with a required SAL of 10–6, the half-cycle must demonstrate the ability to ensure that there are no survivors from BIs that have a certified population of 106. Since the BIs have a greater resistance than the product bioburden, it can be concluded that the time is sufficient to achieve product sterility. However, adding an equivalent sterilization time period increases the margin of safety. The additional 6 log reduction in the population (10–6) is theoretical and is obtained by doubling the half-cycle gas exposure time. In other words, if the half-cycle gas exposure time period was 2 hours, the full-cycle gas exposure time would be a minimum of 4 hours. The processing ranges established for the critical parameters in the full cycle should be based on the ranges determined in the half-cycle. The minimum process parameters in the full cycle, such as temperature, sterilant concentration, pressure, and humidity, may also be greater than those used in the half-cycle to ensure greater lethality with the full cycle. The first full cycle should be included in the validation to complete the profile of the product load. The same type and amount of monitoring should be conducted during the first full cycle as was conducted on each of the half-cycles. Comparisons of temperature distribution throughout the chamber and the load can justify a determination of reliability and repeatability for the process. These data can also be useful for determining the significance of any minor changes that are made to the load. REPORT ON VALIDATION AND CERTIFICATIONThe documentation on the validation and certification should include but not be limited to the following:
ANNUAL REVALIDATIONAn annual revalidation of the process should be conducted to verify the integrity of the original validation data. It may be advisable to conduct this revalidation within 13 months of the initial validation and each year thereafter until sufficient data are obtained to allow that time period to be extended. Afterwards, revalidation should be conducted at least every 2 years. Data from each revalidation should be compared with that from the original validation and any subsequent revalidation to confirm that the original performance specification remains valid. A revalidation consists of a thorough review of all the factors that could affect the sterilization process, including changes to the product or its packaging, the manufacturing methods and facility, and component and materials suppliers. Product bioburden test data should be analyzed and trends noted to ensure that there have been no substantial unacceptable changes in quantity or characterization. The testing conducted of the preconditioning room, the sterilization chamber and ancillary equipment, and the aeration room and equipment should be compared with the data obtained during the original validation as well as to the commissioning data. All associated programs, such as preventive maintenance and calibration, should be reviewed for effectiveness. CONCLUSIONA well-designed and scientifically sound MPQ helps ensure that the process is safe, efficacious, and efficient. Performed well, the MPQ should deliver the required SAL for the product in an economical process and should enable the process to continue without having to be repeated. It should also prevent product reprocessing and delays in product release to the marketplace. A knowledgeable approach that considers all of the various aspects of the MPQ will ensure the success of the process. ACKNOWLEDGMENTThe authors would like to express their gratitude to Aubrey S. Outshoorn, PhD, of the United States Pharmacopeial Convention Inc. for his contributions to this article. REFERENCES1. PJ Sordellini and SE Satter, "EtO Sterilization: Principles of Process Design," Medical Device & Diagnostic Industry 20, no. 12 (1998): 47–59. 2. "Sterilization of Medical Devices—Microbiological Methods—Part 1: Estimation of Population of Microorganisms on Products," ANSI/AAMI/ISO Standard 11737-1 (Arlington, VA: Association for the Advancement of Medical Instrumentation, 1995). 3. "Guidance on the Application of EN 29001 and EN 46001 and of EN 29002 and EN 46002 for Non-Active Medical Devices," BS/EN 724 (London: British Standards Institution, 1995). 4. "Medical Devices—Validation and Routine Control of Ethylene Oxide Sterilization," ANSI/AAMI/ISO Standard 11135 (Arlington, VA: Association for the Advancement of Medical Instrumentation, 1994). 5. EW Nester et al., Microbiology, 2nd ed. (Chicago: Holt, Rinehart, and Winston, 1978). 6. The Center for Devices and Radiological Health, FDA Guide for Validation of Biological Indicator Incubation Time (Rockville, MD, FDA, 1985). 7. United States Pharmacopeia 23, 8th supp., monograph <71>, 1998. 8. "Sterilization of Health Care Products—Biological Indicators—Part 1: General Requirements," ANSI/AAMI/ISO Standard 11138-1 (Arlington, VA: Association for the Advancement of Medical Instrumentation, 1994). 9. "Sterilization of Health Care Products—Biological Indicators—Part 2: Biological Indicators for Ethylene Oxide Sterilization," ANSI/AAMI/ ISO Standard 11138-2 (Arlington, VA: Association for the Advancement of Medical Instrumentation, 1994). 10. "Contract Sterilization for Ethylene Oxide," AAMI TIR no. 14-1997 (Arlington, VA: Association for the Advancement of Medical Instrumentation, 1997). 11. IJ Pflug and RG Holcomb, "Principles of Thermal Destruction of Micro-Organisms," in Disinfection, Sterilization and Preservation, ed. SS Block, 3rd ed. (Philadelphia: Lea and Febiger, 1983). 12. GS Graham and CA Boris, "Chemical and Biological Indicators," in Sterilization Technology: A Practical Guide for Manufacturers and Users of Health Care Products (New York: Van Nostrand Reinhold, 1993). 13. H Shintani et al., "Comparison of D10-Value Accuracy by the Limited Spearman-Karber Procedure (LSKP), the Stumbo-Murphy-Cochran Procedure (SMCP), and the Survival-Curve Method (EN)," Biomedical Instrumentation Technology 29, no. 2 (1995): 113–124. Susan Edel Satter of Satter & Associates Inc. (Boulder, CO) and Paul J. Sordellini of Quality Solutions Inc. (Annandale, NJ) are consultants for the medical device manufacturing industry. Illustration by Brad Hamann |
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