Challenges in validating the sterilisation dose for processed human amniotic membranes

Most of the tissue banks in the Asia Pacific region have been using ionising radiation at 25 kGy to sterilise human tissues for save clinical usage. Under tissue banking quality system, any dose employed for sterilisation has to be validated and the validation exercise has to be a part of quality document. Tissue grafts, unlike medical items, are not produced in large number per each processing batch and tissues relatively have a different microbial population. A Code of Practice established by the International Atomic Energy Agency (IAEA) in 2004 offers several validation methods using smaller number of samples compared to ISO 11137 (1995), which is meant for medical products. The methods emphasise on bioburden determination, followed by sterility test on samples after they were exposed to verification dose for attaining of sterility assurance level (SAL) of 10⁻¹. This paper describes our experience in using the IAEA Code of Practice in conducting the validation exercise for substantiating 25 kGy as sterilisation dose for both air-dried amnion and those preserved in 99% glycerol.     

ISO radiation sterilization standards

This presentation provides an overview of the current status of the ISO radiation sterilization standards. The ISO standards are voluntary standards which detail both the validation and routine control of the sterilization process. ISO 11137 was approved in 1994 and published in 1995. When reviewing the standard you will note that less than 20% of the standard is devoted to requirements and the remainder is guidance on how to comply with the requirements.

Future standards developments in radiation sterilization are being focused on providing additional guidance. The guidance that is currently provided in informative annexes of ISO 11137 includes: device/packaging materials, dose setting methods, and dosimeters and dose measurement, currently, there are four Technical Reports being developed to provide additional guidance:

1. 1. AAMI Draft TIR, “Radiation Sterilization Material Qualification”

2. 2. ISO TR 13409-1996, “Sterilization of health care products — Radiation sterilization — Substantiation of 25 kGy as a sterilization dose for small or infrequent production batches”

3. 3. ISO Draft TR, “Sterilization of health care products — Radiation sterilization Selection of a sterilization dose for a single production batch” li]4. ISO Draft TR, “Sterilization of health care products — Radiation sterilization-Product Families, Plans for Sampling and Frequency of Dose Audits.”

     

New applications of the VDmax approach to substantiation of preselected sterilization doses

The VD_max approach for substantiation of 25 kGy has been in use for more than 10 years. VD_max methods are included in ISO 11137-2:2006 and AAMI TIR33:2005. ISO Technical Specification, 13004, is under development that will include sterilization doses from 15 to 35 kGy.For substantiation of a sterilization dose for very low bioburden products, less than or equal to 0.3, values of VD_max have now been derived and tabulated for a sterilization dose of 12.5 kGy.Products have been encountered that have both low bioburden and a relatively low maximum dose. In several situations, existing tabulated VD_max values could not be effectively used; in one such situation, the average bioburden was too high to substantiate a 15 kGy sterilization dose and the use of a 17.5 kGy sterilization dose was not practicable due to the likelihood of exceeding the product's maximum acceptable dose. For this product, values of VD_max were derived and tabulated for substantiation of a 16.1 kGy sterilization dose.Values of VD_max have been derived and tabulated for the substantiation of sterilization doses linked to a sterility assurance level (SAL) of 10^?3. To offer a potential alternative to aseptic processing, the notion of using an “aseptic processing equivalent dose”, 10^?4 SAL, has been investigated along with the use of alternate model populations for calculation of VD_max values.     

Comments on the Japanese study of the radiation resistance of the bioburden of medical devices

The findings obtained in an extensive study of the radiation resistance of microbes forming the bioburden of a number of medical devices has been carried out by Takehisa et al. and reported in this number of this Journal. The following paper reviews the expected behaviour of the reported populations under the conditions specified in ISO Standard 11137 Method 1, which is widely used in the determination of the radiation sterilization dose required for sterilization of medical devices. The populations reported for “dry” devices contain a higher proportion of more radiation resistant microbes than that found in the standard distribution of resistance (SDR) that is used for setting the sterilization dose in Method 1. A possible alternative dose setting method for more resistant microbial populations is introduced and discussed.     

Field evaluations of the VDmax approach for substantiation of a 25 kGy sterilization dose and its application to other preselected doses

The International and European standards for radiation sterilization require evidence of the effectiveness of a minimum sterilization dose of 25 kGy but do not provide detailed guidance on how this evidence can be generated. An approach, designated VD_max, has recently been described and computer evaluated to provide safe and unambiguous substantiation of a 25 kGy sterilization dose. The approach has been further developed into a practical method, which has been subjected to field evaluations at three manufacturing facilities which produce different types of medical devices. The three facilities each used a different overall evaluation strategy: Facility A used VD_max for quarterly dose audits; Facility B compared VD_max and Method 1 in side-by-side parallel experiments; and Facility C, a new facility at start-up, used VD_max for initial substantiation of 25 kGy and subsequent quarterly dose audits. A common element at all three facilities was the use of 10 product units for irradiation in the verification dose experiment.

The field evaluations of the VD_max method were successful at all three facilities; they included many different types of medical devices/product families with a wide range of average bioburden and sample item portion values used in the verification dose experiments. Overall, around 500 verification dose experiments were performed and no failures were observed. In the side-by-side parallel experiments, the outcomes of the VD_max experiments were consistent with the outcomes observed with Method 1.

The VD_max approach has been extended to sterilization doses >25 and <25 kGy; verification doses have been derived for sterilization doses of 15, 20, 30, and 35 kGy. Widespread application of the VD_max method for doses other than 25 kGy must await controlled field evaluations and the development of appropriate specifications/standards.     

Dose mapping for documentation of radiation sterilization

The radiation sterilization standards EN 552 and ISO 11137 require that dose mapping in real or simulated product be carried in connection with the process qualification. This paper reviews the recommendations given in the standards and discusses the difficulties and limitations of practical dose mapping. The paper further gives recommendations for effective dose mapping including traceable dosimetry, documented procedures for placement of dosimeters, and evaluation of measurement uncertainties.     

Feasibility of in-house sterilization of laboratory plastic consumables using 2 MeV electron beam facility

In-house sterilization by electron beam (EB) radiation of various plastic consumables (plastic petridishes, micro centrifugal tubes and screw-capped vials) used routinely in the lab was studied by use of three microbiological cultures (S. aureus, Bacillus subtilis and Candida albicans). Current international standards (ISO 11137-Part 2 -ISO, 2011) recommend an irradiation dose of 25 kGy as a reference dose for terminal sterilization. All containers were exposed in an ILU-6, 2 MeV, 20 kW, Pulse EB accelerator located in our complex. Sterility test (S.T.) was performed and results revealed that 10⁶ and 10⁷ population of all strains passed whereas 10⁸ population failed S.T. for all micro-organisms indicating the potential of 2 MeV EB for commercial sterilization of plastic lab consumables for up to 10⁷ population of these micro-organisms.     

Characterization of a new photo-fluorescent film dosimeter for high-radiation dose applications

Characterization studies on one of the first versions of the Sunna fluorescent dosimeter™ have been published by Kovács and McLaughlin. This present study describes testing results of a newer version of the dosimeter (Model γ, batch 0399-20). This dosimeter is a 1-cm×3-cm polymeric film of 0.5 mm thickness that emits a green fluorescence component at intensities almost linear with dose. The manufacturing method (injection molding) allows potential batch sizes on the order of a million while maintaining a signal precision on the order of ±1%. Studies include dose response, dose rate dependence, energy dependence, post-irradiation stability, environmental effects, and variation of response within a batch. Data for both food irradiation and sterilization dose levels were obtained. The results indicate that the green signal (0.3–250 kGy) works well for food irradiation dose levels, especially in refrigerated facilities that maintain tight temperature control. The green signal also works well in sterilization facilities because its irradiation temperature coefficient above room temperature is minimal at sterilization doses. If the user requires readout results in < 22 h after room temperature irradiation, the user can either calibrate for a specific post-irradiation readout time(s) or simply heat the dosimeters in a small laboratory oven to quickly stabilize the signal.     

Computer and field evaluations in support of the VDmax approach for selected sterilization doses greater than 25 kGy

Originally, the VD_max approach was developed to substantiate a selected sterilization dose of 25 kGy. Thereafter, computer and field evaluations demonstrated the value of the approach for substantiation of selected sterilization doses less than 25 kGy. Verification of the use of the approach for substantiation of sterilization doses in excess of 25 kGy is now needed.The results of the computer evaluations conducted on the VD_max approach with 35 challenge microbial populations at sterilization doses of 30 and 35 kGy generally gave outcomes consistent with those observed previously, namely, safe and unambiguous. Outcomes perceived as unsafe have been shown to be a peculiarity of the manner of assembling challenge microbial populations. Field evaluations involving substantiation of four selected sterilization doses greater than 25 kGy and associated sterilization dose auditing gave acceptable outcomes. The present findings further affirm the value and reliability of the VD_max approach.     

Sterilization validation for medical devices at IRASM microbiological laboratory—Practical approaches

EN ISO 11137 established regulations for setting or substantiating the dose for achieving the desired sterility assurance level.The validation studies can be designed in particular for different types of products. Each product needs distinct protocols for bioburden determination and sterility testing.The Microbiological Laboratory from Irradiation Processing Center (IRASM) deals with different types of products, mainly for the VD_max²⁵ method. When it comes to microbiological evaluation the most challenging was cotton gauze. A special situation for establishing the sterilization validation method appears in cases of cotton packed in large quantities. The VD_max²⁵ method cannot be applied for items with average bioburden more than 1000 CFU/pack, irrespective of the weight of the package. This is a method limitation and implies increased costs for the manufacturer when choosing other methods.For microbiological tests, culture condition should be selected in both cases of the bioburden and sterility testing. Details about choosing criteria are given.     

Sterilization validation for medical compresses at IRASM multipurpose irradiation facility

In Romania, IRASM Radiation Processing Center is the unique supplier of radiation sterilization services—industrial scale (ISO 9001:2000 and ISO 13485:2003 certified). Its Laboratory of Microbiological Testing is the sole third party competent laboratory (GLPractice License, ISO 17025 certification in progress) for pharmaceutics and medical devices as well.We here refer to medical compresses as a distinct category of sterile products, made from different kind of hydrophilic materials (cotton, non-woven, polyurethane foam) with or without an impregnated ointment base (paraffin, plant extracts). These products are included in the class of medical devices, but for the sterilization validation, from microbiological point of view, there are important differences in testing method compared to the common medical devices (syringes, catheters, etc).In this paper, we present some results and practical solutions chosen to perform a sterilization validation, compliant with ISO 11137: 2006.     

Dose response and post-irradiation characteristics of the Sunna 535-nm photo-fluorescent film dosimeter

Results of characterization studies on one of the first versions of the Sunna photo-fluorescent dosimeter™ have previously been reported, and the performance of the red fluorescence component described. This present paper describes dose response and post-irradiation characteristics of the green fluorescence component from the same dosimeter film (Sunna Model γ), which is manufactured using the injection molding technique. This production method may supply batch sizes on the order of 1 million dosimeter film elements while maintaining a signal precision (1σ) on the order of ±1% without the need to correct for variability of film thickness. The dosimeter is a 1 cm×3 cm polymeric film of 0.5-mm thickness that emits green fluorescence at intensities increasing almost linearly with dose. The data presented include dose response, post-irradiation growth, heat treatment, dosimeter aging, dose rate dependence, energy dependence, dose fractionation, variation of response within a batch, and the stability of the fluorimeter response. The results indicate that, as a routine dosimeter, the green signal provides a broad range of response at food irradiation (0.3–5 kGy), medical sterilization (5–40 kGy), and polymer cross-linking (40–250 kGy) dose levels.     

The radiation resistance of the bioburden from medical devices

An extensive study of the radiation resistance of microbial species constituting the bioburden of a number of different medical devices obtained from Japanese medical device manufacturers has been carried out. A standard protocol for determining radiation resistance was used and validated at the fourteen centres involved in the study. Individual microbial isolates from the bioburden obtained from seven different devices manufactured in these centres were studied. A total of 3742 unselected isolates were obtained, of which 197 failed to survive long enough for subsequent radiation resistance studies. The remainder were subjected to an initial screen test to identify those organisms that were sensitive to the lethal effects of radiation with a D₁₀ of < 1.5kGy. The 465 isolates that survived the screen doses were then tested for survival in an incremental series of radiation doses using methods similar to those of Whitby (1979) and Yan and Tallentire(1995). The isolates from “dry” devices were more resistant than those obtained from the one water filled (“wet”) device studied. The overall distribution of radiation resistance among the isolates was considered to be similar to that forming the “Standard Distribution of Resistance” (SDR) included in the ISO International Standard 11137 “Sterilization of Health Care Products — Requirements for validation and routine control — Radiation sterilization”.     

Radiation sterilization of chitosan sealant for vascular prostheses

Chitosan was used as a sealant of knitted polyester vascular grafts. Three sterilization methods for chitosan-coated prostheses were tested: sterilization with ethylene oxide, formaldehyde and irradiation with gamma rays. Radiation sterilization was found to be the most promising of tested methods. The radiation-induced changes in chitosan irradiated in solid state were investigated. Main chain scission was found as the predominant effect of irradiation. Changes in IR and UV spectra were analyzed. Existence of some post-effects was detected. It seems that the observed increase in biocompatibility of chitosan surface caused by irradiation with sterilizing dose (25 kGy) is due to some structural factor connected with a decrease in molecular weight.     

Characteristics study of clear polymethylmethacrylate dosimeter,Radix W,in several kGy range

Basic characteristics of Radix W, a commercially available undyed polymethylmethacrylate (PMMA) dosimeter conventionally used by readout at 320 nm, were studied in the dose range of 0.5–8 kGy, for its wide application especially for the evaluation of the sterilization dose and the quality assurance of food irradiation. The characteristics of dose response, the effect of irradiation temperature, and its stability after irradiation were examined over candidate readout wavelengths of 270–320 nm. The dose response readout at shorter wavelength is higher than that at longer wavelength, and 280 nm is the suitable readout wavelength for measurement of dose range of 0.5–8 kGy. The post-irradiation stability of dose response for 6 kGy is less than 1% within 24 h after irradiation at an irradiation temperature of 20 °C. Dose response is higher with temperature at irradiation temperatures in the range of −40 to 20 °C.     

Effect of γ-radiation on ointment cold cream

In this paper, Co-60γ ray was used to irradiate the ointment cold cream at room temperature (25°C). We also used FTIR, GC and thin film chromatogram to analyse various irradiated samples. It was found that the ointment cold cream can be irradiated at dose of 5–35 kGy and at dose rate from 0.2 to 0.6 kGy/h at room temperature (25°C) without evident decomposition. At dose of 5–15 kGy, the number of bacteria can be reduced to hygienic standard value. The radiation sterilization is a safe method for killing the bacteria in the ointment cold cream.     

Effect of radiation on disinfection and mechanical properties of Korean traditional paper,Hanji

Fumigants, including methyl bromide and ethylene oxide, are generally used for the preservation of the Korean cultural heritage, especially paper products like letters and books. However, the use of fumigants is banned because of their harmful effects on humans and the environment. Gamma irradiation is being considered as an alternative for the sterilization of insects and fungi in organic products. Therefore, the purpose of this study was to investigate the sterilization effects of radiation and its effect on the mechanical properties of the Korean traditional paper—Hanji. Treatment doses of 9 kGy and 8 kGy of gamma irradiation inactivated 5 log units of Aspergillus niger and Bacillus cereus spores inoculated on Hanji, respectively. The gamma irradiations up to an absorbed dose of 50 kGy resulted in no significant changes in the tensile strength, bursting strength, and appearance of Hanji. These results confirmed that radiation treatment disinfects the Korean traditional paper efficiently without changing its properties and that this treatment could be used to prevent the damage of Korean ancient archives by molds and fungi.     

Comparative study of effect of low and medium dose rate of γ irradiation on microporous polysulfone membrane using spectroscopic and imaging techniques

The effect of low (LDR) and medium dose rate (MDR) of γ irradiation at low doses (0-100 kGy) on the structural and chemical changes of microporous polysulfone (PSf) membrane has been studied using UV-vis, FTIR, SEM and dead-end filtration techniques. PSf membrane was cast by phase inversion method. Irradiation was done at room temperature in air media. The doses chosen were 0-100 kGy for LDR and 0-50 kGy for MDR; they were below and above sterilization dose. Analysis of UV-vis and IR spectra and SEM images obtained suggested that chain scissions and crosslink had occurred simultaneously in the irradiated membranes in both cases. This radio-oxidation effects observed start at a very low dose i.e. 1.66 kGy and increase with increase in dose. It is supported by the flux values obtained; it is increased with increase in dose. The results indicate that a very low dose γ irradiation was able to change the physicochemical characteristics of microporous PSf membrane which depend on dose rate of exposure.     

Influence of electron beam irradiation on physicochemical properties of poly(trimethylene carbonate)

Electron beam (EB) irradiation of poly(trimethylene carbonate) (PTMC), an amorphous, biodegradable polymer used in the field of biomaterials, results in predominant cross-linking and finally in the formation of gel fraction, thus enabling modification of physicochemical properties of this material without significant changes in its chemical structure. PTMC films (M_w: 167-553 kg mol⁻¹) were irradiated with different doses using an electron accelerator. Irradiation with a standard sterilization dose of 25 kGy caused neither significant changes in the chemical composition of the polymer nor significant deterioration of its mechanical properties. Changes in viscosity-, number-, weight-, and z-average molecular weights of PTMC for doses lower than the gelation dose (D_g) as well as gel-sol analysis and swelling tests for doses above D_g indicate domination of cross-linking over degradation. EB irradiation can be considered as an effective tool for increasing the average molecular weight of PTMC and sterilization of PTMC-based biomaterials.