Experiences using IAEA Code of practice for radiation sterilization of tissue allografts: Validation and routine control

Problems of tissue allografts in using International Standard (ISO) 11137 for validation of radiation sterilization dose (RSD) are limited and low numbers of uniform samples per production batch, those are products obtained from one donor. Allograft is a graft transplanted between two different individuals of the same species. The minimum number of uniform samples needed for verification dose (VD) experiment at the selected sterility assurance level (SAL) per production batch according to the IAEA Code is 20, i.e., 10 for bio-burden determination and the remaining 10 for sterilization test. Three methods of the IAEA Code have been used for validation of RSD, i.e., method A1 that is a modification of method 1 of ISO 11137:1995, method B (ISO 13409:1996), and method C (AAMI TIR 27:2001). This paper describes VD experiments using uniform products obtained from one cadaver donor, i.e., cancellous bones, demineralized bone powders and amnion grafts from one life donor. Results of the verification dose experiments show that RSD is 15.4 kGy for cancellous and demineralized bone grafts and 19.2 kGy for amnion grafts according to method A1 and 25 kGy according to methods B and C.     

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.     

Physicochemical and functional characteristics of radiation-processed shrimp chitosan

The effects of gamma irradiation on chitosan samples were determined in terms of physicochemical and functional properties. Shrimp chitosan was extracted from shell using a chemical process involving demineralization, deproteinization, decolorization and deacetylation. Commercial snow chitosan was also used. Samples (in a solid state) were given irradiation dose of 25 kGy at a dose rate of 1.1013 kGy/h in air and 0 kGy samples were used as controls. Results showed that moisture contents were between 8.690% and 13.645%. There were no significant differences (P>0.05) in the degree of deacetylation of the chitosan samples. Significant differences (P<0.05) were observed in the viscosity and viscosity-average molecular weight of the chistosan samples. Viscosity and molecular weight decreased when the samples were given the irradiation dose of 25 kGy. Chitosan samples had low antioxidant activity compared with BHT. Water binding capacity ranged from 582.40% to 656.75% and fat binding capacity was between 431.00% and 560.55%. Irradiation had a major effect on the viscosity and the viscosity-average molecular weight of the chitosan samples.     

DSC and EPR analysis of some radiation sterilized alkaloids

The effect of ionising radiation on the physico-chemical properties of salts of three alkaloids has been studied: codeine phosphate (COD), papaverine hydrochloride (PAP) and pilocarpine hydrochloride (PIL). These compounds in the solid state were irradiated with an e-beam of the energy of 9.96 MeV to achieve doses ranging from 25 to 400 kGy, and then they were subjected to organoleptic analysis, thermal analysis (differential scanning calorimetry, DSC), electron resonance (EPR) spectroscopy, scanning electron microscopy observations and X-ray diffraction study. The most informative were the results provided by the EPR and DSC methods. The EPR spectra revealed the presence of long-lived radicals whose concentration was directly proportional to the dose of irradiation for all the compounds studied. (PIL 2.14 × 10¹⁶ spin/g, COD 6.85 × 10¹⁵ spin/g, PAP 2.50 × 10¹⁴ spin/g—for the dose of 100 kGy). The DSC results revealed a decrease in the melting point by 5.9 °C for COD and by 0.8 °C for PIL after irradiation with 200 kGy, which is indicative of products of radiolysis, of which at least one is non-white, and changes the colour of the compounds. PAP, for which no decrease in the melting point and no colour change was observed and for which the concentration of free radicals was the lowest, was found to be most stable from among the compounds studied. It will probably be suitable for radiation sterilisation. The other two compounds COD and PIL show much lower radiochemical stability and should be subjected to more detailed examination to establish the mechanism of radiolysis and the possibility of radiation sterilisation. Our results have confirmed the earlier reports on high radiochemical stability of PAP, but do not confirm the resistance to ionising radiation of COD and PIL.     

Spectroscopic analysis of pindolol irradiated in the solid state

Pindolol ((2RS)-(1-(1H-indol-4-iloxy)-3- [(1-metyloetylo)amino]-2-propanol) in substantia was exposed to ionising radiation emitted by high energy electrons from an accelerator, in the standard sterilisation dose of 25 kGy and in higher doses from the range 50–400 kGy. The effects of irradiation were checked by spectrometric methods (UV, MS, FT-IR, EPR) and hyphenated methods (HPLC-MS) and the results were referred to those obtained for non-irradiated sample. EPR results indicated the presence of free radicals in irradiated samples, in the amount of 1.36 × 10¹⁶ spin g^?1 for 25 kGy and 3.70×10¹⁶ spin g^?1 for 400 kGy. The loss of pindolol content determined by HPLC was 1.34% after irradiation with 400 kGy, while the radiolytic yield of the total radiolysis for this dose of irradiation was 2.69×10⁷ mol J^?1. By means of HPLC-MS it was possible to separate and identify one product of radiolytic decomposition, which probably is 2-((R)-3-(1H-indol-4-yloxy)-2-hydroxypropylamino)propan-1-ol formed upon oxidation. In the range of sterilisation doses (25–50 kGy), pindolol was found to show high radiochemical stability and would probably be safely sterilised by the standard dose of 25 kGy.      

DSC and spectroscopic studies of disulfiram radiostability in the solid state

The effect of ionising radiation on the physico-chemical properties of disulfiram (Antabuse, Esperal, bisdiethylthiocarbamoil disulphide) has been studied by DSC, FTIR, EPR, MS, TLC and HPLC. Sterilisation was carried out in the solid state, at room temperature and normal air humidity using the electron beam of 9.96 Mev from accelerator. All the measurements were made simultaneously for the irradiated and nonirradiated substance. It has been found that the drug studied in solid phase when subjected to an electron beam corresponding to the irradiation in the doses 10–100 kGy shows the presence of free radicals (EPR), and a change in colour from white to pale green-grey that disappears after solution in water or methanol. After the irradiation with the dose of 100 kGy, its melting point and enthalpy slightly decreased. Also the content of the active substance decreases (HPLC −1.5%, UV −3.6%, iodometric titration method −2.7%) and trace amounts of the radiolysis products appear (HPLC). The substance irradiated with the doses 10–50 kGy does not show changes in the melting point, in the content and presence of the radiolysis products. The EPR results have shown that free radicals disappear after about a year and the discolouring disappears with them. The results of this study have shown that disulfiram can be subjected to sterilisation by irradiation with no deterioration of its physico-chemical properties, but a long time of storage needed to remove free radicals and discolouration questions the economic justification for this type of sterilisation.     

Thermal study of four irradiated imidazoline derivatives in solid state

Four imidazoline derivatives: antazoline (AN), naphazoline (NN), tymazoline (TM), xylometazoline (XM), in the form of hydrochlorides in solid phase have been subjected to high energy e-beam irradiation from an accelerator (~10 MeV) at a dose varied from 25 to 200 kGy. The effects of the irradiation have been assessed by DSC, X-ray diffraction, FTIR, EPR and TLC. The standard sterilisation dose of 25 kGy has been found to produce changes in the properties of one derivative (XM), two other ones (AN and TM) have been found sensitive to doses >100 kGy, whereas NN has been resistant to irradiation in the whole range studied (25–200 kGy). EPR results indicated that the changes taking place in the therapeutic substances studied are related to radical formation. The irradiation induced changes in colour, a decrease or increase in the melting point, changes in the XRD pattern, small changes in the shape of FTIR peaks and the presence of radiolysis products. The XM compounds cannot be sterilised by irradiation because of the radiation induced changes in its physico-chemical properties.     

Chemical, sensory and microbiological changes of gamma irradiated coconut cream powder

A study was carried out to determine optimum decontamination dose for a locally manufactured coconut cream powder. Samples were gamma irradiated (0–15 kGy) and ageing process was achieved using GEER oven at 60 °C for 7 days, which is equivalent to one-year storage at room temperature. Iodine value (IV), ranging from 4.8 to 6.4, was not affected by radiation doses and storage, however peroxide value and thiobarbituric acid (TBA) generally increased with radiation doses. In most samples, peroxide value (meq/kg) reduced after storage, whilst the TBA (mg malonaldehyde/kg), indicator for product quality, slightly increased. The sensory evaluation conducted using 25 taste panellists indicated that scores on odour, creamy taste and overall acceptance for all irradiated samples at more than 5 kGy were significantly lower (P<0.05) than the control. However, the panellists could not detect any significant differences among the irradiation doses (P>0.05). All stored products were significantly different in colour, creamy taste, odour and overall acceptance (P<0.05) when compared to the non-stored non-irradiated control. Microbiological count of the samples prior to irradiation was in the range of 1×10²–1.7×10³ cfu/g with no detection of Salmonella sp. and Escherichia coli. No microbial colonies were detected after irradiation. Based on the TBA and overall sensory acceptance, gamma irradiation of 5 kGy was found to be the optimum dose and lower doses can be considered to decontaminate coconut cream powder.     

Inactivation of prion infectivity by ionizing rays

Inactivation of prion deposits on medical devices or prion contamination in pharmaceutical raw materials is considered as impossible by using gamma irradiation. Early, the guideline WHO/CDS/CSR/APH/2000 has described irradiation as an ineffective process. But, in 2003, S. Miekka et al. noted radiation inactivation of prions in a particular application to purify human albumin, shown by the physical denaturation of the infectious protein (PrP). The aim of our study was to determine the inactivation of prions with a scrapie model (strain C506M3) by irradiating standardised preparations. Results: Gamma irradiation was partially effective, showing a 4–5 log reduction on exposure to 50 kGy. A characteristic effect–dose curve was not observed (25, 50 and 100 kGy), only an increase in the incubation period of the murine disease (229 days with 25 kGy to 290 days with 100 kGy) compared with 170 days without irradiation. Since the inactivation was not a total one, the observed effect is significant. It is proposed that further work be undertaken with the model to investigate the application of gamma radiation known levels of prion contamination.     

Effect of gamma-irradiation and refrigerated storage on the improvement of quality and shelf life of pear (Pyrus communis L., Cv. Bartlett/William)

Gamma-irradiation alone and in combination with refrigeration was tested consecutively for 3 years for extending the shelf life of pear. Matured green pears were irradiated in the dose range of 0.8–2.0 kGy and stored under ambient (temperature 25±2 °C, RH 70%) and refrigerated (temperature 3±1 °C, RH 80%) conditions. Dose range of 1.5–1.7 kGy extended the storage life of pear by 14 days under ambient conditions. Control unirradiated pears were almost fully ripe within 8 days, while as the pears irradiated in the dose range of 1.5–1.7 kGy were fully ripe within 22 days of ambient storage. Irradiation dose of 1.5–1.7 kGy significantly inhibited the decaying of pears upto 16 days of ambient storage. Irradiation in combination with refrigeration prevented the decaying of pears upto 45 days as against the 35% decay in unirradiated samples. Irradiation dose of 1.5–1.7 kGy also gave an extension of 8 and 4 days during additional ambient storage of the pears following 30 and 45 days of refrigeration, respectively.     

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.     

Effect of gamma irradiation on the antimicrobial and free radical scavenging activities of Glycyrrhiza glabra root

The efficacy of gamma irradiation as a method of decontamination for food and herbal materials is well established. In the present study, Glycyrrhiza glabra roots were irradiated at doses 5, 10, 15, 20 and 25 kGy in a cobalt-60 irradiator. The irradiated and un-irradiated control samples were evaluated for phenolic contents, antimicrobial activities and DPPH scavenging properties. The result of the present study showed that radiation treatment up to 20 kGy does not affect the antifungal and antibacterial activity of the plant. While sample irradiated at 25 kGy does showed changes in the antibacterial activity against some selected pathogens. No significant differences in the phenolic contents were observed for control and samples irradiated at 5, 10 and 15 kGy radiation doses. However, phenolic contents increased in samples treated with 20 and 25 kGy doses. The DPPH scavenging activity significantly (p<0.05) increased in all irradiated samples of the plant.     

Quality of fresh-cut Iceberg lettuce and spinach irradiated at doses up to 4 kGy

Fresh-cut Iceberg lettuce packaged in modified atmosphere packages and spinach in perforated film bags were irradiated with gamma rays at doses of 0, 1, 2, 3, and 4 kGy. After irradiation, the samples were stored for 14 days at 4 °C. O₂ levels in the packages of fresh-cut Iceberg lettuce decreased and CO₂ levels increased with increasing radiation dose, suggesting that irradiation increased respiration rates of lettuce. Tissue browning of irradiated cut lettuce was less severe than that of non-irradiated, probably due to the lower O₂ levels in the packages. However, samples irradiated at 3 and 4 kGy had lower maximum force and more severe sogginess than the non-irradiated control. In addition, ascorbic acid content of irradiated lettuce was 22–40% lower than the non-irradiated samples after 14 days of storage. The visual appearance of spinach was not affected by irradiation even at a dose of 4 kGy. Consumer acceptance suggested that more people would dislike and would not buy spinach that was treated at 3 and 4 kGy as compared to the non-irradiated sample. Overall, irradiation at doses of 1 and 2 kGy may be employed to enhance microbial safety of fresh-cut Iceberg lettuce and spinach while maintaining quality.     

X-ray versus gamma irradiation effects on polymers

Today, the most common methods used for medical device sterilisation are by gaseous ethylene oxide and by electron beam or gamma irradiation. With X-ray sterilisation about to enter the market, its material compatibility needs to be assessed at doses typically encountered during a sterilisation process. This paper reports on a study that compares the effects of exposing different types of plastics that are commonly used in medical devices to ⁶⁰Co or to 5 MeV X-rays. The dose rate for both irradiation modalities was of the same order of magnitude. Under these conditions, both types of radiation are found to have similar effects on polymer properties.     

The effect of sterilization methods on the thermo-gelation properties of xyloglucan hydrogels

In this study, the influence of different sterilization methods on the thermo-gelation and structural properties of xyloglucan hydrogels was investigated. Xyloglucan samples were treated by either 70% ethanol, 70% isopropanol, γ-irradiation (10 kGy) at room temperature, γ-irradiation (10 kGy) in dry ice or autoclaving. These samples were tested for sterility by incubation with sterile Lysogeny Broth (LB) at room temperature, 30 °C and 37 °C for 30 days. According to the results obtained, xyloglucan hydrogels were only effectively sterilized by autoclaving or by γ-irradiation either at room temperature or in dry ice. These samples were analyzed by rheology measurements and dynamic and static light scattering analysis. Gamma-irradiation at room temperature markedly changed the polymer structure, preventing thermo-gelation. Only autoclaving and γ-irradiation in dry ice preserved the rheological properties of the polymer. The sol-gel transition as a function of the temperature was similar for these samples and the control sample.     

Effect of electron irradiation on nitrofurans and their metabolites

Research on the degradation of aqueous furazolidone, nitrofurantoine and semicarbazide (SC) solutions, and 3-amino-2-oxazolidinone (AOZ) residues in tissues of chicken and crucian under electron beam irradiation have been carried out. Results showed that about 75% furazolidone and 70% nitrofurantoine degraded at 6 kGy dose, and SC with the initial concentration of 667 μg/L degraded by 94% at 12 kGy dose. While AOZ in the crucian and chicken degraded by 22.5% and 20.7%, respectively, after being irradiated at 12 kGy. The degradation conditions were investigated to provide a reference to improve irradiation techniques.     

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.     

Effect of high-dose irradiation on quality characteristics of ready-to-eat chicken breast

High-dose (higher than 30 kGy) irradiation has been used to sterilize specific-purposed foods for safe and long-term storage. The objective of this study was to investigate the effect of high-dose irradiation on the quality characteristics of ready-to-eat chicken breast in comparison with those of the low-dose irradiation. Ready-to-eat chicken breast was manufactured, vacuum-packaged, and irradiated at 0, 5, and 40 kGy. The populations of total aerobic bacteria were 4.75 and 2.26 Log CFU/g in the samples irradiated at 0 and 5 kGy, respectively. However, no viable cells were detected in the samples irradiated at 40 kGy. On day 10, bacteria were not detected in the samples irradiated at 40 kGy but the number of bacteria in the samples irradiated at 5 kGy was increased. The pH at day 0 was higher in the samples irradiated at 40 kGy than those at 0 and 5 kGy. The 2-thiobarbituric acid reactive substance (TBARS) values of the samples were not significantly different on day 0. However, on day 10, the TBARS value was significantly higher in the samples irradiated at 40 kGy than those at 0 and 5 kGy. There was no difference in the sensory scores of the samples, except for off-flavor, which was stronger in samples irradiated at 5 and 40 kGy than control. However, no difference in off-flavor between the irradiated ones was observed. After 10 days of storage, only the samples irradiated at 40 kGy showed higher off-flavor score. SPME-GC–MS analysis revealed that 5 kGy of irradiation produced 2-methylbutanal and 3-methylbutanal, which were not present in the control, whereas 40 kGy of irradiation produced hexane, heptane, pentanal, dimethly disulfide, heptanal, and nonanal, which were not detected in the control or the samples irradiated at 5 kGy. However, the amount of compounds such as allyl sulfide and diallyl disulfide decreased significantly in the samples irradiated at 5 kGy and 40 kGy.     

Predicting optimal conditions to minimize quality deterioration while maximizing safety and functional properties of irradiated egg

Irradiation is an excellent method for improving the safety and functional properties of egg. However, the internal quality of egg can be deteriorated due to a rapid decrease in Haugh units. In this study, the optimal conditions for maintaining the quality and maximizing the safety and functional properties of egg were determined when combination of irradiation and chitosan coating was treated using response surface methodology (RSM). Independent degradation parameters—irradiation dose (0–2 kGy) and concentration of chitosan coating (0–2%) were assigned (?2,–1, 0, 1, 2), and 10 intervals were set on the basis of central composite design for the degradation experiment. The dependant variables within a confidence level less than 5% were Haugh units, foaming ability, foam stability, and number of Salmonella typhimurium. The predicted maximum values of Haugh units and foaming ability were 82.7 (irradiation dose 0.0006 kGy and concentration of chitosan solution 1.03%) and 62.2 mm (1.99 kGy and 0.86%), respectively. S. typhimurium inoculated on the egg surface was not detected after 1.86 kGy and 0.48%. Based on superimposing four-dimensional RSM with respect to freshness (Haugh units), functional property (foaming capacity and foam stability), and reduction of S. typhimurium, the predicted optimum ranges for irradiation dose and chitosan solution concentration were 0.35–0.65 kGy and 0.25–0.85%, respectively. The predicted optimum values were obtained from 0.45 kGy and 0.525%. This methodology can be used to predict egg quality and safety when different combination treatments were applied.