A. Tallentire

Radiation sterilization — evaluation of a new approach for substantiation of 25 kGy

Abstract The fundamentals of a prototype method for substantiation of a sterilization dose of 25 kGy have been described in a recent report (Kowalski, J.B., Tallentire, A., 1999. Radiat. Phys. Chem. 54, 55). The method, whose basis is the conservative Standard Distribution of Resistances (SDR), incorporates an approach to identifying the maximal dose to be used in the verification experiment (VD max ). The approach has been subjected to computer evaluations involving challenging with hypothetical microbial populations of various sizes and resistances. The evaluations utilised the SDR (Distribution C), Distributions A and B which both have resistances less than that of the SDR, and Distribution D that is greater in resistance than the SDR. Fifty-four modified distributions, which varied in resistance in a graded and systematic manner from that of the SDR, were also utilised. Where appropriate, the outcomes from challenging the VD max approach were compared with those of the dose substantiation method specified in ISO TR 13409 (Method TR) and dose setting Method 1. The results of the evaluations revealed uniformly safe and unambiguous outcomes with Distributions A, B and the SDR. For Distribution D, such outcomes were observed at low and high bioburden levels, whereas, at intermediate bioburden levels, ambiguous outcomes were observed. From an analysis of the evaluations that employed Distribution D and the modified distributions, the latter outcomes were found to be inevitable with challenges having widely differing bioburden levels and resistances which exceed that of the SDR. To examine the influence of the individual components of the SDR on substantiation outcomes, ‘impact factors’ were calculated. Differences in the most influential components were seen for the VD max approach and Method TR. A comparison of such factors for the VD max approach and Method 1 has affirmed the conservativeness and credibility of VD max .

BACTERIAL SURVIVAL IN SYSTEMS OF LOW MOISTURE CONTENT: Part IV. The Effects of Increasing Moisture Content on Heat Resistance, Viability and Growth of Spores ofB. subtilis

INTRODUCTION PREVIOUS papers112 in this series have shown that the spores of B. subtilis will remain viable in dry spray-dried powders for long periods. This is true for peptone powders, yet if such powders are dissolved in a suitable quantity of water they form an excellent medium for bacterial growth ; the spores germinate, and the resultant vegetative cells rapidly multiply. Some intermediate proportion of water must be just sufficient to cause the spores to initiate the germination process. It was the purpose of this present work to determine this critical water content and to investigate the fate of the spores. Obviously they will lose their heat resistance at some point, but, the interesting question is, “will the resultant vegetative cells multiply or will they die off due to unfavourable environmental conditions?’ Such questions have an intrinsic interest of their own, but also it might be possible that advantage could be taken of the facts to devise conditions under which sterility would be attained with or without the use of minimal quantities of heat or antiseptics. Methods might then be elaborated which could have direct pharmaceutical applications in the preparation of sterile powders. Previous work related to this subject seems to have been of a qualitative rather than a quantitative nature. Thus Tompkins3 found that the range of humidities, over which germination of certain species of mould spores is possible, varies with the temperature ; the further the temperature was removed from the optimum for growth, the narrower the range of humidity over which germination occurred. When a spore germinates a series of changes is initiated. (a ) The sporecase cracks or is absorbed or is gelatinised and then di~solved.~ (b) The spore contents enlarge, a germination tube may grow out through the envelope, or the cell may simply enlarge and divide, giving rise directly to vegetative multiplication. (c ) The resistance to adverse environmental conditions, such as heat and harmful chemical substances f a k 4 All these changes have been used as criteria of germination, Observation of (a) and (b) involves direct microscopical examination of the individual spores. This can easily be carried out in the case of moulds where the spores are relatively large. This method was employed by Tornpkin~.~ Bacterial spores are approximately one-tenth the size of mould spores and morphological changes on germination are indistinct and difficult to observe. On the other hand, as pointed out by Wynne and F o ~ t e r , ~

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

Abstract The International and European standards for radiation sterilization require evidence of the effectiveness of a minimum sterilization dose of 25xa0kGy but do not provide detailed guidance on how this evidence can be generated. An approach, designated VDmax, has recently been described and computer evaluated to provide safe and unambiguous substantiation of a 25xa0kGy 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 VDmax for quarterly dose audits; Facility B compared VDmax and Method 1 in side-by-side parallel experiments; and Facility C, a new facility at start-up, used VDmax for initial substantiation of 25xa0kGy 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 VDmax 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 VDmax experiments were consistent with the outcomes observed with Method 1. The VDmax approach has been extended to sterilization doses >25 and

The spectrum of microbial radiation sensitivity

Abstract The innate sensitivity of micro-organisms towards high energy radiation varies widely; different types, species and strains exhibit greatly differing sensitivities. Certain environmental factors are also able to influence the response of a given type of microorganism to radiation, e.g., the presence of oxygen, water or adventitious water-soluble organic material. Such environmental influences hold equally for all types of microorganisms, irrespective of the innate sensitivities which they possess.

Radiation sterilization—computer evaluation of ISO 11137 dose setting method 1 and ISO/TR 13409 method for substantiation of 25 kGy

Abstract Computer evaluations of ISO dose setting/substantiation methods, involving challenging them with hypothetical microbial populations of various sizes and resistances, have shown the methods to be generally reliable and safe. However, the ability to Method 1 to detect at a sterility assurance level (SAL) of 10 −2 the occurrence of populations of resistances greater than that of the standard distribution of resistances (SDR) decreases with decreasing bioburden level. The ISO method for substantiation of 25 kGy operates correctly for populations of high bioburden levels whose resistances are either greater or lower than that of the SDR. At low bioburden levels, however, the method can provide “incorrect” results—substantiation is rejected even though an SAL of 10 −6 or less is obtained at 25 kGy. This occurs because there is no direct relationship between the outcome of the verification dose experiment and attainment at 25 kGy of an SAL of 10 −6 .

A DEPENDENCE ON WATER CONTENT OF BACTERICIDAL EFFICIENCY OF GAMMA-RADIATION.

Gamma irradiation of Bacillus megaterium spore samples of different water content was carried out at 22 deg in the absence of oxygen. To avoid lethal damage from postirradiation oxygen effect, all samples were given an anoxic postirradiation soaking with liquid water. Surviving fractions based on colony counts gave exponential dose/survival curves that were used as a measure of radintion lethal efficiency. The equilibrinm water vapor pressure range tested was from 5 x 10-4 to 21 torr. In changing from a condition in which spores are driest to one in which they are in equilibrium with water vapor at a partial pressure equivalent to 100 percent relative humidity, the lethal efficiency of the gamma radiation increases by about 35 percent.

Protection of protein A-sepharose columns irradiated to sterilization doses

Abstract The effects of ionizing radiation on protein A-sepharose mini columns have been investigated. Radiolysis to a dose of 25 kGy is accompanied by a 55% loss of binding capacity to IgG. Various OH. radical scavengers have been used to render them radiation resistant at the high doses commonly used for the purpose of sterilization. Inclusion of mannitol at high concentrations (1 mol dm−3) partially protects the columns as does ascorbate (10−2 mol dm−3), the loss of binding being only ∼15% at 25 kGy. A mixture of mannitol (1 mol dm−3) and ascorbate (10−2 mol dm−3) however protects to ∼5% decomposition. This value is not effected by the presence of oxygen at the beginning of irradiation.

Distribution of radiation resistances of microbiological contaminants of a cotton-based medical product

Abstract A distribution of radiation resistances of microorganisms has been compiled from the results of D10 determinations of isolates recovered from a cotton-based medical product. In all, 250 organisms were isolated from a total microbial population of around 21,000 organisms present on about 170 g of product. D10 values of the isolates fall within the range of 0.5 to 3.6 kGy. The findings indicate that organisms having a D10 value greater than 3.6 kGy occur amongst contaminants on this cotton product at a probability of less than 1 in 5000. The overall resistance of the population of organisms found in the present study is somewhat less than that of the ‘Standard Distribution of Resistances’ used in ‘AAMI dose setting methods’.

Studies on the postirradiation oxygen effect in bacterial spores.

Experiments were designed to elucidate the mechanisms of postirradiation effects of oxygen in bacterial spores. Kaolin powder contaminated with spores of Bacillus subtilis was dried, sealed under vacuum, and treated at 22 un. Concent 85% with up to 800 krad of gamma radiation from a Co/sup 60/ source. The criterion of lethal damage was the inability of the spore to give rise to a colony on incubation in nutrient agar. From colony counts of samples yielding surviving fractions less than 0.5, exponential dose/survival curves were constructed and the slopes estimated using the expression: surviving fraction = e/ sup -kD/ where k is the slope and D the dose in krad. The slope is used as a measure of the lethal efficiency of the radiation, higher values of k indicating greater efficiency. The highest level of lethal efficiency shown is that resulting from postirradiation storage of spores in oxygen for 48 hr (k = 0.045 krad/sup -1/). The lowest efficiency is that for identical oxygen treatment preceded by exposure to nitric oxide for 15 min (k = 0.010 krad/sup -1/), showing the prevention of the postirradiation oxygen effect by nitric oxide. Also, the development of the postirradiation oxygen effect can bemorexa0» arrested by removing the oxygen and can be restarted by re-admitting oxygen to the dried spore system. Treatment with nitric oxide between exposures to oxygen, however, prevents further development of the oxygen effect. (TCO)«xa0less

Sub-process irradiation of naturally contaminated hypodermic needles

Abstract The proportion of nonsterile hypodermic needles within a population of needles ( P ), contaminated initially with native microflora, has been measured as a function of gamma-radiation dose ( D ) over the range 0 – 4 kGy. The relationship between P and D was regular and allowed extrapolation to higher doses corresponding to the generally accepted probability of occurence of a nonsterile item or sterility assurance level (SAL), and beyond. An absorbed dose of 7.5 kGy was estimated to be sufficient to give a value of P of 10 -6 , whereas a dose of 25 kGy would be expected to give a P of 10 -18 .