— Zentr Steril 2002; 10 (3): 158-167 Keywords Bacillus subtilis * ethylene oxide © sterilisation process * kill kineties AREER mon Kill Kinetics Study of Bacillus subtilis Spores in Ethylene Oxide Sterilisation Processes D. Heider’, J. Gémanr?, U. Junghans’, U, Kaiser? kill kites of Bacillus subilis spores in ethylene oxide sterilisation process- es were determined while varying te process parameters ethylene oxide concentration, rel- ative humidity (RH) temperature and inert gas type. The experiments were carried outin an style oxide rsistometer in accordance with EN 886 Part2. The D values inferred from ‘the kill kinetios ware used to determine the roaction velocity changes of the various processes conducted. The study showed, on changing the eth- von oxide concentration rom 0 1200 mgl, that the reaction velocity ofthe process retly influenced bythe athylone oxide centration and thatit corresponds somewhat toareaction ofthe firstordar kinetics across the entire concentration ange. On varying the relative humidity from 100 10%, we found that there was a corolation botwoon the ro- action velocity inthe range between 10 and {60% RH, whereas no further changes were observed at higher levels. On varying thetem- perature between 30 and 64°C, it was ob- ‘thylone oxide concentration between 0 and 150 mg/l for sterilisation, no changes were observed on adding air, whereas on adding carbon dioxide the reaction velocity declined by up to 32%. Introduction and Task Definition Ethylene oxide sterilisation processes are boing used worldwide on a broad scale in the medical devices industry when ther- ‘mal or irradiation sterilisation processes cannot be used. To minimise the con- sumption, release of ethylene oxide into the environment as well as the residual amount of the gas in the devices, attempts are being made to operate with minimal ethylene oxide concentrations and short storlisation times. Whereas formerly con centrations between 600 maf up to 1000 magll were used, concentrations of even less than 300 mgf are being used at pres- ent, Moreover, inert gases are added to the pure sterilisation gas in order to curtail the Fisk of explosions. To guarantee the safety of the ster sation process, the influence exerted by all relevant process parameters must be ‘quantitatively elucidated, as required by the standard EN-ISO 14937, In our study we investigated the influence generated by he parameters ethylene oxide concen. tration, relative humidity and inert gas type (air and carbon dioxide} on the reaction velocity in sterilisation processes with et ylene oxide, In order to be able to evalu ‘ate the influence generated by these pa: remeters, one parameter was varied in leach case while keeping the others con- stant in aceordance with the requirements ‘specified in the standards EN 866-2 and ISO 111382, The reaction kinetics was ‘experimentally determined by plotting sur- vival curves in accardance with the Euro pean standard EN 866-1 and the interna- tional standard |SO 11138-1 inan ethylene oxide resistometer which conformed to the standards EN 866-2 and ISO 11138-2, A mathematical model of the reaction k netics was inferred from the results. Material and Methods ‘The investigations were conducted in an ethylene oxide resistometer designed by the firm gke-mbH, The principle coms nent of this resistometer was a tworltre lass reaction vessel. An external water thermostat via a water jacket with a pre- Cision of 0.1 K af the set point was used 1683 10 provide for temperature control of this vessel. A vacuum-tight asitator device in the vessel made provision for homoge: nous conditions within the reaction chem- bor, The active ingredients were fed via supply pines, controled by magnet valves, from supply containers into the chamber, A vacuurn pump was connected for evac: uation of the reaction vessel. The steril- sation conditions were controlled via @ Pt 100 temperature sensor of pracision class 8 (90.2221, manufactured by Juchheim GmbH & Co., Fulda) and a pressure sen- sor with a characteristic deviation of 0.5% (4753, manufactured by Juchheim GmbH & Co,, Fulda); a recorder (L 250, manu- factured by Linseis, Selb) was used for data registration. A sample holder was Used to secure the samples in the reaction chamber. The test organisms employad were bi- ‘logical indicator strips from gko-nbH con- taining the test organism Bacilus subtilis (ATCC 9372}. The mean population was 2.0.x 10* cfulstrip. AD, value of 2.9 min- utes was obtained at 84 °C under the ster ilisation conditions specified in EN 868-2. The test procedure set out inthe stan- dards EN 866-2 and ISO 11138-2 was used as a reference test condition to as: certain the D value at 64°C (soe table 1). ‘Then one process parameter was changed in each case and the change in the reac- tion velocity was obtained by measuring the D value, 1 Hossshue Anta, Brburyer St 52-57, 0-05966 Kotter 2 gkesnbH, Ast der Lind 10, 05578 Waltons Esch CENTRAL SERVICE Volume 10-2002D. Heider et al. STERILISATION ) Parameter | Reference conditions as por EN ‘Variation range ofthe parameters used inthis study E10 Cone 600 mgi + 30mg 50-1200 mg = 10 mg Relative amnos- | 60% AH = 10% 10 100% RH 3% phere humidity Temperature 058°C 201°C Inert gas- rnonet, ar CO, added Table 1 The stndard process condons set outin EN 66-2 andthe variation range of the paramotorsinvostigatod inthis study, * due tothe nature ofthe process, minimum quanttos of residual romain inthe reaction chamber sul \ Ss 6 0 s - tylene oxide concentration ‘As a preparation for measurements, and having used a thermostat to maintain the temperature of the resistometer at the ‘operating temperature, the biological in- dicators to be measured were fixed on ‘the sample holder in the upper section of the reaction vessel, The agitator was ‘switched on to ensure hamageneous con- ditions throughout the entire reaction ves- sel. The reaction vessel was evacuated 1060 mbar to remove ai. Since it was nat possible to fully remove the ait, @ residual air concentration of 2.2 mmol/l was pres tent for each measurement, Having evac- uated the reaction vessel and reached the set point temperature, the requisite ac- tive ingredients were supplied. For pre- conditioning, the amount of steam re- Quired for setting the relative atmospher- ic humidity was supplied from a thermo- stat-controlled water reservoir and kept e1 Correlation betwoen te D valve and the ethylene oxide concentration at °C 60% Rl) constant for 30 min. Then the other gas- es were added. The concentration of the gases was set by measuring their partial pressure values. For sterilisation with the inert gases “sir” as ambient ait or “cat bon dioxide”, the gases were added un- {il the ambient pressure of 1000 mB had ‘been reached, in order to achieve as high {a8 possible a concentration of the inert ‘gases. The equipment settings remained Unchanged until the end of the gassing duration, The pressure and temperature values were continually monitored for con- {orrmance with the set point. At the end of the experiments, the gases were with- drawn from the reaction chamber and the chamber was ventilated. Air removal was repeated on four occasions 0s to com- pletely remove the £0. The biological in- dicators were removed from the reaction vessel and their population assessed im- 164 mediately. To datermine the population, the biological indicators were _ho- ‘mogenised in a blender filed with 100 ml water. The suspension obtained was sub- jected to a defined dilution process and plated onto atrypticase solid medium in accordance with the requirements of the USP 24 and the German Pharmacopoeia {DAB) 10, Colonies were counted after a two-day incubation period at 35 °C.The population of surviving organisms wes cal culated by multiplying with the dilution factors. ‘Based on the requirements of EN 886- 2, itignot necessary to neutralise the eth- ylone oxide. The ethylene oxide is com pletely removed by the ensuing anc re- peated evacuation of the reaction chamber and any residual amounts of ethylene ox- ide are greatly diluted by the subsequent treatment of the bilogical indicators, The experiments were evaluated by determining the B values ofthe biological indicators employed. The D value is de- fined as the time in minutes needed 10 r= ‘duce the baseline microbial count ofa par- ticular microorganism by one power of tenin accordance withthe conditions stip- Ulatedin te standard, orto reduce the mi- crobial count by 90%, The reaction ve- locity of the sterilisation process can bein ferred. by measuring the D value. The D value is inversely proportionsl to the re action velocity r of the process (© ~ VN Having pitted the experimental D val us against the experimental varabies, the influence exerted by the measured parameters is illustrated. For the kinetics study, the influence ‘generated by the parameters ethylene ox- ide concentration, relative atmospheric humidity, temperature and addition of at ‘and carbon dioxide, as inert gases, was in vestigated. Tre) o 4 8 oF Dvaluo(nin) | 135 572818 Table? Correlation batwoon D values and temperature (50 mg/ Et, 60% RH)Kill Kinetics of B. subtilis Spores in Ethylene Oxide Sterilisation Siktanow — Results + Influence Exerted by the Ethylene Oxide Concentration ‘The correlation between the D value and the ethylene oxide concentration was meas- uted over a concentration range between ‘50 mg/l and 1200 mgf (see Figure 1). The D value was somewhat inversely propor- tional to the ethylene oxide concentration ‘28cf0ss the entire measuring range. Dvalue ° = ° ny ry 1 ¥20 | Influence Exerted by the Relative Humidity Relate atmospheric huniiy ‘The correlation between the D value and Figure 2 Correlation between the D value and tha relative atmospheric humidity at 84°C, 600 mg/l E{0) the relative humidity in the range 10 ~ 100% RH was measured (Figure 2), For a relative humidity between 10nd 30 %, it ‘was demonstrated that there was a strong correlation between the D value and the relative humidity. In the range between ‘30 and 50% relative humidity it was pos- sible to observe a sort of transition range, ‘while the D value approached a saturation value when the relative humidity increased ‘to above 60% (see Figure 2). Toa 0 values) Influence Exertod by the Temperature Inaddition, the correlation between the D value and the temperature in the range Figure3Ascortainmont of z value (at O% AH, 600 mg EO) 30 to 64°C wes studied. An Increese in ‘temperature led toa reduction inthe D vat tue and thus to an inerease in the reaction velocity, ‘The z value gives the temperature in °C or K required to change the D value by cone power of ten. The Q,, value is given fas the quotient reaction velocity constant for each 10 k change in temperature. A zvalue of 35.4 °C (Figure 3) and a, vat ‘Tomperature Expein. series x | Ue of 2.0 wore caleviated from the re- 2O{mgt) | ruationvesso mbar] tein sults. The latter means that the reaction ms velocity doubles if the temperature is A < ar changed by 10 K, ° ion = a6 Influence Exerted by Ar or Carbon 1000 sors | an The correlation between the reaction vo- 22 am fan [am | - 180 locity and the addition of air or carbon 10 1000 zat | sar | sia | - 188 dioxide wes measured. To tis effect, the 1000 2 | ssi | 2a | amas | isa D values obtained when conducting ster 28 aw Tas a eto wh amour of eens oie, i" ‘ aa [ am faut] — | mt) tea tana wre compared wth ud SL a bon ye ‘those obtained on using on the one hand, Correlation between the D values and the adian of sir or carton dowde. Thee saries of ® Mixture ethylene oxide, steam and air experiments were conducted with an athylone oxide concentration of50, 100 and 160mgil___ 84, on the other hand, @ mixture of eth- ‘and diferont concentrations ofthe gases air and carbon dioxide at constant tempe ylene oxide, steam and carbon dioxide of 4°C and RH of 60%, ‘The results (see Table 3) demonstrate thet 165 | GanmAat SERVICE Voie 10-3002)