Radiation Sterilization EFFECT OF HIGH-ENERGY GAMMA RADIATION FROM KILOCURIE RADIOACTIVE SOURCES ON STEROID HORMONES WILLIAM TARPLEYI AND RfILTON YUDIS Physical Qrgunic C h e m i s t q D e p a r t m e n t , Scienti$c Research Division, Schering Corp., Bloomjield, N . J .
BERSARD IVANOWITZ, ROBERT V. MORRIGAN, AND JEROME W'EISS Reactor Science and Engineering D e p a r t m e n t , Brookhaven National Laborutory, Upton, N . Y .
T
HE absorption of high energy ionizing radiation such as
that from x-ray machines, cathode ray (electron) acceleraBots, or gamma photon-emitting radioisotopes, although producing only negligible temperature rise (3) has been shown t o Be extremely effective in the destruction of microorganisms &, 8, 9, SO, 34). When these radiations interact with matter, fonization occurs, usually of the more abundant solvent moleeules, but with no induction of radioactivity in the materials exposed (26). The penetration of x- or gamma radiation (energy = 1,000,000 electron volt's) is very great (31) and aqueous ayeterns may be effect>ivelysterilized through a volume several feet in diameter. In contrast, cathode rays (energy = 3,000,000 electron volts) n-ill penetrate only about 3 / 8 inch (13),and ultraviolet radiation is effective for only a fraction of a millimeter (17). Thus, x- or gamma radiation mag be used to sterilize many guccessive thicknesses of material a t one exposure. Such sterilization of heat-labile materials in the final container might compete successfully with the more laborious aseptic procedures nomemployed (4). The use of various electromagnetic radiations to effect steriliEation is not new. The commercial application of ultraviolet radiation for surface sterilization has become widely accepted 489). A recent review article ( 2 6 ) discusses t,he work of a number of investigators Tvho have studied the action of ionizing radiation on various t,ypeB of microorganisms. Application of more penetrating radiation such a8 x- and gamma rays, however, has awaited the availabilit,y of relat,ively inexpensive and reliable aourceR, capable of delivering the dose required for sterilization. The recent availability of intensely radioactive sources (1000curie) (ai),and t,he potential availability of even more intensely radioactive bp-products from the atomic energy program, malie investigation of the feasibility of radiation sterilization of great interest at the present, time. Preliminary studies using radioactive cobalt (cobalt-60) as a Zypical mock fission by-product source have indicated that an exposure of from 2.7 to 3.2 megarep. (million roentgen equivalent ghysicttl) vas necessary to accomplish sterilization of steroid auspensions heavily contaminated wit,h a variety of microorganisma (as). (One rep. is a unit of absorbed radiation corresponding to t,he absorption of energy equivalent to 84 ergs in 1 gram of air.) C l o . d d i u ? nsporogenes, a spore-forming organism, proved the most difficult to kill. Molds and heat-resistant organisms required much less irradiation. A large percentage of the organisms originally present viere destroyed by radiation doses of 600.000 rep., and those not killed failed to reproduce in normal fashion. If there are caees where only a significantly lower total radiation t h e e can be applied, i t would seem possible to reduce the number of orgmisms below Some critical value and thus to attain a more readily stored product. The potcantis1 commercial application of radioactive sources of lonizing gamma radiation may t,ake the form of passing the prod1
P-nmnt address, 119 Kline Blvd., Frederick, N d .
uct through the radiation field around the source by means of conveyors (51). Alternatively, if apparatus were designed t o remove the source by remote control from its lead shield inside a shielded cubicle, much larger volumes of product could be irradiated on a batch basis. As gamma radiation intensity decreases with distance from the source (just as does light), a knowledge of the intensity dependence of the sterilization dose was necessary Bacteriological experiments, carried out in the underwater irradiator (described here), x-ere reported in the first paper in this series (sa). I t was concluded that within the range of 14 to 377 hours needed for irradiation. the intensity a t which the radiation was applied had no effect on the magnitude of the total radiation dose necessary to accomplish sterilization. I n terms of pilot plant design, this means that gamma radiation may be allowed to pass through many successive layers of packages of product, and the time of exposure need be increased only for packages that are more remote from the source. I n any sterilization procedure, it is necessary to evaluate the effect of the procedure not only upon the microorganisms, but as well on the substances and vehicles to be treated. Because of newness of the technique, more extensive proof of stability and lack of toxicity will probably be required for products sterilized by means of radiation than for those given heat sterilization (WO). It is hoped that ultimately knowledge of the principles of radiation sterilization will become widely disseminated and will allow acceptance as in the case of heat sterilization. The present communication describes the relatively simple apparatus requirements and evaluates changes produced by gamma radiation acting upon the steroid hormones, cortisone acetate and pregnenolone acetate. The choice of these substances was dictated by the relative instability of cortisone acetate suspensions to heat sterilization. EXPERIMENTAL
G-~AIMA RAY
.4 practical study of radiation sterilization should be conducted with the waste fission products accumulating as a result of the atomic energy program. HOKever, as their preparation present's great enginecring iroblenis, such sources are not yet available. Accordingly, it w:ts decided to use the more readily available mock fission pr,xluct sources prepared by neutron activation of cobalt or tantalum tuhing ( 2 1 ) . The result'irig radioact,ive isotopes (cobalt-60 and tantalum-182) emit gamma rays with average energies of 1.23 and 1.15 m.e.v., respectively. Two radioactive source geometries were used in the present study. Most of the steroid samples were irradinteti inpide tubular sources rvhich are kept inside of lead containore with 7-inch walls ($8). Although t,his apparatus is admirably niiitod for research, only rmall volumes may be irradiat,ed, and a large percentage of the radiation is lost in the Iead shield. An rinderwater irradiator (Figure 1) was constructed from a radial arrangement of aluminum tubes around a kilocurie source, with simple po3i1458
80URCES.
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DOSIMETRY MEASUREMENTS IN UNDERTABLEI. GAMMA-RAY WATER IRRADIATOR ( a t various distances in water from a tantalum souroe) Distance from Intensity, Intensity, Source Tube KiloTube KiloCenter, Inches Position5 No.6 rep./Hr. N0.b rep./Hr. 112 1 112 F 2 2.2 116 E 118 118 119 D 120 120 C 116 113 B 111 111 A
4.2
F
E
D C B
A
7
32 34 36 35 34 32
8
38 39 41 41 38 32
7.0
a Vertical location in tube. (12 inches higher). b See Figure 2.
A , opposite bottom of souroe; F, opposite top
tions as shown in Figure 2. This device was used t o irradiate certain steroid samples, and was particularly useful for the large number of bacteriological samples required for the sterilization studies (58). Intensity levels corresponding to those used in that study are summarized in Table I for the various positions. From measurement of the intensity of radiation in the various sources and positions, the appropriate length of exposure to produce the desired total radiation dose can be computed. Among the many systems available RaDI.4TION DOSIMETRY. for measuring the intensity of gamma radiation are those based on physical instruments, biological changes, and chemical changes (68). The latter include oxidations, reductions, and destruction of dyes. The radiation-induced conversion of ferrous to ferric ion was used. This system has been extensively studied (IO, 15, 23) and precisely related to the calorimetric dejinition of the roentgen (12). This oxidation was unsatisfactory for total dose measurements in excess of 50,000 rep., but dose rates could readily be obtained. For measuring the total exposure, the reduction of ceric to cerous ion was used. This reaction has been found to be linear with respect to total radiation and intensity up to a t least 7 megarep (36). I n the bacteriological studies (38) a ceric dosimeter vial was incorporated a t the center of each bundle that was irradiated. The conversion in these dosimeters supplied assurance that all bundles received thedesired amount of radiation. CONSIDER4TIONS O F IRRADIATION GEOMETRY.Bacteriological samples were contained in 0.6-ml. rubber-capped pellet vials; samples irradiated for the evaluation of chemical changes were contained in 15-ml. multiple-dose vials sealed with rubber stoppers which were lined with nylon sleeves. All samples were irradiated in the same geometrical arrangement as used for dosimeters; blank vials filled with water were used to fill any empty spaces. The 0.6-ml. pellet vials were grou ed in bundles of seven. The radiation intensity measured in t i e central vial of each bundle was found to be within 2 ~ 1 5 %of that received in any of the other vials. T o eliminate the pcssibility of accidental contamination from other experiments, all packages which were irradiated were checked and proved free of low-level surface contamination, thus supporting the prediction that no radioactivity would be induced in the samples. CONTAINER SIZE AND RADIATIOX DOSE. Because Of secondary electron production during the absorption of gamma rays, an
Figure 1. Underwater Irradiation Apparatus
TABLE 11. EFFECTOF CONTAINERSIZE ON RADIATION Dosas (Ferrous dosimeter system, in central volume of source) Container 0.6-ml. pellet r i a l Bundle of 7 pellet vials 6-ml. vial 15-ml. vial
Dimensions 40 X 8 X 1.5 mm. thick 42 X 19 X 1.5 mm. thick 57 X 26 X 2 mm. thick
Dose Rate, Kilorep./Hr. 180 rt 9 180 zk 9 100 f 5 100 5
*
increase in radiation dose inside the containers might be expected if the ratio of surface to volume was increased. The data OQ Table I1 indicate this to be the case, but the effect is not signiftcant with container sizes greater than 6 ml. A similar incream might also come about if the heavier elenleiits were used in fab& cating the container. PHYSICAL AND ANALYTICAL PROCEDURES
MELTIYQPOINT. Melting points were determined by a modified U.S.P. method ( 8 ) in which the capillary was inserted 10" below the expected beginning of melt, while the bath temperature was increased a t the rate of 1" per minute. Liquefaetion and end of melting points listed in Tables I11 and IV are the average of three observations obtained on different days. Because of polymorphism and solvation complications (14), l ~ 9 well as decomposition which occurs a t the melting temperature, the 2ulimits were large (rt2" to f4' on pregnenolone acetate an4 cortisone acetate, respectively). (Throughout this communios-
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diation were determined in carbon disulfide solution (30 mg. per
ml.) in a 1-mm. cell and the regions obscured by this solvent were TABLE 111. EFFECT OF GAMMA IRRADIATION ON PREGNEXOLONE investigated in purified bromoform
ACETATE
Appearance Melting range, C. Optical rotation, C. Infrared spectra
Starting Material
Comminuted with Water
Comminuted and Irradiated 3.4 Megarep.
Off-white crystalline powder
Off-white crystalline powder
Off-white crystalline powder
148-140.4
143.4-148.2
145.5-148.4
18.3 zt 1 . 6
1 4 . 3 dz 1 . 6
18.6 i 1.8
All identical in solution
(NUMBERS TO
SOURCE
REFER
POSITIONS
POSITION
)
ULTRAVIOLET SPECTRA.The ultraviolet spectra were obtained with an Applied Physics Model 11 double-beam recording spectrophotometer, equipped with quartz prism. A study of the reproducibility of this instrument when the 3-keto-A4 chromophore present in cortisone acetate was measured indicated a dayto-day absorption reproducibility of 1 2 % . In this determination 8 to 10 mg. of sample were accurately weighed on a microbalance and made up to 500 ml. with methanol (25' C.). Spectra were obtained for the region from 220 to 350 mp and the complete analysis was repeated on a t least three successive days. The absorbance value a t the maximum was read from each of the curves and is shown in Table IF7. Typical spectra are shown in Figure 3. The recording spectrophotometer is of particular value, as continuous curves are obtained and the appearance of new absorption bands is readily detected. The ultraviolct method is an extremely sensitive test for traces of impurities with intense absorption bands-e.g., condensed ring, polynuclcar hydrocarbons such as chrysene, cholanthrene, and other aromatic phenanthrene derivatives. The spectra of highly concentrated solutions of cortisone acetate (0.8 mg. of cortisone acetate per ml. of methanol) m r c ohtained in the regions of 270 to 330 mp (Figure 4).
W
Figure 2.
Plan I'iew of Underwater Source (
tion, 20 limits are used to express the precision of measurement. This means that 95% of a series of measurements will be found within such limits, if the variability is due to random errors.) OPTICAL ROTATIONS.Optical rotations (25' C., sodium vapor lamp) were obtained on all samples using 2% solutions.
2 MG./ 100 M L M E T H A N O L )
X X X X
BEFORE IRRADIATION AFTER IRRAOIATION ( 13.7 MEGAREP) [BOTH CURVES CONTINUOUS)
The path length was 2 dm. Samples of 300 mg. of cortisone acetate or of pregnenolone acetate were accurately weighed out into 15-ml. volumetric flasks. Chloroform was used as solvent for cortisone acetate, while abEolute ethyl alcohol was used for WAVELENGTH MJJ pregnenolone acetate. The final volume adjustment was made after the solution had been equilibrated for 30 minutes in a constant temperature bath (25' (2.). Twenty readings of the match Figure 3. Ultraviolet S p e c t r u m of Cortisone Acetate of field were made on the polarimeter for each .ample. Precision ( 2 u limits) of the order of 11.4' was obtained. TABLEIV. EFFECTOF GAMXA IRRADIATION O N CORTISONE ACETATE INFRARED S P E C T R A .The infrared spectra xere obtained ( 2 a limits used throughout to indicate precision of measurements) with a Perkin-Elmer &f ode1 21 double-beam spectrophoStarting Comminuted G a m m s Irradiated after Comminuting tometer ( 3 7 ) e m p l o y i n g a Material with Water 3 4 megarep. 6.4 megarep 13.7 megarep. sodium chloride prism. InOff-white Off-white Creamy white Off-white Off-white termediate response and rescrystalline crystalline crystalline Appearance crystalline crystalline olution were used throughout. powder powder powder powder poivder The spcctra were scanned a t 222-224 222-224 226-234 225-230 Melting range, C. 237-240 a , r a t e of 1.5 minutes per 222.2h1.0 217.13~2.5 221.1f1.4 224.711.4 Opticalrotation, [a]*,, 227 i 1 . 4 micron. Mineral oil spectra 97.4% of 58%.0f 9 5 . 6 %, of 58.8%.of were obtained using weighed original origlnal original original samples of steroid in a suitPhaee solubility 57.6f0.5% 95.5&1.0% 94.62~1.070 0 3 . 6 1 1 . 0 % ... pure pure pure pure able volume of mineral oil to p r o d u c e fingerprint r e g i o n spectra as nearly comparable All solution spectra identical Infrared solution spectra in intensity as possible. ResInfrared solid state Slight differThese solid-state spectra identical olution of intense carbonyl spectra ence in finger a b s o r p t i o n bands was impoint region proved by dilution of the Dihydroxyacetone side 19,820 1360 1'2 780 f 350 1 9 , G50 i 350 ... 19,230 & 350 samples with additional minchain 99!6,% of 95%. of 97%. ?f original original original eral oil. The spectra of cor15,620 f 370 16,580 & 370 15,310 370 15 460 1370 3-Kcto-A4structure 15,620 1370 tisone acetate were determined 98%. o! 08:9% of 90 7%. of original original original in purified bromoform soluAbsorbanceat208mp 0 , 4 8 3 i. 0.016 0.498 10 . 0 1 6 ... 0.483 10.016a 0.498 10.016 tions a t a concentration of 50 mg. per ml. Cells of 0.2-mm. a I m urities from 3.4- and 6.4-megarep. experiments were treated by solubility analysis techniques under condipath length were used. The tions wtich would concentrate impuntie& if present, eightfold. spectra of pregnenolone acetate both before and after irra-
*
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1461
of total impurity present. This procedure will allow detection of as little as 0.5% of other components without previous knon-ledge of the nature of the impurity. Analysis of the fractions with the most and least amounts of impurity, which may be isolated in the course of the procedure, will detect significantly smaller amounts of impurity (83). STEROID IRRADIATION PROCEDURE
280
290
300
310
320
WAVELENGTH MU Figure 4. Ultraviolet Spectrum of Cortisone Acetate
Suspensions were prepared by comminuting separately 6 grams each of cortisone acetate and pregnenolone acetate in water. The concentration of suspended solids was 133 and 143 mg. per ml., respectively. The resulting mixtures were very thick foamy suspensions which did not settle during the course of the experiment. Microscopic examination demonstrated that 98% of the particles were less than 10 microns in their
COLORIMETRIC ASSAYOF DIHYDROXYACETONE SIDE CHAIN. The method of Porter and Silber (n), slightly modified to fit the requirements of the double-beam recording spectrophotometer, was used for determining this functional group in the cortisone acetate molecule. Day-to-day reproducibility of absorbance values measured at the maximum (413 mp) gave 2u limits of &2%. Results of this analysis are shown in Table
z
IV.
t - 3 I
SOLUBILITY ANALYSISO F CORTISONE ACETATE.Solubility analysis was applied t o the cortisone acetate samples both before and after irradiation as a measure of the amount of degradation produced. The procedure has been described (33). A series of sealed glass ampoules was prepared t o contain various amounts of excess solid cortisone acetate in equilibrium with bennene solutions. After equilibration for 14 days, aliquots of the clear supernatant solution were evaporated to constant weight in tared weighing bottles. Curves were drawn in which the amount of solid per milliliter of solution was plotted against the amount of solid initially added per milliliter of solvent (Figure 5 ) . The slope of this curve times 100 is equal t o the percentage
Figure 6.
SOLVE N T: BENZ E NE
-
0
t-
7
4,-
t t t
NON-IRRADIATED
5
IO
15
20
WEIGHT OF S A M P L E MG./ML. SOLVENT Figure 5.
Solubility Analysis of Cortisone Acetate
major dimension. Each of the preparations was divided into two parts, one for irradiation and one t o be used as a control. Two 15-ml. multiple dose vials of cortisone acetate and two of pregnenolone acetate were sent to the Brookhaven National
Infrared Spectra of Pregnenolone Acetate before and after Irradiation
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INDUSTRIAL AND ENGINEERING CHEMISTRY
Laboratory for irradiation. They yere exposed for 24 hours in a radioactive tantalum source a t an intensit,y of 1.4 X 106 rep. per hour, thus receiving a total irradiation dose of 3.4 megarep. This total dose corresponds t o that necessary for sterilization (52). The control vials were stored a t room temperature. The irradiated samples appeared exactly like the controls, except, for an intense brownish colorat,ion of the glass. The glass faded rapidly during the first few hours after its removal from the radiation field, and more s l o \ ~ l yover an extended period. The colorat,ion persists for several years and could he used to distinguish irradiated from nonirradiated vials. The irradiated suspensions were quantitatively transferred to evaporating dishes and dried to constant weight in a vacuum oven a t 40" C. (small nitrogen stream). The total residues were ground and mixed until each was uniform. The unirradiated control samples were similarly treated. The results of physical and chemical measurement are summarized in Tables 111and IV. As it was felt that cortisone acetate should be the more labile substance, it was decided t o overirradiate this steroid and prepare concentrates of any degradation products for biological testing. Accordingly, one half of the cort>isone acetate which had received 3.4 megarep. was further irradiated in a tantalum source. This portion received an additional 3 megarep. a t 1.2 X 105 rep. per hour, increasing the total dose to 6.4 megarep. (twice the radiation dose necessary for sterilization). I n addition, a new sample of cortisone acetate suspension, prepared as before, was irradiated in a cobalt source for a total irradiation dose of 13.7 megarep. (approximately 4.25 t,imes the radiation dose necessary for sterilization, at 1 X 105 rep. per hour). The data on the products from these irradiations are in Tables I11 and IT'. RESULTS ARD DISCUSSIOY
The data presented in Tables 111 and IV clearly establish that less change in the steroid molecule wax produced by gamma irradiation than in the preparation of the suspension. The effects of radiation on other substances of biological interest have been extensively explored ( 2 8 ) . I n general, these changes involve oxidation or other reactions leading to loss of activity. However, in the present investigation, general tests for unpredicted products were included in addition to tests for specific chemical changes and loss of the major constituent.
Vol. 46, No. 7
SPECIFICTESTS. This laboratory as well as ot,hers ( 1 ) has found that among the most specific tests for the presence of impurities is the appearance of new absorption bands in the infrared spectra or the disappearance of absorption bands charact'eristic of the steroid in question. This test is particularly valuable for cortisone acetate and for pregnenolone acetate, as there is a multiplicit,y of absorption bands, permitt'ing many points of comparison. From the infrared spectra of Figures 6, 7 , and 8, it may be seen that irradiation with as much as 6.4 megarep. did not change t,he infrared spectrum of pregnenolone acetate and that irradiation with as much as 13.7 megarep. did not change the spectrum of cortisone acetate. Since t)hese spectra were obtained in solution, quantitat,ive comparison is possible. The spectra of cortisone acetate before and after irradiation 6 t h 6.4 megarep. were also obtained in the solid state as mineral oil mulls. No detectable absorption band changes resulted from irradiation. The comparison of solid state spectra of otherwise ident>icalsamples is somet,imes complicated by differences in crystal orientation, particle size, polymorphism, and solvation. Such differences in solid state spectra, observed when start,ing material and comminuted material were compared, disappeared in solution and the samples may therefore be considered identical by this criterion ( 7 , 18). UELTING RAZGE. The melting ranges of both cortisone acetate and pregnenolone acet,ate are lowered during the prolonged comminuting with m-ater. This lowering is believed t o be due to changes in the crystalline structure, and in part to slight hydrolysis of the acetate group. The melting range of steroids is sometimes very sensitive to such minor changes, Significantly, within the limits of this measurement, there is no further alteration in melting points upon irradiation. COLORIMETRIC ASSAYFOR DIHYDROXYACETONE SIDE CHAIN. The reaction described by Porter and Silber has been found very specific, as either oxidation or reduction of t'he side chain will lead to changes which can be detected ( 2 7 ) . Furthermore, the absence of the eleven-carbonyl group in the cortisone acet'ate molecule will lead t o failure of t'he test. However, hydrolysis of the 21-acetoxy group will not, be detected. This reaction may then be rega.rded as both a test for the presence of these functional groups in the cortisone acetate molecule and a specific identification. The data of Table 11- indicate no detectable alteration
July 1954
INDUSTRIAL AND ENGINEERING CHEMISTRY
Figure 8.
1463
Infrared Spectra of Cortisone Acetate before and after Irradiation 13.7 megarep.
by this criterion, in that the molecular extinction coefficient values did not change upon irradiation. OPTICAL ROTATIOX.The optical rotation of steroids is a very specific property, although changes may sometimes be simulated by appropriate mixtures of impurities. When considered in conjunction with the other data of this investigation, the specific optical rotation serves as both a qualitative and quantitative indication that little or no change has resulted. ULTRAVIOLET SPECTRA.The destruction of unsatupted systems through acetoxylation or hydroxylation has been reported as the chief reaction induced by the irradiation of other steroids (19). In the case of cortisone acetate, the ultraviolet absorption spectrum provided a very sensitive method for detecting any alteration in the unsaturated 3-keto-A4 chromophore normally present in this molecule. The data shown in Table IV clearly indicate no loss of this very important functional group. It can also be seen from Figures 3 and 4 that the ultraviolet absorption curves may be superimposed. Thus it may be concluded that no new substances have been formed with absorption bands in the ultraviolet region. Further evidence on this point is available from the spectra of highly concentrated solutions of cortisone acetate in the region from 270 to 330 mp. The absorbance of cortisone acetate in this region is very low ( A = 150 to 420), although a certain amount of absorbance is normally present, owing to the tail of the intense absorption band a t 237 nip. On the other hand, the absorbance of condensed ring polynuclear hydrocarbons is extremely high ( e = 50,000 to 160,000) ( 1 1 ) . Unlikely as the formation of such hydrocarbons as chrysene, picene, cholanthrene, and pyrene may br, since complex dehydrogenation processes would necessarily have to take place, it was felt important to prove their absence. Correcting for the background absorption of cortisone acetate, it has hren calculated that as little as 0.07% would be detectable b.v change in shape of the absorption curve. The curves found and thP absorption reported a t 297 mp cIcarly indicate no detectahlr change (Figure
4). Furthermore, the least pure fraction mas isolated by the solubility analysis technique (33) from the 3.4- and 6.4-megarep. experiments. In this way impurities were concentrated eightfold. Therefore, as little as 0.009% should easily be detected in the ultraviolet spectra of these concentrated impurities, but no absorbance increase was observed. GENERALCRITERIAOF CHAXGE.Solubility analysis is the most general test for the presence of impurity; no previous knowledge of the nature of the impurity is required. It is based on the thermodynamic requirement of the phase rule that a pure substance (a single phase) has a constant solubility regardless of the amount of excess solid phase present in the system (%$,Si). This technique has been adapted to steroids in the laboratory and found to be extremely useful (33). Because of the pressure of other work, it was not possible to apply this criterion t o the less interesting pregnenolone acetate. However, analysis of the 3 . 4 and 6.4-megarep. irradiated cortisone acetate indicated no significant change in purity. The experimental error of the technique was found to be 5 0 . 7 % . Analysis of the starting material after comminuting with water indicated the presence of about 4.5% of impurities believed to be the result of partial hydrolysis of the 21-acetate group to free cortisone. These results indicate a high degree of stability for the steroid molecule and are in general agreement with the resistance of such materials to oxidation by chemical reagents (16). English investigators ( 1 9 ) have reported some change in steroid solutions exposed to 1 to 2 megarep. of x-rays. In general, these changes involved interaction with free radicals formed in the solvent (dilute aqucous acetic acid), producing hydroxy and acetoxy groups a t or adjacent to double bond systems. The substances produced appear to be innocuous. The greater stability observed in the present investigation may be due to the low solubility of steroids in thp aqueous system which the authors used. This simulation of dry sterilization may be onr method for minimizing radiation-induced decomposition and Htill accomplishing com-
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I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
plete sterilization (%), as it is well established that the solvent plays a large role in radiation chemistry ( 5 ) . In any investigation of this sort, the final criteria must be the retention of biological activity and the failure to develop toxicity. Sccordingly, long-range biological tcsting of irradiated pregnenolone acetate has begun. Because of t,he diverse biological actions of cortisone acetate, however, it was felt desirable to concentrate the degradation products before biological testing. kttempts have been made to obtain concentrates of any traces of degradation products. These Irere to be studied by the criteria of Table IV and also examined for toxicity and biological activity. The relatively small amount of decomposition indicates that insufficient material was irradiated to yield significant quantities of such degradation products. Accordingly, thia work should be repeated on a larger scale with prolonged irradiation t,ime in order to prepare quantities of degradation products sufficient to study their toxicity. I t is ultimately desirable, although perhaps not immediately necessary, to ascertain the nat.ure of the chemical changes produced in the steroid molecules by over-irradiation. Preliminary computations rvere based on penetration of many successive layers of packages of material by gamma rag's, and on the assumption that 15-ml. multiple-dose vials may be packed into layers with an average density approximately half that of water. By removing and replacing the vials at different distances from the source at appropriate time intervals, approximately 1500 vials per week may be sterilized by irradiation with a 1kilocurie source of cobalt-60. I t is estimated that within a reasonable time period (1 week, minimum radiation intensity level 12,030 roentgens per hr.), the greatest distance from such sources for economical irradiations will be 18 inches. This will involve utilization of approximatcly 75% of the radiation produced by such a source and Frill produce a sufficieiit number of vials for a pilot plant operation. Until data on pilot plant cost can be obtained and evaluated, it will be impossible to discuss the economic aepect,s of the gamma ray sterilization. However, the data accumulated so far in t,his study indicate that the application to st,eroid sterilization may be one of the earliest practical examples of the utilization of waste fission products. SU>I>\IARY
Irradiation of steroid solutions has been reported by others to lead to structural changes. In contrast, irradiation of suspensions of cortisone acetate with up to 13.7 megarep. (approximately 4.5 times the sterilization dose) and of pregnenolone acetate with up to 6.4 megarep. (trvice the sterilization dose) produced no change in t,he molecules which can be detected by appearancc or by the physical and chemical criteria applied. Gamma-ray irradiation, which like x-irradiation cannot induce radioactivity, is successful in the sterilization of aqueous suspensions of cortisone acetate and pregnenolone acetate. Irradiated cortisonc acctatc and pregnenolone acetate suspensions satisfy all normal specifications, except melting range, and meet rigorous general and specific tests designed to detect minute changes. These t,ests include ultraviolet and infrared spectrophotometry, optical rotat,ion, melting range, solubilit'y analysis, and a specific colorimetric test. A suggested explanation for the melting point lorvering, observed upon comminuting of the steroids within water, has been advanced on the basis of solid state changes and slight hydrolysis of the acetate groups. Significantly, no further change in melting point was observcd upon irradiating the comminuted steroids. There has been no loss of the functional groups believed t o be responsible for the biological activity. The gamma-ray intensity fields around a kilocurie source have been determined. As the radiation dose necessary for sterilization is independent of the distance from the source, it was possible to forecast pilot plant sterilization rates.
Vol. 46, No. 7
Biological experiments are under way to determine whether any change in the irradiated steroids can be detected in animals. On the basis of the chemical and physical tests performed, none are to be expected. ACKXOW LEDGM ER'T
The authors wish to acknowledge the assiet,ance of Cecelis Vitir~llo, Betty Blasko, and EdTvard Townley of the Schering Corp., and Otto Kuhl of the Brookhaven National Laboratory. The counsel of J . H. Hayner, Engineering Division, Atomic Energy Commission, and of R. E. Waterman and E. B. Hershberg of the Schering Carp. is grat'efully acknowledged. LITERATURE CITED
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ACCEPTED
>larch 15, 1954.
